Citrus species, which have high economic importance and are frequent targets for emerging pathogens. In this study, we performed in silico analysis to identify and classify LRR-RLK homologues in the predicted proteomes of Citrus clementina (clementine) and Citrus sinensis (sweet orange). In addition, we used large-scale phylogenetic approaches to elucidate the evolutionary relationships of the LRR-RLKs and further narrowed the analysis to the LRR-XII group, which contains several previously described cell surface immune receptors.Leucine-rich repeat receptor-like kinases (LRR-RLKs) represent the largest subfamily of plant RLKs. The functions of most LRR-RLKs have remained undiscovered, and a few that have been experimentally characterized have been shown to have important roles in growth and development as well as in defense responses. Although RLK subfamilies have been previously studied in many plants, no comprehensive study has been performed on this gene family in Citrus clementina and Citrus sinensis using all predicted protein sequences obtained from whole genomes. A total of 300 and 297 proteins were identified as LRR-RLKs in C. clementina and C. sinensis, respectively. Maximum-likelihood phylogenetic trees were estimated using Arabidopsis LRR-RLK as a template and they allowed us to classify Citrus LRR-RLKs into 16 groups. The LRR-XII group showed a remarkable expansion, containing approximately 150 paralogs encoded in each Citrus genome. Phylogenetic analysis also demonstrated the existence of two distinct LRR-XII clades, each one constituted mainly by RD and non-RD kinases. We identified 68 orthologous pairs from the C. clementina and C. sinensis LRR-XII genes. In addition, among the paralogs, we identified a subset of 78 and 62 clustered genes probably derived from tandem duplication events in the genomes of C. clementina and C. sinensis, respectively.We built integrative protein signature databases for Citrus. A large expansion of LRR-XII in Citrus genomes suggests that it might play a key role in adaptive responses in host-pathogen co-evolution, related to the perennial life cycle and domestication of the citrus crop species.This work provided the first comprehensive evolutionary analysis of the LRR-RLKs in The online version of this article (doi:10.1186/s12864-016-2930-9) contains supplementary material, which is available to authorized users. Arabidopsis [Arabidopsis RLKs based on their ECDs, with the largest one containing leucine-rich repeats (LRRs) [Arabidopsis RLKs using the KDs and structural comparison of their ECDs allowed the identification of more than 40 subfamilies [Signaling through cell surface receptors is essential for cells to communicate and interact with the environment. Plant cells are able to perceive and transduce a wide range of signals mainly through receptor-like kinases (RLKs), which mediate cell-to-cell communication by binding to extracellular ligands or forming heteromeric complexes to mediate intracellular signaling . These mbidopsis . RLKs arbidopsis . The ECDbidopsis . More ths (LRRs) . Phylogefamilies .Arabidopsis genome [Arabidopsis FLAGELLIN SENSING 2 (FLS2) [In plants, LRR-RLK proteins constitute a diverse group of transmembrane receptors involved in many biological functions usually associated with growth and development –9 and res genome –16. Concs genome , 18. LRRs genome . Receptos genome  and reprs genome . The Ara2 (FLS2) , EF-TU R2 (FLS2)  and rice2 (FLS2)  are amonCitrus comprise some of the most economically important crops in the world, and the species of this group produce fruits with great commercial value, such as oranges, mandarins, lemons, grapefruits and pummelos. While Citrus clementina represents one of the major species of mandarins, consumed as fresh fruit [C. sinensis) has the largest commercial importance, mainly due to the orange juice market [C. maxima and C. reticulata [Citrus species are mostly diploid and display a basic chromosome number of x = 9. Substantial segmental synteny is observed with other eudicots and an orthology relation of one to one across oranges and plants such as grape, strawberry and cacao suggests the inexistence of recent whole genome duplication (WGD), with the exception of a triplication genome shared by all eudicots [Citrus is part of the Sapindales order, a sister order of Brassicales into the Malvidae family, which allows the performance of studies