Hypera postica), a major herbivorous pest feeding on legumes, and its host plant alfalfa (Medicago sativa). Insect and plant samples were collected from a crop field and an adjacent meadow. We characterised the diversity and abundance of viruses associated with weevils and alfalfa, and described nine putative new virus species, including four associated with alfalfa and five with weevils. In addition, we found that trophic accumulation may result in a higher diversity of plant viruses in phytophagous pests compared to host plants.Advances in viral metagenomics have paved the way of virus discovery by making the exploration of viruses in any ecosystem possible. Applied to agroecosystems, such an approach opens new possibilities to explore how viruses circulate between insects and plants, which may help to optimise their management. It could also lead to identifying novel entomopathogenic viral resources potentially suitable for biocontrol strategies. We sampled the larvae of a natural population of alfalfa weevils ( Our knowledge of viruses infecting insects is largely skewed towards a small number of species of economical or health concerns in humans and their domesticated plants and animals . Yet, inViral metagenomics has revolutionised virus discovery from samples of any nature and at all scales, i.e., from the scale of an individual to populations living in the same environment ,5,6,7,8.Insects are integral components of agrosystems, where they fulfil crucial functions, if only to cite pollination. However, the abundance of food and favourable trophic conditions can make some herbivorous or sap-feeding species switch to real “pests of concern”. Management plans to reduce insect pests’ pressure on food production have long relied on chemical insecticides, which are easy to use and efficient, making made them success . As a coBiocontrol refers to the use of natural enemies to control pests, among which microbial pesticides and viruses have proven efficient and successful, although their use remains limited to a few cropping systems . EntomopHypera postica  is a major widespread pest that can reach high densities on leguminous plants. This pest particularly affects alfalfa (Medicago sativa L.) yield production, resulting in significant economic losses in Europe and the USA [H. postica, although few biocontrol trials with parasitic wasps (Bathyplectes species), generalist predators  and fungal pathogens have been reported [Anthonomus grandis) [The weevil  the USA . Damages the USA . Chemicareported ,17. Howegrandis) .The objective of this work was to explore the diversity of viruses associated with the alfalfa weevil using a viral metagenomic approach. We characterized for the first time the diversity of viruses that can be found in this pest, including four new putative virus species infecting alfalfa and five new virus species infecting weevils.H. postica) and alfalfa leaves (M. sativa) species were collected on the 1st April 2015 in Domaine de Restinclières, Prades-le-Lez, France . The sampling took place in an experimental plot including an alfalfa field that did not receive any insecticide and an adjacent meadow. Insects were collected with nets on a surface of 100 m2/sample in both crop and meadow. Alfalfa leaves were randomly collected from plants located in the crop field and in the neighbouring meadow. One leaf was sampled per individual. Samples were maintained and transported on ice to the lab and stored at −80 °C until processing. Larvae of alfalfa weevils  were degraded by DNase and RNase incubation at 37 °C for 1.5 h. Total RNA and DNA were then extracted using a NucleoSpin kit . Reverse transcription was performed by SuperScript III reverse transcriptase (Invitrogen), cDNAs were purified by a QIAquick PCR Purification Kit  and complementary strands synthesised by Klenow DNA polymerase I. Double-stranded DNA was amplified by random PCR amplification. Samples were barcoded during reverse transcription and PCR steps using homemade 26-nt Dodeca Linkers and PCR multiplex identifier primers. PCR products were purified using NucleoSpin gel and PCR clean-up . Three negative controls were added during (1) viral purification, (2) nucleic acid extraction, and (3) cDNA purification. Finally, libraries were prepared from purified amplicons and sequenced on an Illumina MiSeq V3 chemistry to generate 300 nt paired-end (PE) reads .Viromes were obtained from 14 alfalfa weevil samples  and 2 alfalfa plant samples  as described previously  Supplem. Briefly−5 [−3 in order to eliminate likely false positives. Viral contigs completion and coverage was assessed by iterative mapping using BOWTIE2 2.3.4.3 with the options end−to-end and very-sensitive [Illumina adaptors were removed, and reads were filtered for quality  using cutadapt 1.18 . Cleaned−5 . All conensitive . Putativensitive . In all ensitive . Mutatiohttps://talk.ictvonline.org/ accessed on 21 March 2021).To determine if viral contigs belonged to new species, their nucleotidic sequences or their predicted protein sequences were aligned and compared with the 10 closest related viral genomes found by similarity searches performed above using MAFFT v7.450 with the G-INS-i algorithm ,30 or MUIflaviridae and Alphaflexiviridae family trees for which 100 bootstrapped replicates were used. Trees were mid-point rooted and visualised with FigTree 1.4 (http://tree.bio.ed.ac.uk/software%20/figtree/ accessed on 21 March 2021).Phylogenetic trees were built using maximum likelihood methods for all reconstructed genomes corresponding to new species in order to place them within the currently known viral diversity and to infer their possible host range. Representative sets of replication and capsid proteins, or polyproteins, were extracted from the NCBI GenBank non-redundant (nr) database for each taxonomic group in which the genomes were classified . Amino acid sequences were aligned using MUSCLE 3.8.425 (16 iterations) with default settings . SequencHypera postica samples using specific PCR and Sanger sequencing. Purified DNA was screened for the presence of this virus using a specific primer pair targeting a 1127 nt long region of the polyprotein gene . A PCR mix was prepared using the HotStarTaq Master Mix kit  according to the manufacturer′s protocol. Amplification conditions were as follows: 95 °C for 2 min, then 35 cycles of 95 °C for 2 min, 55 °C for 1 min, 72 °C for 2 min and a final extension step of 72 °C for 5 min. Amplicons were Sanger sequenced , and sequences were aligned with the reference genome sequence.The presence of the most abundant virus species found in weevils (Hypera postica associated iflavirus 1) was confirmed in H. postica viromes were made using the Vegan package [H. postica and M. sativa were visualised using barplots and a heatmap.Statistical analyses were performed using R and RStudio 1.2.5019 software ,35. Data package . DiffereThe taxonomic assignment of contigs obtained from 16 samples  and arthropod-infecting virus belonging to nine families . Sequences belonging to three families of bacteriophages  were also detected but at low abundance , Permutotetraviridae, Sinhaliviridae, Phenuiviridae, Qinviridae, Reoviridae, Birnaviridae and Mesoniviridae, were detected at low abundance and low frequency, in one or two samples only  , where they can represent high read counts, like members of the family Partitiviridae (>1000 reads), in five weevil samples ; this result is likely to be due to the low number of collected plants  compared to collected arthropods. No arthropod-infecting virus sequences were found in the viromes of alfalfa plant samples. Interestingly, members of families Partitiviridae and Alphaflexiviridae were present in most, if not all, weevil samples, although at various abundances. Members of the family Partitiviridae were found to be also highly abundant in alfalfa samples, unlike Alphaflexiviridae family members that were not detected in alfalfa samples, which might be explained by their high and low prevalence in the agrosystem, respectively. Finally, viruses belonging to the family Amalgaviridae displayed higher read counts in the alfalfa collected from the meadow compared to the crop. The low abundance and presence of the members of this virus family in the weevils sampled in the crop further support that amalgaviruses displayed a low prevalence and/or cause covert infection in alfalfa  . More pre family . They reles only . Plant/fviridae) , where t samples . On the  alfalfa .We reconstructed 17 contigs from the 16 samples, ranging from 1712 to 12,255 nt and we obtained the full coding sequences for all but one, for which we recovered > 95% of its coding sequence . The depnt identity). These viruses likely represent new isolates belonging to six plant virus species , Medicago sativa amalgavirus 1 , Medicago sativa alphapartitiviruses 1 and 2 , Bean leafroll virus  and Alfalfa virus F  [Eight of these contigs correspond to six virus species infecting alfalfa that have already been reported . Their g species , namely,fivirus) .Alphaflexiviridae, Platypuvirus; species demarcation is <80% aa sequence identity in the capsid or polymerase proteins [Iflaviridae, Iflavirus; species demarcation is <90% aa sequence identity in the capsid proteins [Solemoviridae, Sinhaliviridae and Permutotetraviridae) and could not be proposed for classification, although they likely represent new species given their low aa identity with their closest relatives  and Hypproteins )  and three Hypera postica-associated sobemoviruses . The Alphaflexiviridae family phylogenetic tree indicates that HpaAV clustered with viruses isolated from plants , these viruses displayed low abundance/absence in alfalfa, which could suggest that their infection level in the plant and/or their prevalence in the crop is probably low , which comprises viruses that only infect arthropods. Both iflaviruses clustered with viruses discovered in Coleoptera (Aulacophora lewisii and Lampirys noctiluca), their closest relative being Aulacophora lewisii iflavirus 1 that was isolated from a beetle species  and leafhoppers (Scaphoideus titanus), suggesting that the genetic diversity and the host range of this virus family are probably more diverse than currently recognised. Hypera postica-associated sinaivirus is placed at the base of the three species members of the Sinaivirus genus  currently reported to infect bees , HpaV1 encodes for a long single polyprotein . The closest relatives of this virus are the Wuhan house centipede virus 3 (NC_033458) and the Diabrotica undecimpunctata virus 1 (MN646770), which have been isolated recently from two arthropod species, including the house centipede (Scutigera coleoptrata) and the spotted cucumber beetle (Diabrotica undecimpunctata). Since these viruses all infect arthropods, we speculate that HpaV1 likely infects H. postica  ssRNA viruses, whose closest relatives belong to the Picornavirales order. Like members in this virus order  ssRNA viruses. We found reads classified in only three families of DNA viruses, two belonging to virus families whose host range comprises plants (Geminiviridae and Caulimoviridae) and one to a family comprising arthropod-infecting viruses (Parvoviridae), although they represent low numbers of reads so we could not reconstruct any of their full coding sequences. We cannot exclude that the greater abundance of small viruses could be due to the sample preparation method, based on virus-like particle enrichment and involving filtration and ultracentrifugation steps that are known to bias virome composition toward small viruses [Second, it is noticeable that most of the virus sequences reported here correspond to small RNA viruses (16 out of 20), with viral particle ranging in size from 30 to 80 nm, except for the members of the  viruses ,45. MethThe results we obtained from this field screening allowed us to identify arthropod- and plant/fungus-infecting viruses that potentially correspond to nine novel virus species. Interestingly, arthropod viruses were more divergent from their closest relatives than the putative plant/fungus-infecting viruses that we found in weevils. Moreover, six plant-infecting virus species described in this paper belong to already known species. These findings illustrate the poor knowledge we have of the diversity of arthropod-infecting viruses including from “well known” insect pests that threaten major cropping systems ,46. The Two novel RNA virus species  were relatively abundant in most of the insect samples, suggestive of a covert or an overt infection. Interestingly, the alfalfa weevil larvae complete their whole development on the same plant. This biological feature would make virus horizontal transmission and spreading to the population poorly efficient, limited to the few individuals living on the same plant, which is an argument for persistent infections and virus vertical transmission. Indeed, iflaviruses can often result in covert, sublethal infections in their insect hosts , althougIn addition to arthropod viruses, this dataset highlights that the weevil samples also contain a higher diversity, although a low abundance, of putative plant/fungal viruses compared to plant samples. The richness of plant/fungal viruses in weevils can be attributed to sampling bias, both in the number of samples from pooled individuals (14 weevils versus two plant samples) and the number of individuals per sample . However, the higher richness of plant viruses in insects can also be explained by (1) trophic accumulation, as already reported in dragonflies , or by (Today, viral metagenomics and high-throughput sequencing allow us to fully integrate viruses into the dynamics of agrosystems. Such approach allows us to detect infections in insect communities that have largely been ignored to date, in particular, concerning insects of concern, such as pests and vectors. Major challenges remain ahead, both conceptual and methodological: first, in basic virology, to integrate co-infections and virus communities into the understanding of the functioning of agrosystems; second, in applied virology, to better integrate “beneficial” viruses into sustainable management programmes.


Brassica species possesses sporophytic self-incompatibility (SSI), which is controlled by the pollen- and stigma-determinant factors SP11/SCR and SRK. However, the mysterious molecular mechanism of SI remains largely unknown. Here, a new class II S haplotype, named BrS-325, was identified in a pak choi line ‘325’, which was responsible for the completely self-compatible phenotype. To obtain the entire S locus sequences, a complete pak choi genome was gained through Nanopore sequencing and de novo assembly, which provided a good reference genome for breeding and molecular research in B. rapa. S locus comparative analysis showed that the closest relatives to BrS-325 was BrS-60, and high sequence polymorphism existed in the S locus. Meanwhile, two duplicated SRKs (BrSRK-325a and BrSRK-325b) were distributed in the BrS-325 locus with opposite transcription directions. BrSRK-325b and BrSCR-325 were expressed normally at the transcriptional level. The multiple sequence alignment of SCRs and SRKs in class II S haplotypes showed that a number of amino acid variations were present in the contact regions (CR II and CR III) of BrSCR-325 and the hypervariable regions (HV I and HV II) of BrSRK-325s, which may influence the binding and interaction between the ligand and the receptor. Thus, these results suggested that amino acid variations in contact sites may lead to the SI destruction of a new class II S haplotype BrS-325 in B. rapa. The complete SC phenotype of ‘325’ showed the potential for practical breeding application value in B. rapa.Most flowering plants have evolved a self-incompatibility (SI) system to maintain genetic diversity by preventing self-pollination. The  Brassica species, the SI system is characterized by sporophytic self-incompatibility (SSI), in which the diploid parental genotype determines the pollen grain self-incompatible phenotype, and is controlled by a single Mendelian locus (named S locus) [S locus contains multiple physically linked genes and segregates with the SI phenotype [S locus genes have been identified, including S locus Glycoprotein (SLG), S locus receptor kinase (SRK), and S locus cysteine-rich protein (SCR)/S-locus protein 11 (SP11). SLG was the first identified S locus gene, encoding a protein secreted in stigmatic papilla cells [Brassica [SRK was the second identified gene, and encodes a transmembrane serine/threonine receptor kinase located at the stigmatic papilla cell plasma membrane, which had been testified as being the female determinant factor [SP11/SCR was the third identified gene in the S locus, and encodes a peptide protein located in anther tapetum cells and/or pollen grains, which had been testified as being the male determinant factor [S haplotype, SRK is activated and a signaling cascade is elicited within the stigmatic papilla cell to reject self-pollen [In flowering plants, self-incompatibility (SI) evolved to maintain genetic diversity and prevent self-pollination, in which pistils inhibit pollen germination or pollen tube growth after self-pollination . In BrasS locus) . The S lhenotype . The S lla cells  and may Brassica ,6,7. SRKt factor ,9,10,11.t factor ,13,14. Wf-pollen ,16. S haplotypes were classified into two groups, class I and class II [S haplotypes are dominant over class II in pollen and the dominance relationship of S haplotypes is involved in SCR/SP11 expression suppression, which is controlled by the SCR methylation inducer (Smi) [S haplotypes is observed in pistils [S haplotypes have been identified in B. rapa [S haplotypes have been reported in B. oleracea [S haplotypes have been identified in B. napus, including BnS-1 to BnS-7 and BnS-1300 [S haplotypes in B. rapa, including BrS-40, BrS-60, BrS-29, and BrS-44 and three class II S haplotypes in B. oleracea, including BoS-2b, BoS-5, and BoS-15 [S haplotypes has been observed through reciprocal test pollinations in B. rapa (BrS-44 > BrS-60 > BrS-40 > BrS-29) [SCR methylation inducer 2 (Smi2) [SCR and SRK genes in the recessive class II S haplotypes have been characterized in B. rapa [S locus organization of the class II S haplotypes was limited to the BrS-60 haplotype [S locus.Based on the nucleotide sequence similarity, class II . Class Ier (Smi) ,20,21,22 pistils ,24. More B. rapa , and abooleracea , while sBnS-1300 ,28. Howe BrS-29) ,23, and  B. rapa . Howeveraplotype ,33, whicS locus genes, including SCR genes [SRK genes [S locus gene disruption also changes the SI phenotypes, such as M-locus protein kinase (MLPK) [EXO70A1), Phospholipase D α1(PLDα1), and Glyoxalase1(GLO1), were identified as self-compatible factors working downstream of ARC1 [In the last few decades, researchers have concentrated on identifying the SI reaction factors; however, the self-incompatible mechanism and self-compatible pathway remain unexplained. Mutant screening and research are very valuable to identify new factors involved in the SI system ,36,37,38CR genes ,39,40 anRK genes ,42. On te (MLPK) ,44 and Ae (MLPK) ,46,47,48e (MLPK) ,50,51. S of ARC1 ,53,54,55 of ARC1 . Thus, tBrassica. Some SC lines have been identified from SI populations [Brassica rapa subspecies, including heading Chinese cabbage, yellow sarson, and no-heading pak choi [Brassica genomic variation research. In this study, a completely self-compatible line with a single S locus mutation was identified, which had a new class II S haplotype, named BrS-325. Complete genome assembly was gained by means of Nanopore sequencing strategy and the S-intergenic region was determined. The S locus genes were identified, and BrSCR-325 and BrSRK-325b had normal expression levels. Previous reports investigated leaf development, flowering, and LTR-RT expansion based on the assembled genomes in B. rapa [S locus of two class II S haplotypes, BrS-60 and BrS-325, which are each other’s closest relatives. The S locus of BrS-60 and BrS-325 showed high sequence polymorphism with few genes’ distribution. Meanwhile, multiple sequence alignment of class II SCRs and SRKs was carried out to reveal amino acid variations in the contact regions (CR II and CR III) of BrSCR-325 and the hypervariable regions (HV I and HV II) of BrSRK-325s, which may influence the interaction between the ligand and the receptor. Here, a complete pak choi genome was obtained and many amino acid variations were identified in the contact regions of BrSCR-325 and the hypervariable regions of BrSRK-325s, which may lead to self-incompatibility destruction.The screening and establishment of new self-compatible (SC) lines are critical for SI system research in ulations ,35,38,57ulations ,59,60,61ulations ,63,64,65pak choi ,33,66, aS haplotype by cloning and analyzing S locus genes. The line ‘325’ could be amplified by universal primer pairs of class II, but not class I, indicating that a class II S haplotype existed  were obtained by the means of PCR and sequenced (SCR and SRK E1 sequences in ‘325’ were different from the reported class II S locus genes and showed the highest sequence similarity with that of BrS-60 (79% sequence similarity of SCR and 91% sequence similarity of SRK E1)  . Phylogef BrS-60 D,E. Thus1 hybrids were created from the SI parent ‘326’ (BrS-12) and SC parent ‘325’ (BrS-325), and pollination behavior displayed self-incompatibility (1 hybrids (1 hybrids (1.29 and 1.48 cm per silique)  , indicating that the SC trait in ‘325’ was controlled by a single genetic locus. S haplotypes in the F2 population were segregated into S-12S-12: S-12S-325: S-325S-325 =20:50:22 , and all the pollination phenotypes were consistent with the genotypes  B,C. Aversilique) B,D. Baseenotypes . All theBrS-325 and discover the underlying mechanism of SC in ‘325’, we carried out Nanopore sequencing and next de novo assembly to obtain the pak choi genome [B. rapa genome assemblies , retrotransposons (42.41%), and unclassified elements (3.41%). The annotated retrotransposons mainly consisted of the long terminal repeat retrotransposon (LTR-TE), which covered 120 Mb and occupied 31.96% of the assembled genome . In totaSCR and SRK from ‘325’, homologous genes were blasted in our assembled genome. Finally, we obtained a 145 kb sequence from contig, named ctg00061, which covered the whole BrS-325 locus and boundary regions   . BrS-325 showed the highest sequence similarity with those of BrS-60 , and their F1 hybrid. BrSCR-325 showed a higher expression level in the anther of the SC line ‘325’ than that of BrSCR-12 in the SI line ‘326’, while in the F1 hybrid, the expression of BrSCR-325 was hardly detected  and amino acid deletion . As we know, the hypervariable region of SRK and contact regions were necessary for the interaction between SCRs and SRKs with the same plotypes ,74,75. BS domain of BrSRK-325  and three class II S haplotypes in B. oleracea  [S haplotype, named BrS-325, was identified in a pak choi line‘325’   A. The sine ‘326’ . All theS haplotype BrS-325  and two duplicated SRK genes (BrSRK-325a and BrSRK-325b) exist in the BrS-325 locus; however, expression analysis results showed that the expression of BrSCR-325 and BrSRK-325b are normal, which may not be responsible for the SC phenotype  was performed in a 10 μL reaction system, including 1 μL of DNA, 0.5 μL of each primer at 10 μM, 5 μL of 2× Taq Reaction Buffer  , and 3 μL of distilled water. The amplification program involved one cycle of 95 °C for 5 min, followed by 35 cycles of 95 °C for 30 s, 56 °C for 30 s, and 72 °C for 1 min. PCR products were electrophoretically-fractionated in a 1% agarose gel, stained with ethidium bromide, and then viewed under a UV illuminator. The SCR sequence was determined using the II SCR-1Land II SCR-1R primer pair, and the partial sequence (included first exon and four to seven exons) of SRK was determined using SRK II E1L and E1R and SRK II E4L&E7R in the self-compatible line “325”. PCR products were sequenced to determine the S haplotype. To explain the mutation locus of the self-compatible inbred line ‘325’, the F1 hybrid was created by crossing the SI parent ‘326’ (BrS-12) and the SC parent ‘325’, and self-pollinated to produce the F2 segregation population. S haplotypes were determined with the specific primer pairs: PK1and PK4 for detecting class I S haplotypes and SRK II E4L and SRK II E7R for detecting class II S haplotypes. PCR was performed under the same conditions mentioned above. Primer information for detecting S haplotypes is listed in Genomic DNA from all individuals was extracted from young leaves as described by Murray and Thompson . The S hand PS15  and PK1aand PS15  to identand PS21  and SRK Genomic DNA was extracted from the first young leaves of the self-compatible inbred line ‘325’, according to the procedure described by Belser et al . Genomichttp://www.girinst.org/repbase; accessed on 21 March 2020) (software:RepeatMasker [http://www.repeatmasker.org/RepeatModeler/ accessed on 21 March 2020), Piler [http://bix.ucsd.edu/repeatscout/ accessed on 21 March 2020) and repetitive sequence features (software: TRF (https://tandem.bu.edu/trf/trf.html accessed on 21 March 2020) and LTR-Finder [In our assembled genome, we carried out genomic annotation, including the prediction of repeated sequences, the prediction of non-coding RNA, and the prediction of gene structure and functional annotation. Repetitive sequence annotation is combined with the homologous prediction method and the de novo ab initio prediction