involving genomic comparisons with Arabidopsis thaliana [sh fruit , sweet oe market . The genticulata , 27. Citeudicots . Citrus thaliana .Citrus species for different diseases, such as the Citrus canker [Xylella fastidiosa, for instance, causes CVC in all commercial sweet orange varieties, but not in C. clementina, despite both species being derived from the same ancestors [C. clementina and C. sinensis and the involvement of LRR-RLKs in defense responses, we performed in silico analyses to elucidate and compare the structural organization of LRR-RLK members from the Citrus databases. The LRR-RLK subfamily has been characterized in plants such as Arabidopsis, rice, Populus, tomato, and others [Citrus species.The main problem that affects the citrus culture worldwide is the huge amount of phytopathogens , which cs canker –29, Altes canker , Huanglos canker  and Citrs canker , 33. Xylncestors . The comd others –39, but C. sinensis and C. clementina genomes, we used a combined computational approach. The predicted proteomes of each Citrus species and A. thaliana were used as inputs  and C. sinensis (4.0 %). These percentages of genes encoding kinase proteins are close to what was found in poplar and rice [C. clementina, C. sinensis and A. thaliana, respectively, contained the KD and TM helices . As reported by Shiu et al. [C. clementina and C. sinensis, respectively, in addition to the 209 KDs from the A. thaliana LRR-RLKs  phylogenetic trees allowed us to estimate the evolutionary relationships among the sequences  previously described for A. thaliana [Citrus classification  [A. thaliana was due to Brassicaceae expansion and subsequent retention in this species. For Citrus, in contrast, the number of LRR-I remained the same as suggested by LCAA, perhaps because no recent WGD was detected in Citrus plants [In general, the number of LRR-RLK receptors for most of the subfamily groups among the r genome , A. thal1 (IOS1)  and FLG21 (IOS1) , the maj1 (IOS1) , the lass plants .C. clementina and C. sinensis showed 148 and 140 members, respectively, while in A. thaliana there were only 10 corresponding members. However, one of them (At2g24130) was not included in our analysis because it did not show a predicted transmembrane helix domain in TMHMM v 2.0. If again we compare this with the number of receptors in LCAA LRR-XII (13 genes) [Citrus species, the LRR-XII had a stronger numerical expansion. Interestingly, as observed for C. clementina and C. sinensis, the LRR-XII group of different plant species also underwent an expansion [A. thaliana LRR-XII contains two of the most studied PRRs, i.e., FLS2 and EFR, which are involved in the perception of the bacterial PAMPs flagellin and EF-Tu, respectively [Oryza longistaminata is another important well-studied PRR [In relation to LRR-XII, 3 genes) , it is pxpansion , 39. Theectively , 22. In died PRR , which rdied PRR .Table 4Tde novo creation of genes and deletions [Citrus and other crop species, the number of LRR-XII is increased in relation to LCAA  method were used to detect the LRR-XII orthologs. A total of 68 paired sequences were identified whose similarity was confirmed through 13 well-supported clade grouping of the sequences from the reconstructed phylogenetic tree , related to the LRR-XIIb members and the other formed by the remaining eleven clades, which represent the LRR-XIIa  or a glycine (G) amino acid residue in the substitution of the highly conserved arginine (R) in the HRD motif [Citrus species, in which non-RD carrying C or G in place of R accounts for over 95 %  kinases are kinases that lack the highly conserved arginine (R) that precedes the catalytic aspartate (D), which is typical of most kinases . This suRD motif  and the C. clementina and 9 members from C. sinensis have a kinase with an alternative catalytic function (ACF), which did not show either RD or non-RD motifs. The non-RD kinases identified in Citrus LRR-XII open new possibilities for further studies involving the function of these receptors in defense responses by recognition of microbial signatures.These changes can affect the charge of the molecules and potentially modify the kinase regulatory and catalytic mechanisms . Only 7 Citrus LRR-XII group, we analyzed the LRR-XII RD and non-RD kinase motifs in A. thaliana. Only two sequences showed the RD motif, while all the other seven were non-RD kinases, as already identified by other