method . The homatMasker  and Repe), Piler , RepeatSR-Finder ).http://rfam.xfam.org/ accessed on 21 March 2020) [The structure prediction of coding genes was usually combined with a variety of prediction methods, such as homolog prediction , de novoch 2020) .3PO4) for 3 h. The stained pistils were placed on the glass slide in distilled water to detect the pollen germination and pollen tube growth using the blue channel (UV 340–380 nm) of a fluorescence microscope .The aniline blue staining assay was performed according to the established procedure with minor modifications . The antTM RT reagent kit . The real-time quantitative polymerase chain reaction (RT-qPCR) was performed using 2× SYBR Green master mix  with the 10 μL reaction system, including 5 μL of 2× SYBR Green master mix, 0.4 μL of each primer at 10 μM and 4.2 μL of 50× diluted cDNA. Amplification was performed using the CFX96 Touch Real-Time PCR Detection System . The amplification program involved one cycle of 95 °C for 5 min, followed by 45 cycles at 95 °C for 15 s, 60 °C for 20 s, and 72 °C for 30 s. After each run, a melting curve was performed by heating up the samples from 60 to 95 °C. All analyses were repeated with three biological replicates. Actin gene (GenBank accession No: AF111812) was used as an internal control and actin was used to normalize transcript levels for all expression analyses [t-test. The primer pairs coming from S locus genes (SRK and SCR) are developed. All primers are listed in Anthers were cut from medium-size buds (2 to 3 days before flowering) and stigmas were gained from mature buds (on the day of flowering). Total RNA of stigmas and anthers was isolated from more than 30 stigmas and from six anthers of more than 20 buds using the SV Total RNA Isolation System kit  following the manufacturer’s instructions. One microgram of total RNA was used for the cDNA synthesis using a PrimeScriptanalyses . SignifiBrSRK-60 came from the genome of ‘Chiifu’ [http://nrbsc.org/gfx/genedoc 26 August 2015). Class II SCR drafts were drawn referring to the research results [CDS and amino acid sequences of class II SRK and SCR were obtained from the NCBI and their GenBank or entry name are listed in ‘Chiifu’ . MEGA-X ‘Chiifu’  program  results  and clas results . Phyloge results  using thS haplotypes, BrS-60 was obtained from the genome of ‘Chiifu’ [S haplotype and a contrasting SI phenotype, and BrS-325 was obtained from our assembled genome. Collinearity analysis of S locus sequences was performed using the software: MuMmer [S locus collinearity analysis of BrS-325 and BrS-60 was performed with the MuMmer tool. Then, visualizations of the S locus collinearity analysis were displayed using a JCVI tool.To display the synteny relationships of two class II ‘Chiifu’  and ‘Z1’‘Chiifu’ , which p: MuMmer  and JCVI: MuMmer . The S lt-test.The self-incompatibility phenotype was measured as in . When thS haplotype in Brassica, which displayed completely self-compatibility. It is a good resource for breeding practices due to its recessiveness to all class I S haplotypes [S haplotype in B. rapa.In conclusion, we identified a new class II plotypes . Here, wplotypes . In this


Diabetes mellitus (DM) is expected to reach US$ 745 billion in 2030. The growing prevalence of the disease, mainly type 2 diabetes, is the result of population aging, nutritional transition, which include growing rates of obesity and consumption of foods high in sugar and fat. Brazil is the fourth country in the number of patients with diabetes globally and follows the global trends, with a continuous increase in prevalence.The global economic burden of To estimate the economic burden of DM in Brazil, including all direct and indirect costs.We used a cost-of-illness approach to calculate the total economic burden of DM. We used official healthcare-related statistics referring to 2016.We estimated the Brazilian economic burden to reach US$ 2.15 billion in 2016, of which 70.6% are indirect costs related to premature deaths, absenteeism, and early retirement. We estimate that if the rate of growth of diabetes prevalence remains in Brazil, direct and indirect costs of diabetes will more than double by 2030 (an increase of 133.4% or 6.2% per year).Our results are in accordance with the literature that shows that indirect costs are more relevant in low- and middle-income countries due to poorer access to health care, which result in higher mortality rates from non-communicable diseases. However, due to the potentially underestimated prevalence of diabetes in Brazil and the lack of access to nationwide private healthcare costs, we estimate costs of diabetes in Brazil to be higher than the conservative results we found. The onset of the COVID-19 pandemic is likely to result in even greater costs than what we estimated. Diabetes mellitus (DM) is one of the most prevalent non-communicable diseases (NCDs) in the world, and the third highest risk factor for premature mortality after high blood pressure and tobacco use [2527acco use . The gloacco use 35. The racco use 34. In thacco use 252728.In this sense, DM is associated with growing costs to health systems (disease and co-morbidity treatment), and therefore to society as a whole . In 20104Whether direct or indirect costs are the main drivers of the economic burden of DM differs by economic development and access to health care. In developed countries, the direct costs tend to be higher than non-medical and indirect costs 43 – and 121891629Despite the efforts of the Brazilian public health care system (SUS), Brazil is the fourth country in the world in number of adults with DM (7.7% of Brazilians 18 or older in 2019) 1822. The1817Diabetes-related health expenditures with hospitalizations in Brazil were estimated to be around US$265 million in 2014 . In 200713We employed a prevalence-based approach that combines the most recent nationwide estimates of diabetes prevalence, epidemiological data, health care cost, and economic data using a cost-of-illness method.1We estimated DM prevalence and total population with DM using the most recent National Health Survey, conducted in 2013, which provides nationwide self-reported estimates of DM for the Brazilian adult population stratified by sex  and age groups  .To estimate diabetes prevalence in 2016, we scaled the estimates based on population growth between 2013 and 2016 by sex and age groups using population projection estimates conducted by the Brazilian National Bureau of Statistics for 2016 .The method we used to measure the economic burden of DM in Brazil is similar to previous studies on the economic burden of DM. Unless otherwise specified, we defined an event caused by DM when the primary cause of hospitalization was classified between E10 and E14 of the International Classification of Diseases (ICD 10). We excluded DM acquired during pregnancy 11.To calculate all the economic costs associated with DM, we accounted for the relationship between DM and the incidence of other diseases. We reviewed the medical literature involving the increase in risks of some conditions due to DM  to underThe concept of Population Attributable Risk is used to measure the relationship between DM and other diseases to assess the important second-order effect of DM prevalence. The Population Attributable Risk indicates the proportion of cases that would not occur if DM was absent. This concept depends on the prevalence of the risk factor and the risk of exposure  of the disease:The fraction is calculated using DM prevalence rate (P) and the relative ratio risk (RR) for each chronic complication of DM . The subWe considered only adults older than 35 years old to calculate the costs associated with DM-related diseases because information on the relative risk of such conditions associated with DM is scarce for younger individuals due to the lower prevalence of DM among this group. The parameters are calculated based on the National Health Survey for 2013 (PNS-2013), stratified by gender and age groups.The economic costs of DM were calculated using the cost-of-illness (COI) approach, adding the medical and non-medical costs of DM and other diseases associated with DM for Brazil. Direct costs refer to health sector costs for disease prevention, diagnosis, and treatment. In this paper, the following expenditures are included: expenses for hospital care , medical services (outpatient), and pharmaceutical expenses. All the costs were disaggregated by gender (men and women) and age . Costs related to hospitalization and ambulatory procedures were also divided into costs incurred by the Brazilian public health system (SUS) and private costs, which include both out-of-pocket and insured medical services.Hospitalization cost: Hospitalizations were categorized into two groups: (1) those in which the main diagnosis was reported as DM and coded as ICD10 E10 to E14; (2) those in which the main diagnosis was reported as any chronic complication of DM and related diseases, including infectious and neoplastic diseases for which DM is considered to be an important risk factor.We collected hospitalization data from the National Public Healthcare Hospitalization System (SIHSUS) for the public health system and from the Communication System of Hospital and Ambulatory Information (CIHA) for private health system costs. Both datasets are generated by DATASUS , and provide data on the hospitalization date, length of stay, days in an intensive care unit (ICU), gender, age, among others. It is noteworthy that only SIHSUS presents the average costs per hospitalization.Costs in SIHSUS were disaggregated into expenses for healthcare professionals and materials. These data also provided the cost of days spent in ICUs. Therefore, we use SIHSUS costs as a lower-bound approximation for private average costs, as public prices of services and medication are lower than market prices in Brazil.Ambulatory Cost: We collected data from two sources: (i) the Ambulatory Information System (SIASUS), which considers all outpatient procedures paid by the public health system; and (ii) the Private Healthcare Ambulatory Information System (CIHA), which includes costs associated with the private health system. Considering CIHA does not provide cost information, we used the average cost per utilization stratified by age and gender provided by the Ambulatory Information System (SIASUS) to estimate the costs of the procedures and utilization in the private health system.Ambulatory costs were also categorized into two groups: (1) those in which the main diagnosis was reported as DM and coded as ICD10 E10 to E14; (2) those in which the main diagnosis was reported as any chronic complication of DM and related diseases, including infectious and neoplastic diseases for which DM is considered to be an important risk factor.Medication costs: We collected data from the Popular Pharmacy program (PP). This Brazilian government program provides commonly used drugs at a reduced price or free, including several free diabetes drugs. The entire Brazilian population is eligible for the program, but monthly purchases are capped to the amount to be used by the individual per month. The medication can be purchased at any accredited private pharmacy or program pharmacy. Microdata for 2016 were obtained by invoking the Brazilian Information Access Law. The pharmaceuticals include human insulin and oral medicines [edicines .Other out-of-pocket costs for materials: We collected data from the last available National Household Budget Survey (POF) to calculate direct costs related to materials associated with capillary blood glucose testing. No other material costs related to diabetes care were available at POF. POF is conducted by the Brazilian Institute of Geography and Statistics (IBGE). Since the last data available refer to January 2009, we inflated the costs to 2016 prices using the National Consumer Price Index (IPCA-e) between 02/2009 and 12/2016. We did not use the cost of the Popular Pharmacy Program to measure expenditures with materials to avoid double-counting, since it was still incipient when the last household survey was conducted [onducted .We considered the following indirect costs in the analysis.Absenteeism: Denotes the lost productivity due to sickness requiring absence from work. We used the number of workdays missed (WDM) per year per person due to the disease. This measure corresponds to the number of workdays missed (WDM) multiplied by the daily average minimum wage. In our data, absenteeism is a direct implication of hospitalization, even for children and the elderly, since they may require the accompaniment of some adult (possibly of working age). Children and adolescents up to 18 years old and elderly (above 60 years) have the right to have one companion in the hospital while hospitalized [italized 13.We used the minimum monthly salary of US$441.10 (in 2016) to evaluate a missed day for people aged between 18 and 64. For the younger and older than 65, we attributed the average salary of those aged between 18 and 64 as an opportunity cost.Premature death (PD): Denotes the economic losses due to labor decreases as a result of terminal conditions. We calculated PD losses from the present value of the labor market outcomes lost prior to retirement based on age. According to the Brazilian pension system (in 2016), private-sector employees are entitled to retirement if they meet one of two conditions:(i) retirement based on age 65 for men and 60 for women with a minimum length of the contribution of at least 15 years, and (ii) retirement based on length of contribution – 35 years for men and 30 years for women:ijN is the number of deaths per year of individuals of gender i and age j, w is the minimum salary established in January 2016, r is the real interest rate in Brazil (average between 2012 and 2016). The rate was calculated by subtracting the average Brazilian benchmark interest rate  by the accumulated inflation for the year .In which T = (ij –ARiL) is the difference between the age of mortality, ijL, and age of retirement of the gender, iAR, for those above the minimum legal age (18 years old).The To calculate YLL, we used data on premature deaths available at the Mortality Information System (SIM) in 2016. This publicly available dataset is disclosed annually and contains information on all deaths in Brazil. We attributed a minimum value to the lost years of life in productivity. In this calculation, we considered deaths of which DM was the cause. We understand that our decision to consider deaths attributable to DM potentially underestimates the number of deaths due to diabetes. When admitted to in-patient or out-patient care, patients may either fail to mention they have diabetes or may not know they have the disease . Also, hDisability or early retirement (DIS): Represents the value of interruption of employment due to sickness related to DM. The cost is estimated using the average amount paid by the government to new retirees and sickness aid due to DM in 2015. The number of beneficiaries is determined as the number of people retiring early due to disabilities related to severe diabetes.Finally, to calculate the cost due to early retirement related to DM complications, we proxied the number of beneficiaries by people under the legal age of retirement who have DM in the most recent Brazilian National Health Survey (PNS 2013). We considered data of new disability benefits granted, which include retirement for disability due to injury and sickness. We have also restricted the data to benefits related to DM according to ICD-10. As the most recent data are for 2015, we inflated the values to 2016 using the price consumer index and the real interest rate. We only considered men until 65 years and women until 60 years, as these are the legal ages for retirement in Brazil.We projected future direct and indirect costs of diabetes to 2030 based on two scenarios. The first is a conservative scenario in which we considered that the DM prevalence would remain the same as it was found in the 2013 Brazilian National Health Survey (6.2%). In the second scenario, which we judge to be more realistic, we considered that the prevalence of diabetes in Brazil will grow from 2016 to 2030 at the same annual rate it grew between 2003 and 2013 . In both situations, we consider the prevalence by sex and age groups. We did not include people under 18 years old since data on diabetes prevalence for children and adolescents are not available in either survey. In this scenario, the diabetes prevalence in Brazil is a weighted average of the groups’ prevalence. On the cost side, we used our estimate of diabetes cost, maintaining it constant over time.Finally, the number of people with diabetes was calculated by multiplying the estimated prevalence of diabetes in 2030 by the projected population in 2030 according to IBGE. The future cost is given by the product of the cost per person calculated in this study and the estimated number of diabetics in 2030 across age groups.We ran sensitivity analysis to try to estimate the costs of the private health care that covers 23% of the Brazilian population by using data from hospitalization provided by a health care system in a Brazilian state located in the Southeast of the country [We use hospitalization prices from Diagnosis Related Group (DRG-Brazil) – a dataset composed of data from more than 200 hospitals in the country. In 2016, this dataset included 4,909 patients that were hospitalized due to DM or complications of DM .We estimated total costs of US$2,153.05 million: US$633.03 million (29.4%) in direct costs and US$1,520.02 million (70.6%) in indirect costs in 2016.Table 1 shows all components of the direct costs. When it comes to hospitalizations, we estimated total expenses to be US$232.8 million . When accounting for DM expenses and related conditions, women’s hospitalization expenses were US$108.6 million (46.7%), while the expense for men were US$124.1 million (53.3%). Direct costs of hospitalizations, only related to DM, represented US$50.2 million for the public health system. On the other hand, DM-related conditions summed US$139.7 million in the public health system.In 2016, the total direct costs related to DM were US$633 million. Regarding outpatient costs, we calculated a total of US$ 86 million in 2016 . Most of costs were related to women , while men’s costs totaled US$37 million (43%). The total costs of outpatient care attributed to DM (US$ 82.1 million) were 21 times higher than total DM outpatient costs when DM was the main cause of admission (US$ 3.9 million).Table 1 are also disaggregated by age groups in Table 2. Costs for DM as the main cause was calculated in seven different age groups, while costs attributed to DM were calculated by five age groups due to the availability of information on relative risks. In both cases, we observed some heterogeneity. The age groups 55–64, 65–74, and 75 years and older are responsible for 80.7% of total hospitalization costs and 86.0% of ambulatory costs.The same results of Finally, the total direct costs include drugs (US$304 million) and materials (US$10 million). Due to data limitations, these total direct costs could not be divided by gender or age group.Total indirect costs are calculated by the sum of economic losses due to absenteeism, premature deaths, and early retirement caused by DM and its complications. In 2016, we estimated that they represent US$1.52 billion.Table 3), which has a small share of total indirect costs (2.6%), amounting to US$39.8 million . The results are homogeneous by gender but not by age group. The groups above 55 years old are responsible for more than 75% of total absenteeism costs.We use hospitalization data to calculate absenteeism (Table 4) were US$1.18 billion : US$409 million for women and US$ 775 million for men. We did not estimate the indirect costs of premature death for chronic complications of DM and related diseases.The indirect costs due to premature deaths due to DM , of which US$186.4 million (7.3%) is for women and US$110.4 (12.3%) for men.Table 5 shows that the total cost per patient diagnosed in Brazil with DM is US$223.54: US$65.72 of direct costs , and US$157.81 of indirect costs .In 2016, 9,631,664 Brazilians over 18 were estimated to be diagnosed with diabetes . Therefore, When we calculated those costs for the Brazilian population, the total costs per capita represented US$10.49: US$3.09 as direct costs per capita, and US$7.41 as indirect costs.Table 6 shows. In the first scenario, we considered that the prevalence of DM would remain the same as the latest available estimate for 2013 and would only increase as the population age. In this case, the average prevalence rate would increase from 6.4% in 2016 to 7.9% in 2030. The total economic burden would increase from US$ 2,15 billion to US$ 3.06 billion in 2030 . In the second scenario we considered, which we believe to be more realistic, the prevalence rate would increase to 13.0% in 2030. In this scenario, the total economic burden would increase to US$ 5.03 billion, an increase of 133.4% or 6.2% a year.In order to project our estimates to 2030, we considered two different scenarios, as We ran a sensitivity analysis using hospitalization prices provided by DRG-Brazil. Total costs of hospitalization using DRG prices are US$422.5 million. When using such prices, the total diabetes costs estimated reached US$2,342.7 million: US$822.7 million in direct costs (35.1%) and US$1,520.01 million in indirect costs (64.9%) in 2016.Using conservative estimates, we calculated that the Brazilian burden of diabetes in 2016 was US$2.15 billion, or 0.12% of the country’s GDP. Most of the burden is attributed to indirect costs 70.6%), as previous studies in Brazil indicate 0.6%, as . The recAnother limitation usually referred to in the literature  includes713We were also able to disaggregate the results by gender and age group. In this case, we assessed the heterogeneity of the results, including hospitalization and ambulatory costs. For direct costs, DM is the main diagnosis across seven of the age and gender groups and costs attributed to DM in five of these groups. The elderly individuals (above 65 years) are responsible for 40% to 70% of total hospitalization and ambulatory costs. This result is similar to the literature, which also shows that individuals older than 65 are responsible for a much larger proportion of hospital resources  4.The analysis of gender costs provides interesting results. We find that ambulatory costs are more relevant among women, probably due to the greater attention that women give to their health than men 42. The sOur conservative analyses also did not consider costs associated with