authors [C. clementina and C. sinensis, respectively, were grouped across LRR-XIIb. For the non-RD kinases, 97 % and 99 % from C. clementina and C. sinensis, respectively, were grouped across LRR-XIIa. This separation of the Citrus RD and non-RD kinases in two distinct clades, as observed in A. thaliana, suggests a possible common phylogenetic origin for each of the RD and non-RD kinases in the LRR-XII group.In addition to the identification of RD and non-RD kinases in the  authors , 50. TheC. clementina and C. sinensis LRR-XII group because they can evolve new functions in relation to the ancestor proteins [We analyzed the paralogous sequences in the proteins   for C. sinensis. Chromosomes 4 and Un from C. clementina and C. sinensis, respectively, displayed the majority of the tandem duplicated genes. Duplication events seem to be pronounced in domesticated plant species [Citrus LRR-XII might be regarded as a plant-specific adaptation to extracellular signal perception, for example, to recognize different PAMPs [The  species . Segment species . Consident PAMPs .Citrus genome into the same cluster if they were arranged in a genomic fragment with a maximum of 200 Kb. LRR-XII gene clusters are present in all chromosomes, with the exception of chromosomes 1 and 3 from C. clementina and C. sinensis, respectively  were found in 33 cluster regions, and for C. sinensis, 94 of 140 LRR-XII genes (67.1 %) were distributed in 30 clusters. Tandem duplications seem to be an important mechanism for expansion of the LRR-XII group, which could be confirmed by the presence of the LRR-XII gene in clusters. Approximately 70 % and 63 % of these clusters are formed by tandem duplicated paralogs in the C. clementina and C. sinensis genomes, respectively. Wang et al. [Citrus nucleotide binding site (NBS) genes, which is a large class of intracellular immune receptor genes that also display LRR domains beyond the nucleotide-binding site domain. Clustering in NBS gene loci has been reported in many species, including Arabidopsis and rice [Gene duplication is an important strategy for adaptive evolution in plants . To idenely Fig. . A distrg et al.  also demand rice . On the and rice  and citrC. clementina and C. sinensis, 46 and 38 genes from C. clementina and C. sinensis, respectively, were located in cluster regions . This miscorrelation of some LRR-XII genes and SBs in the same chromosomes could be a result of chromosomal rearrangements in the genomes.The establishment of synteny relations between species is an important tool to improve the understanding of genome evolution and the conserved biological functions among species . To bettmes Fig. . When anina Fig. . Of the ina Fig. . The difmes Fig. . The higmes Fig. . Curioussis Fig. . In the Citrus. Lineage-specific expansion was observed in the Citrus LRR-XII group that might have occurred due to tandem duplications making the number of individuals larger compared to the majority of plant species. Considering the diverse number of pathogens affecting the domesticated citrus culture, the independent expansion of a defense-related group could be associated with an adaptive process related to plant-pathogen co-evolution. Our comparative data provided valuable information concerning these RLKs, opening new perspectives in the study of their function in diverse processes, such as development and defense responses in two worldwide important economic crops, specifically, sweet oranges and clementines.This work provides the first comprehensive evolutionary analysis of the LRR-RLKs in Citrus clementina , Citrus sinensis  [Arabidopsis thaliana (http://arabidopsis.org) were selected and downloaded. The InterProScan 4 package software (http://www.ebi.ac.uk/interpro/download.html) was used to identify different protein signatures in each proteome dataset [http://dbvis.com/).Predicted proteomes from ata.php)  and Arab dataset . To recohttp://www.cbs.dtu.dk/services/TMHMM/) hosted at the Center for Biological Sequence Analysis, Technical University of Denmark. The prediction of transmembrane helices in the protein sequences were conducted according to the default parameters of version 2.0, without considering the old model option (version 1).The LRR-RLK homologues were retrieved from the relational databases by searching for protein sequences with kinase, transmembrane and leucine-rich repeat domains. To recover the identifiers with