the management of renal diseases alone, which can be responsible for a large share of the costs associated with the complications of diabetes .One important limitation of all the economic burden of DM analysis carried out in Brazil is the lack of price information of the medical services provided by the private sector. Previous studies assume that the service prices are the same as those paid by the government, which is a very conservative hypothesis that underestimates direct cost estimates. In this sense, we ran a sensitivity analysis using price estimates provided by a private healthcare provider located in the State of Minas Gerais, Brazil. The average price of medical services of this private system is 30% higher than that paid by the public health system. Despite our inherent limitation associated with using data from a single provider, our results show that the current estimates of direct costs of diabetes in Brazil in the literature – along with ours – are underestimated. This provider, however, serves 200 hospitals and covers 1.5 million lives.Another limitation of our analysis includes the lack of other negative externalities of DM in Brazil. Negative externalities might occur when the health system does not absorb the increased demand for DM patients, generating spillovers for other patients.Finally, we could not include judicialization costs of diabetes treatment in our analyses. The list of medicines available at the public healthcare system in Brazil is limited. For instance, insulin analogs and new oral drugs are not widely for the population. Because patients can file lawsuits to assert their constitutional right to health and thus request access to medicines and treatments not covered by the national public healthcare system at no cost. Although nationwide data are not available, findings from the state of S˜ão Paulo – the most populated and affluent in the country – show that 25% of all lawsuits of this type involve DM-related drugs .Diabetes mellitus in Brazil using a cost-of-illness approach using several data sources. Our conservative analyses show that the cost of the disease represented at least 0.12% of the country’s GDP in 2016, with a potential to more than double in the next 14 years. The indirect costs accounted for the highest proportion of total DM costs. The undiagnosed cases of diabetes, the unidentified deaths by diabetes, and our limitations in accessing the unit and per capita cost associated with the complementary private healthcare system that cover a fifth to a quarter of the Brazilian population are the reasons why our estimates are conservative and that the total DM economic burden in Brazil is even higher that what we calculated.We calculated the total economic burden of The additional files for this article can be found as follows:10.5334/aogh.3000.s1Appendix A.Costs attributed to DM – Hospitalization and ambulatory.10.5334/aogh.3000.s2Appendix B.Direct Costs – DM and Morbidities.The majority of the data used are open sourced. Part of the complementary data is the Diagnosis Related Group (DRG) dataset. These data are not publicly available and were kindly provided by Ana Claudia Abreu, Tania Grillo, and Renato Couto at the Federal University of Minas Gerais.


Virus spreading problems in wireless rechargeable sensor networks (WSNs) are becoming a hot topic, and the problem has been studied and discussed in recent years. Many epidemic spreading models have been introduced for revealing how a virus spreads and how a virus is suppressed. However, most of them assumed the sensors are not rechargeable sensors. In addition, most of existing works do not consider virus mutation problems. This paper proposes a novel epidemic model, including susceptible, infected, variant, low-energy and dead states, which considers the rechargeable sensors and the virus mutation factor. The stability of the proposed model is first analyzed by adopting the characteristic equation and constructing Lyapunov functions methods. Then, an optimal control problem is formulated to control the virus spread and decrease the cost of the networks by applying Pontryagin’s maximum principle. Finally, all of the theoretical results are confirmed by numerical simulation. In recent years, wireless sensor networks (WSNs) have played a crucial role in the internet of things (IoT), and WSNs has been widely developed and applied in many fields, for example: industrial, military and healthcare applications. Benefiting from the recent breakthrough in WSNs, they have attracted increasingly attention ,2. WSNs WSNs suffer from the issue of the vulnerability of the network and energy limitation. Wireless rechargeable sensor networks (WRSNs)  are consThus, this paper includes virus mutation to establish a new model, which is very effective to reduce the spreading of the virus, reduce the harm of the virus mutation to WRSNs and greatly improve the security of WRSNs.In order to improve the security in WRSNs, the efficient scheme and energy efficient secured ring routing (E2SR2) protocol was first proposed by Shafie et al. and Bhushan et al. ,13. For Applying epidemic dynamics analysis to the study of WSNs, Kephart et al. ,15 firstAs shown in In the previous studies, the charging behavior and mutation virus activity of wireless sensor networks has rarely been investigated. In this paper, the main goal is to introduce a model that considers the low-energy states and virus mutation. Combined with a practical application, the system can be applied when the stability of the system is proven and the other conditions are not taken into account, and the cost can be controlled by an optimal strategy. It is divided into five variable states. Our contributions are as follows:An epidemic model suitable for WRSNs is established to describe the propagation process of malwares (the virus and mutated virus).To analyze and calculate the basic reproductive numbers By constructing the objective function and applying Pontryagin’s maximum principle, we can obtain the optimal control variable which satisfies the optimal control objective of the security problem.The rest of the paper is as follows: In In this section, the epidemic model of virus mutation in WRSNs is introduced. Sensors are divided into five sensor node states. The five sensor node states include:As shown in Obviously, adding Equation (1), we obtain:As Then, considering the limit of (3), we can obtain Above, In order to discuss the existence of equilibria, let the left-hand side of Equation (1) be zero. Obviously, there are three solutions that satisfy this situation. The disease-free equilibrium is  is a nonlinear system, and we must turn it into a linear system so as to prove the local stability of this system. In order to reduce the calculated amount of the system, Then, the Jacobian matrix of (15) is given by:Theorem 2.Ifand, the disease-free equilibrium is locally asymptotically stable.Proof. According to the Lyapunov criterion , if all Hence, we have the following characteristic Equation (18):Obviously, if Theorem 3.Ifand, the individual plant virus equilibrium is locally asymptotically stable.Proof. The Jacobian matrix is given as:Hence, the characteristic equation is given by:Then, the characteristic value is Theorem 4.Ifand, the endemic equilibrium is locally asymptotically stable.Proof. The Jacobian matrix is given by:By the transformation of (21), we obtainWe have the following characteristic equation:Obviously, According to Vieta’s formulas , the zerIn this part, three Lyapunov functions are constructed to verify the global stability of the disease-free equilibrium Theorem 5.If,and, the disease-free equilibrium is globally stable.Proof. The Lyapunov function is constructed as Obviously, if Theorem 6.If, the individual plant virus equilibrium is globally stable.Proof. According to the second Equation (1), the number of infected nodes is When As The derivative of Obviously, ifTheorem 7.Ifand, the endemic equilibrium is globally stable.Proof. It is noted that the rate of infection is higher than the rate of virus mutation. If the rate of virus mutation is enough small and is equal to be zero, it is assumed that The Lyapunov function Obviously, If In this subsection, the cost of cleaning the virus and the cost of charging low-energy nodes are considered. For this purpose, the control It is clear that the feasible region of the control variable set At the moment, the transversality condition of optimal control satisfies the following equation:According to differential equations of the covariant variable and transversal condition of optimal control, the optimization condition is given by:Finally, Pontryagin’s maximum principle is applied to obtain the optimal control variable set. The result is given as: In this section, all of the theoretical analyses are proved and the numerical results of the system (1) are presented to support the analytic results. The result of the numerical simulation is given as follows.The parameters are given so that The relationship between 1 < 1 and R2 < 1, the parameters are set as β1 = 0.003, β2 = 0.002, γ1 = 0.2, γ2 = 0.4, c = 0.5, u = 0.2, d = 0.1, ε = 0.2 and A = 10. Hence, the basic reproductive numbers are calculated as R1 = 0.1125 < 1 and R2 = 0.075 < 1. N = S + I1 + I2 + L = 100. The disease-free equilibrium E0  can been calculated as S0 = 37.5, L0 = 62.5, I1 = 0, I2 = 0, as shown in If R1(0), I2(0) and L(0) do not have any influence on system stability and the system (1) will be stable at the disease-free equilibrium E0 eventually.As shown in 2 > 1 and R2 > R1, β1 = 0.003, β2 = 0.04, γ1 = 0.2, γ2 = 0.4, c = 0.5, u = 0.2, d = 0.1, ε = 0.2, A = 10, we have R1 = 0.1125 < 1 and R2 = 1.5 > 1 > R1. The individual plant virus equilibrium is calculated to be If the parameters satisfy RAccording to Theorem 3, it is noted that the system (1) will be stable at the individual plant virus equilibrium eventually. From If the parameters satisfyObviously, as shown in Briefly, the relationship between the system and parameters is shown as a simulation diagram. The rate of As for the optimal control strategy, the control variable set a method . FirstlyThe parameter values are given as As shown in It is noted that the cost under the optimal strategy is greatly reduced, as shown in In this paper, a novel epidemic model in WRSNs is proposed, including susceptible, infected, variant and low-energy states. The model considers rechargeable sensors and the virus mutation factor. By analyzing the basic reproductive number, the existence of equilibriums is first proved, and the local stability and global asymptotic stability are proved by the Lyapunov stability criterion. Meanwhile, the influence of the rate of virus mutation and the number of mutated nodes on the endemic equilibrium is revealed. In addition, an optimal strategy is proposed to minimize the numbers of the infected nodes and the virus mutation nodes, the cost of cleaning the virus and the cost of charging low-energy nodes. Finally, the numerical simulation validates the theoretical results.Future research will attempt to consider the time delay to move closer to practical application. For WRSNs, it is a key topic of future work to consider the time delay of the virus mutation and the time delay of charging at the same time. Additionally, the model can become closer to reality when the time delay is proposed.


Lumpy skin disease virus (LSDV) causes a severe systemic disease characterized by cutaneous nodules in cattle. LSDV is a rapidly emerging pathogen, having spread since 2012 into Europe and Russia and across Asia. Aedes aegypti, Culex quinquefasciatus, Stomoxys calcitrans, and Culicoides nubeculosus) on LSDV-inoculated cattle in order to examine their acquisition and retention of LSDV. Subclinical disease was a more common outcome than clinical disease in the inoculated cattle. Importantly, the probability of vectors acquiring LSDV from a subclinical animal (0.006) was very low compared with that from a clinical animal (0.23), meaning an insect feeding on a subclinical animal was 97% less likely to acquire LSDV than one feeding on a clinical animal. All four potential vector species studied acquired LSDV from the host at a similar rate, but Aedes aegypti and Stomoxys calcitrans retained the virus for a longer time, up to 8 days. There was no evidence of virus replication in the vector, consistent with mechanical rather than biological transmission. The parameters obtained in this study were combined with data from studies of LSDV transmission and vector life history parameters to determine the basic reproduction number of LSDV in cattle mediated by each of the model species. This reproduction number was highest for Stomoxys calcitrans (19.1), followed by C. nubeculosus (7.1) and Ae. aegypti (2.4), indicating that these three species are potentially efficient transmitters of LSDV; this information can be used to inform LSD control programs.Lumpy skin disease virus (LSDV) is a vector-transmitted poxvirus that causes disease in cattle. Vector species involved in LSDV transmission and their ability to acquire and transmit the virus are poorly characterized. Using a highly representative bovine experimental model of lumpy skin disease, we fed four model vector species (IMPORTANCE Lumpy skin disease virus (LSDV) causes a severe systemic disease characterized by cutaneous nodules in cattle. LSDV is a rapidly emerging pathogen, having spread since 2012 into Europe and Russia and across Asia. The vector-borne nature of LSDV transmission is believed to have promoted this rapid geographic spread of the virus; however, a lack of quantitative evidence about LSDV transmission has hampered effective control of the disease during the current epidemic. Our research shows subclinical cattle play little part in virus transmission relative to clinical cattle and reveals a low probability of virus acquisition by insects at the preclinical stage. We have also calculated the reproductive number of different insect species, therefore identifying efficient transmitters of LSDV. This information is of utmost importance, as it will help to define epidemiological control measures during LSDV epidemics and of particular consequence in resource-poor regions where LSD vaccination may be less than adequate. Poxviridae and the etiological agent for lumpy skin disease (LSD) in cattle. LSDV is a rapidly emerging pathogen that is believed to be mechanically transmitted by arthropod vectors. First described in Zambia in cattle in 1929, LSDV subsequently spread throughout Africa and into the Middle East (2211––Lumpy skin disease virus (LSDV) is a large DNA virus of the family le East 28. LSD isle East 2. Morbidie East 224, 11–13.ast 2211––16 and est 2211–– situatioTo date, the mode of LSDV arthropod transmission has been assumed to be mechanical, as no evidence of active virus replication in insects or ticks has been found . This meStomoxys calcitrans, have been associated with outbreaks of LSDV  (Culicoides punctatus) (Hematophagus dipterans (referred to in this work as “blood-feeding insects”), particularly  of LSDV , 19–21,  aegypti ; the ticiculatus –25, Rhipoloratus , and Ambhebraeum ; the sta species , 29. LSDculosus)  or in finctatus) . HoweverOverall, these studies provide growing evidence of the potential participation of different arthropods in the transmission of the LSDV. Unfortunately, previous studies had design limitations, including reduced number of donor cattle and limited times postinfection, the use of virus-spiked blood meals, and/or reduced number of insects assayed. As a consequence, the results obtained do not fulfil quantitative requirements to assess the risk of transmission. This shortcoming is demonstrated by large uncertainty in parameter estimates. Furthermore, the vital knowledge gap of understanding how efficient each vector is at contributing epidemiologically to the transmission of LSDV remains.LSDV can be detected in skin lesions; blood ; and nasal, oral, and ocular secretions of infected cattle , 31, 32.34–S. calcitrans, Ae. aegypti, Cx. quinquefasciatus, and C. nubeculosus). We obtained quantitative data describing critical, biologically relevant parameters of the mechanical transmission of LSDV in unprecedented detail. These transmission parameters subsequently enabled advanced mathematical modeling to understand the risk of transmission of the virus from experimentally infected cattle to each vector insect species. Furthermore, our in vivo transmission studies provided much-needed evidence that subclinically infected cattle do not contribute efficiently to virus acquisition by a blood-feeding insect and also further defined the infectious period of the cattle host. In combination with published data on insect ecology parameters and the feasibility of LSDV transmission, our results ultimately allowed the determination of the basic reproduction number for each insect vector species. These studies highlight the powerful combination of natural host infection/transmission studies and subsequent mathematical modeling to enable maximum relevance and transferability of data to the in-field situation and direct applicability to improve control measures.In this study, we used a highly relevant experimental LSD infection model in the natural cattle host and four representative blood-feeding insect species previously reported to be capable of acquiring LSDV ((i) Experimental inoculation of calves with LSDV results in clinical and subclinical disease. Eight calves were challenged by intravenous and intradermal inoculation of LSDV in order to act as donors on which blood-feeding insects could feed. The clinical and pathological findings have been described previously  LSDV DNA can be detected in blood and skin of clinical and subclinical calves. In the three clinically affected calves, viral DNA was first detected in the blood by quantitative PCR (qPCR) at 5 dpc and remained detectable in all subsequent blood samples (up to 19 dpc). Peak viral DNA levels in the blood  were reached at 11 dpc  in subclinically infected calves than those in clinically affected calves  or 9 dpc  contained abundant viral genomes as measured by qPCR . Viral D(iii) Infectious LSDV is present in larger quantities in the skin than in the blood. Both skin homogenate and PBMC suspension collected between 5 and 19 dpc from clinical calves were titrated to determine the quantity of live virus in these tissues. Although units of measurement are not directly comparable between sample types , they are representative of the magnitude of exposure that hematophagous insects may encounter during feeding . In all calves, the viral titer from skin homogenate was higher and more constant than from PBMC suspension  (Data Set S1) suggesting the virus is highly concentrated in the skin lesions of clinical animals. Live virus was not detected in blood or skin samples from subclinical animals (including samples which were qPCR positive).spension . Live vi(iv) Humoral response to LSDV inoculation. Serum from the three clinically affected calves contained antibodies to LSDV at 15 to 17 dpc, as determined by a commercial enzyme-linked immunosorbent assay (ELISA). By the end of the study period, all subclinical animals had also developed detectable LSDV antibodies at levels lower than those observed in the clinical animals but above those of the nonchallenged controls (data not shown). The presence of detectable levels of antibodies confirmed exposure to the virus in all eight challenged animals, although the clinical outcome of challenge varied widely between the eight calves.Ae. aegypti and Cx. quinquefasciatus; one species of biting midge, C. nubeculosus; and the stable fly S. calcitrans on days 5, 7, 9, 11, 15, 17, and 19 postchallenge. The selected species are potential mechanical vectors with different feeding mechanisms , as well as species models for Culex and Aedes mosquitoes  the probability of transmission from bovine to insect  among insect species and between clinical and subclinical donors, and (ii) the duration of viral retention among insect species (see Appendix). Models were compared using the deviance information criterion (DIC), with a model having a lower DIC preferred to one with a higher DIC. Positive insects were those with LSDV DNA amplification by qPCR.A total of 3,178 insects were fed on the 8 donor calves (over 7 feeding sessions), of which 180 were positive for viral DNA. A higher proportion of insects were positive after feeding on a clinical donor  than after feeding on a subclinical donor  . CompariThe relationship between the level of viral DNA in the skin or blood of a calf and the proportion of virus-positive insects resulting from a feeding session was examined. For each feeding session that took place on the three clinical calves, the proportion of insects containing viral DNA postfeeding was calculated and compared with the viral DNA copy number present in both the blood sample and the skin biopsy sample taken from the calf on that day . This coCombining the dose-response relationship  with theAe. aegypti and S. calcitrans up to 8 dpf, in C. nubeculosus up to 4 dpf, and in Cx. quinquefasciatus up to 2 dpf (Cx. quinquefasciatus mosquitoes survived to 4 or 8 dpf (Ae. aegypti (5.9 days) and S. calcitrans (5.5 days), followed by Cx. quinquefasciatus (4.5 days), and C. nubeculosus (2.4 days)  ; Table 1Ae. aegypti , Cx. quinquefasciatus , and S. calcitrans    species:  = 0.60) . However = 0.01) . These rAe. aegypti (0.56), intermediate for C. nubeculosus (0.19) and Cx. quinquefasciatus (0.11), and lowest for S. calcitrans (0.05)  . Howeverasciatus , which mR0) is defined as “the average number of secondary cases caused by an average primary case in an entirely susceptible population”   and Ae. aegypti  are also potentially efficient vectors of LSDV  and would be able to sustain transmission if either were the sole vector in a region. Finally, Cx. quinquefasciatus  is likely to be inefficient at transmitting LSDV  ; Fig. 8,ing LSDV ; Fig. 8.R0 for clinical animals alone is very close to that for both clinical and subclinical animals combined . This aCattle experimentally infected with LSDV, including in our study, have higher concentrations of LSDV in skin lesions than in blood  and 3. IIt is not clear if the source of the virus acquired by the insects is skin nodules or viremic blood or a combination of both. Given the higher concentration of virus in the skin lesions than in blood , we can As reported in this study and others , 31, LSDWe found that subclinical donors were much less likely than clinical animals to transmit virus to vectors  and 5, iCulicoides midges also disrupt the skin barrier using their proboscis (0.1 to 0.2 mm long). Midges serrate the skin using saw-like blades on their proboscis that cross over each other to produce a pool of blood  or the following day   . Since wAe. aegypti and S. calcitrans at levels similar to those acquired during feeding deserves further investigation . This result suggests low numbers of infectious virions are present on each insect. In previous work, live LSDV was detected in individual Ae. aegypti for up to 6 days following exposure to an infectious calf , the resulting estimates for the probability of transmission from bovine to insect  are robust, as indicated by the narrow credible intervals for these parameters  and 2. TAe. aegypti, Cx. quinquefasciatus, and C. nubeculosus  and the difficulty in providing a value for each parameter that is applicable in all situations. In particular, vector life parameters will depend on environmental factors, including temperature, precipitation, and habitat suitability. Consequently, the basic reproduction number and, hence, the risk of transmission are unlikely to be constant over space or time but will vary both geographically and seasonally. For a more complete discussion of the role of vector life history parameters, see the earlier study by Gubbins  Ethical statement, housing, and husbandry. The experimental study was conducted under the project license P2137C5BC from the UK Home Office according to the Animals (Scientific Procedures) Act 1986. The study was approved by the Pirbright Institute Animal Welfare and Ethical Review Board. Cattle were housed in the primary high-containment animal facilities (biosafety level 3 agriculture) at The Pirbright Institute. The husbandry of the animals during the study was described previously ((ii) Challenge study and experimental procedures. Ten Holstein-Friesian male cattle  were used for the study, which was done in two experimental replicates of five animals each. The median age and weight of the calves were 104 days old and 145 kg in replicate one and 124 days old and 176 kg in replicate two. Eight calves were challenged by intravenous and intradermal inoculation with a suspension of LSDV containing 106 PFU/ml (6 PFU/ml . More sp6 PFU/ml  were col(iii) Insect exposure. Blood-feeding insects used in the study were Aedes aegypti “Liverpool” strain, Culex quinquefasciatus TPRI line , Stomoxys calcitrans , and Culicoides nubeculosus , and 12:12 light/dark cycle. Cx. quinquefasciatus was reared in pans of 500 to 800 larvae per larval bowl, containing approximately 1.5 liter of water supplemented with ground guinea pig food and maintained at 26°C, 50% RH, and 16:8 light/dark cycle. S. calcitrans was reared in approximately 200 eggs per pot and incubated for 12 to 13 days in larval pots containing a ratio of 3:2:1  and a tablespoon of yeast. C. nubeculosus was reared in approximately 10,000 larvae per pan containing 2 liters of dechlorinated water supplemented with Oxoid broth and dried grass/wheat germ mix. Pots of 800 Culicoides pupae were made with males and females and allowed to emerge. Both S. calcitrans and C. nubeculosus were maintained in insectaries at 27 ± 2°C, 50% RH, with a 16:8 light/dark cycle.eculosus . All insC. nubeculosus between 0 and 2 days posteclosion, female Cx. quinquefasciatus and Ae. aegypti at 5 to 7 days posteclosion, and male and female S. calcitrans at an average of 4 days posteclosion . All adult insects were maintained on 10% sucrose and starved 18 to 24 hours before exposure to the calves.The age and sex composition of the insects at exposure were female and male 2, unfed individuals were discarded, and blood-engorged individuals were collected.All eight challenged calves, independent of clinical status, were exposed (for between 5 and 20 minutes) to each of the four insect species on days 5, 7, 9, 11, 15, 17, and 19 postchallenge. At each time point, each pot of insects was placed on a cutaneous nodule on a clinical animal and a corresponding area of normal skin on a subclinical animal. The hair of the calf at each feeding site was clipped and/or shaved, and the insects were held in close contact with the skin of the calves in a container covered by mesh. Around 2 hours after exposure, insects were anesthetized under COAe. aegypti, Cx. quinquefasciatus and C. nubeculosus blood engorgement was assessed visually by the presence of blood in the abdominal cavity. However, S. calcitrans adults were all collected in a blind manner, and blood engorgement was confirmed by the detection of the bovine cytochrome b gene  after 2 dpf. All insects were kept in a temperature-controlled room at biocontainment level 3, with a 10:14 light/dark cycle. For the incubation, cardboard/waxed pots containing the insects were placed inside plastic boxes covered by mesh which were kept under a plastic shelter to minimize temperature and humidity fluctuations. Temperature  and RH  of the room and of the incubation area were recorded approximately every 15 minutes .Samples from each insect group taken immediately following blood-feeding assessment (dpf 0) were stored at −80°C, and the rest of the insects were maintained for 1, 2, 4 or 8 dpf. After this incubation period, surviving individuals were collected and stored at −80°C after the incubation period. Throughout incubation, all insects were maintained on 10% sucrose solution, except (iv) Samples. Skin biopsy samples were weighed on a calibrated scale  and homogenized in 500 μl high-glucose Dulbecco’s modified Eagle’s medium  supplemented with 5% fetal bovine serum , 100 U/ml penicillin and 100 μg/ml streptomycin , and 2.5 μg/ml amphotericin B  in a Lysing Matrix A tube  using a portable homogenizer . Whole insects were homogenized using a TissueLyser  with one or two steel beads of 3 mm   supplemented with 10% fetal bovine serum, and penicillin-streptomycin as above. Blood collected without anticoagulants was allowed to clot and then was spun at 1,000 × g to 2,000 × g for 10 minutes in a refrigerated centrifuge, and the serum was collected. All samples were stored at −80°C until analyzed.ion, UK)  in 200 μ(v) Laboratory assays. Nucleic acid from 200 μl of whole blood, PBMC suspension, or skin homogenate or 100 μl of insect homogenate was extracted in a 96-well plate with the MagMAX CORE nucleic acid purification kit , using protocol A in a KingFisher Flex magnetic particle processor (Applied Biosystems), and eluted in 50 μl of buffer. qPCR for LSDV ORF074 detection was performed using a modification of the TaqMan assay described by Bowden et al. (AAATGAAACCAATGGATGGGATACATAGTAAGAAAAATCAGGAAATCTATGAGCCATCCATTTTCCAACTCTATTCCATATACCGTTTT). Bovine blood intake by insects was determined using a SYBR green assay  for the detection of bovine mitochondrial cytochrome b as described by Van Der Saag et al.  using the manufacturer cycling conditions for primers with melting temperature (mT) of >60°C. The assay was performed in a 10-μl reaction mix using 2 μl of the template. This assay was specific for bovine cytochrome b, and a melt curve analysis was performed to confirm that only specific amplification occurred. For all qPCR assays, a constant fluorescence threshold was set which produced reproducible quantification cycle (qC) values for the positive-control samples between runs. A double-antigen ELISA  was used to detect circulating antibodies for LSDV in serum samples following the manufacturer’s protocol and analyzed with the Multiskan FC microplate photometer (Thermo Scientific). Infectious virus titer determinations of PBMC suspension and insect and skin homogenate was performed by viral plaque quantification in Madin-Darby bovine kidney (MDBK) cells.n et al.  with theg et al.  with somFull details of parameter estimation are provided in the Appendix but are briefly described below.β is the probability of transmission from bovine to insect, γ is the virus inactivation rate , and t is the time postfeeding at which the insect was tested. This probability  and the probability that the insect retained the virus until it was tested at t days postfeeding [exp(−γt)].The numbers of insects positive for viral DNA after feeding on cattle infected with LSDV were used to estimate the probability of transmission from bovine to insect and the virus inactivation rate. The probability that an insect would be positive when tested isbability  combines0d and 1d are the dose-response parameters and V is the level of viral DNA (log10 copies/ml in blood or log10 copies/mg in skin) in the donor when the insect fed. The different models were compared using the deviance information criterion  in the donor animal, so thatriterion . The twob is the probability of transmission from insect to bovine, β is the probability of transmission from bovine to insect, γ is the virus inactivation rate, T is the time interval between feeding on the donor and refeeding on the recipient, and n is the number of insects which refed. This probability (n refeeding) transmitted LSDV, where the probability that an individual insect will transmit is the product of the probabilities that it acquired the virus during the initial feed (β), retained it until refeeding [exp(−γT)], and subsequently transmitted LSDV at refeeding (b).Data on transmission of LSDV from insect to bovine were extracted from the published literature , 28, 30.bability , is the Previous estimates for the latent and infectious periods of LSDV  were updhttps://github.com/SimonGubbins/LSDVAcquisitionAndRetentionByInsects. Two chains were allowed to burn-in and then were run to generate an effective sample size of around 5,000 samples . ConverR0, is the “average number of secondary cases arising from the introduction of a single infected individual into an otherwise susceptible population” . The mean duration of infection for clinical animals (1/Cr) is based on the detection of virus or viral DNA in skin, while that for subclinical animals (1/Sr) is based on detection of viral DNA in blood (see Appendix).Replicated Latin hypercube sampling was used to compute the median and 95% prediction interval for  species . Paramethttps://github.com/SimonGubbins/LSDVAcquisitionAndRetentionByInsects.We declare that the main data supporting the findings of this study are available within the article and in Data Set S1. The code and the data used are available online for readers to access with no restriction at


The aim of this study was to assess costs and health service use associated with tendon injuries after the use of fluoroquinolone antimicrobials in Finland during 2002–2012. This retrospective observational study included data from the Finnish Pharmaceutical Insurance Pool's pharmaceutical injury claims. In total, 145 compensated claimants aged ≥18 years presenting tendon injuries after the use of fluoroquinolones (FQs) were included in the study. Outcomes of interest were the number of outpatient visits to primary, secondary, tertiary, and private healthcare services, hospital days, rehabilitation and their costs. Regression models were used to analyze the impact of patient characteristics on hospital days, as well as the relationship between patient characteristics and tendon ruptures. Direct costs of a tendon injury averaged 14,800€ and indirect costs were estimated to be 9,077€ for employed claimants. Fifty‐one percent of the claimants were hospitalized, with an average duration of 21 days. Hospitalization was the costliest form of health service use with an average of 9,915€ per hospital episode. Hospital days and direct costs increased with the severity of the injury. Tendon ruptures, in particular bilateral ruptures, required substantially more hospital days and their direct costs were significantly higher than those of uncomplicated tendinitis. Concurrent use of oral corticosteroids and increasing age were associated with a higher likelihood of tendon ruptures. Although rare, FQ‐related tendon injuries can result in considerable costs and health service use. Medical staff should remain vigilant when prescribing FQs, especially in groups at increased risk for tendon injuries. Impact of tendinitis and tendon rupture on hospital days and total direct costs in the 145 claimants who received compensation based on the Finnish Pharmaceutical Insurance Pool's pharmaceutical injury claims 2002–2012.  AEsadverse eventsEMAEuropean Medicines AgencyFDAFood and Drugs AdministrationFQsFluoroquinolones1Fluoroquinolones (FQs) are a class of broad‐spectrum antimicrobials that have been in wide clinical use since the 1980 and are used to treat bacterial infections including genitourinary, respiratory, gastrointestinal, skin, and soft tissue infections.The specific molecular pathway causing FQ‐related tendon injuries remains unidentified. The biological antimicrobial mechanism of FQs is based on targeting bacterial DNA gyrase (DNA topoisomerase),The aim of this study was to assess costs and health service use associated with tendon injuries relating to FQ antimicrobial use in Finland during 2002–2012. Although FQ‐related tendon injuries were first reported in 1983,2This retrospective observational study analyzed data obtained from the Finnish Pharmaceutical Insurance Pool's pharmaceutical injury claims. In Finland, patients, who suffer a pharmaceutical injury are entitled to compensation, if a causality can be detected between the pharmaceutical and the injury. A pharmaceutical injury is a legal term defined as any bodily illness or injury or a psychiatric disease likely to result from a pharmaceutical taken by the injured party.The costs of FQ‐related tendon injuries were evaluated from the Insurance Pool's compensated insurance claims from 2002 to 2012. FQ‐related tendon injury costs were borne by the Social Insurance Institution of Finland, municipalities, employers, patients, and the Finnish Pharmaceutical Insurance Pool. Of these payers, the latter is a secondary insurance system and only compensates patient costs relating to pharmaceutical injuries, which are not covered by other institutions. These costs include excess healthcare and travel costs, temporary incapacity compensation (pain and suffering), permanent functional and cosmetic incapacity, loss of income, and loss of life compensation. In order to receive compensation, the pharmaceutical injury claim must adhere to the terms and conditions of the Finnish Pharmaceutical Insurance Pool. For example, the pharmaceutical injury claim must be submitted within 3 years of the claimant becoming aware of the injury or no later than 10 years after discontinuing taking the pharmaceutical. Additionally, no compensation is rewarded if the pharmaceutical injury is regarded as tolerable in relation to the nature and severity of the illness being treated.Direct societal costs of a FQ‐related tendon injury episode were the primary outcome variable of the study, while other outcomes included the amount of outpatient visits to primary and private healthcare, visits to secondary and tertiary care, hospital days, rehabilitation and their costs in addition to travel costs. Besides direct tangible costs, intangible costs consisting of temporary and permanent incapacity compensation and time costs were quantified and added to direct costs. Outpatient visits and hospitalizations were valued with the unit costs of social and healthcare services in Finland.Indirect costs of the employed claimants were estimated according to the friction cost approach.Previous studies have shown hospitalization to be a costly form of health service use.33.1n = 52) or tendon ruptures (n = 93) after the use of fluoroquinolones were included in the study, after excluding 25 + 14 Figure n = 23) suffered bilateral ruptures. Ninety‐nine of the claimants were men and 46 were women and their average age was 66.8 years (range 21–87). Tendon injuries were reported in connection with all FQs currently on the market in Finland. In all, 121 cases (83%) were associated with levofloxacin, 13 (9%) with ciprofloxacin, 7 (5%) with norfloxacin, 2 (1%) with ofloxacin, and 2 (1%) with moxifloxacin. The majority of the claimants (52%) were treated for respiratory infections Figure In total, 145 compensated claimants aged ≥18 years presenting tendon injuries classified as tendinitis n = 5 or tendo3.2n = 93) were most often treated conservatively (equinus cast for 3–9 weeks), while 33 (23%) claimants’ tendon injuries were treated with surgery.The estimated average direct societal cost of a FQ‐related tendon injury episode per claimant, including incapacity compensation, was 14,800€. Without incapacity compensation, a FQ‐related tendon injury amounted to 8744€. The total direct costs associated with FQ‐related tendon injuries among claimants were 2,146,057€. Hospitalization was the costliest form of health service use. The claimants frequently visited a primary care facility (331 visits) and rehabilitation, such as physical and lymphatic drainage therapy (686 visits). The averages of health service use and costs are presented in Table 3.3Fourteen percent (20) of the claimants were employed at the time of their FQ‐related AEs and the Insurance Pool compensated losses of income due to FQ‐related tendon injuries in the case of six claimants (aged 39–66 years), which amounted to an average cost of almost 50,000€. In one case, the tendon injury led to permanent disability pension payments. Sick leave data were available for 16 claimants, whose sick leave lasted for an average of 78 days (range 1–331 days).The average productive work time of an employee in Finland is estimated to be 7.25 h per day, 5 days a week. The estimated friction period was thus 8 + 4 weeks, which amounted to 12 × 5 × 7.25 productive hours, that is 435 h. The average loss of productivity cost estimate was 26.08€/hour. Consequently, the indirect costs of a friction period would have amounted to 11,346€. When these costs were multiplied by the elasticity measurement 0.8, indirect costs of a FQ‐related tendon injury for an employed person amounted to 9077€ per episode.3.4n = 145). The second part, a zero‐truncated negative binomial model, predicted the length of hospital stay for those claimants (n = 74) who had at least one day of hospitalization. Negative and positive coefficient estimate variation is explained by the logit model modeling the probability of zero and the zero‐truncated modeling a positive count. The results of the two‐part model indicated that claimants who had a tendon rupture had significantly smaller odds to avoid hospitalization that those with tendinitis  as well as a 4.063 time increase in length of hospital stay , given that the other variables are held constant in the model. Additionally, advancing age and having co‐morbidities made claimants more likely to have longer hospital days or less likely to avoid hospitalization, respectively. The prescribed FQ, use of oral steroids, or the claimant's gender had no significant influence on hospitalization in this model. Norfloxacin was used as a reference variable in categorizing FQs. Changing the reference FQ did not alter the results.Dichotomous and continuous independent variables were selected to determine the impact of known risk factors, AE severity and AE year on hospitalization. As approximately half of the claimants had no hospital days and the data exhibited over dispersion, a two‐part model was built to combine the presence of excess zeros and the negative binomial distribution of counts in the data Table . The firn = 23) were the costliest subgroup. These claimants required hospitalization for an average of 25 days and their total direct costs averaged 25,731€. There were no substantial differences between males and females, different ages, treatment options, or AE years. However, claimants with co‐morbidities had on average 11 hospital days in comparison to healthy claimants who had only 2 hospital days. Figure Subgroup analyses were consistent with the results of the above models. Claimants with tendon ruptures required a mean of 15 days in hospital, and their total direct costs amounted to an average of 19,183€, whereas claimants with tendinitis were hospitalized less frequently, for an average of 2 days and with an average direct cost of 6963€. Bilateral tendon ruptures (p = .00808)  (Table 8) Table . The num4Our study found that the 145 claimants with tendon injuries had an average of 14,800€ direct costs in addition to the indirect costs that were estimated to be 9077€ for employed claimants. Fifty‐one percent were hospitalized, with an average duration of 21 hospital days. Hospitalization was the costliest form of health service use amounting to an average of 9915€ per hospital episode. Advancing age and having co‐morbidities made claimants more likely to have longer hospitalizations or less likely to avoid hospitalization altogether. The prescribed FQ, use of oral corticosteroids, or the claimant's gender had no significant influence on hospitalization.The number of FQ prescriptions has almost halved during the 2010s with all antibiotic prescribing steadily declining in Finland, and during the past few years, FQs have comprised approximately 5% of antibiotic prescriptions.As studies have shown drug‐related AEs to be a major clinical and economic problem,At present, clinicians are more likely to avoid prescribing FQs to risk groups than in 2012, due to EMA and FDA recommendations, as well as improved general awareness of the AEs. In addition, treatment practices of tendon injuries have changed slightly toward favoring conservative treatment.Although the number of levofloxacin‐based claims might give the impression that its tendency to cause tendon injuries exceeds that of other FQs, this is likely not the case. Forty‐five percent of the claimants had previously been diagnosed with chronic lung diseases, which made them more susceptible to respiratory infections that were often treated with levofloxacin. Patients with chronic lung disease are frequently treated with inhaled glucocorticoids, as well as systemic glucocorticoids, which strongly increase the risk of FQ‐associated tendon injuries. Moreover, it is possible that some of these patients had received fluoroquinolones on several occasions. The risk of tendon injuries appears to rise with an increasing number of fluoroquinolone prescriptions and with increasing cumulative doseEMA has recommended restricting FQ use in infections that are not severe, as a first‐line treatment and to avoid prescribing to high‐risk groups, such as those using corticosteroids.Medical staff needs to stay wary of the risks in order to prevent and reduce fluoroquinolone‐related tendon injuries, in addition to health service use and costs associated with them. However, due to the growing amount of antimicrobial‐resistant bacteria, the rapid decline of effective antimicrobials, and alarmingly small number of new discoveries, AEs and resulting costs might have to be increasingly tolerated in the future, in order to combat bacteria. Additionally, decision makers need to be aware of the economic burden of AEs from a societal perspective as its extent is often underestimated.The authors of this manuscript have no conflicts of interest to declare.All persons who meet authorship criteria are listed as authors. All authors took part in conceptualizing the idea, designing the study, analyzing the data and writing the manuscript. All authors have seen and approved the final version of the manuscript.This is a retrospective observational study with register data, and neither an ethics approval nor patient consent was required.