KD, we considered Pkinase (PF00069) and Pkinase_Tyr (PF07714), according to the Pfam platform , as diagThe following LRR diagnostic domains were searched: LRR_1 - Leucine Rich Repeat (PF00560), LRRNT - Leucine rich repeat N-terminal domain (PF01462), LRV - Leucine rich repeat variant (PF01816), LRRNT_2 - Leucine rich repeat N-terminal domain (PF08263), LRR_4 - Leucine rich repeats (2 copies) (PF12799), LRR_5 - Leucine rich repeats (6 copies) (PF13306), LRR_8 - Leucine rich repeat (PF13855), LRR_9 - Leucine-rich repeat (PF14580), LRRCT - Leucine rich repeat C-terminal domain (PF01463), LRR_2 - Leucine Rich repeat (PF07723), and LRR_3 - Leucine Rich repeat (PF07725). Only proteins containing LRRs, TM and KD were then considered to be putative LRR-RLK, and for this reason, At2g24130, which did not show TM, was not included in our analyses. Alternative splicing variants were excluded from our analysis.Arabidopsis and Citrus LRR-RLK proteins were extracted by an in-house Pearl script that consider KD coordinates annotation from the Pfam database. In addition, six human kinase protein sequences were used as an outgroup  with G-INS-i strategy and default parameters [www.expasy.org), was used to remove identical or distantly related sequences. The Decrease Redundancy parameters were set as 99 for “% max similarity” and 30 for “% min similarity”. Phylogenetic analyses were performed using the Maximum-likelihood method, as implemented in PhyML [Sequences of conserved KDs from rameters . The alirameters . The prorameters . To optiin PhyML . Twelve in PhyML . The evoCitrus RD motif in the catalytic loop from the LRR-XII kinase subdomain was performed using multiple expectation maximization for motif (MEME) suite web server using default parameters [Identification of the rameters . The kinC. clementina and C. sinensis were used to determine their distribution in the Citrus chromosomes. The coordinates were retrieved accessing the genome browser from each Citrus database. The MapChart graphical tool [The genomic coordinates of each LRR-XII gene from cal tool  was usedCitrus species was achieved through grouping in the phylogenetic tree and the BBH method. The Blastp searches were performed using all the C. sinensis and C. clementina LRR-RLK sequence proteins from group XII. For tandem duplicated paralogs searches, the results from Blasp were analyzed together with well-supported clades from the LRR-XII phylogenetic trees. The tandem duplicated paralogs were eligible when they formed the same clade and showed proximity in their chromosomal location. The identification of the LRR-XII gene clusters was performed from the arrangement of these genes in the chromosomes of each species. The LRR-XII genes were grouped in the same cluster if the genome location between two genes was within 200 kb in the chromosomes of C. sinensis and C. clementina.The identification of orthologous pairwise sequences among Sibelia software. Although this tool was originally optimized to efficiently identify syntenic blocks between closely related microbial genomes [Citrus species with evolutionary proximity.The synteny analyses were performed using  genomes , this toThe minimal nucleotide length considered in the syntenic block was adjusted to 1,000 pb. Iterative de Bruijn graphs were used to show the homology results found across the LRR-XII and the genomic regions in the chromosomes.ACF, alternative catalytic function; ALRT, approximate likelihood ratio test; BBH, bidirectional best hits; BLAST, basic local alignment search tool; C, cysteine; CsiDB, citrus sinensis database; ECD, extracellular domain; EFR, Ef-Tu receptor; Ef-Tu, elongation factor thermo unstable; FLS2, flagellin sensing 2; FRK1, flg22-induced receptor-like kinase 1; G, glycine; HRD, histidine-arginine-aspartate; IOS1, impaired oomycete susceptibility 1; Kb, kilobase; KD, kinase domain; LRRCT, leucine-rich repeat C-terminal; LRRNT, leucine-rich repeat N-terminal; LRR-RLK, leucine-rich repeat receptor-like kinases; LRV, leucine-rich repeat variant; MAFFT, multiple alignment using fast fourier transform; MAMP, microbe-associated molecular pattern; MEME, multiple expectation maximization for motif elicitation; ML, maximum-likelihood; NBS, nucleotide binding site; PAMP, pathogen-associated molecular pattern; PRR, pattern-recognition receptor; R, arginine; RD, arginine-aspartate; RLCK, receptor-like cytoplasmic kinase; RLK, receptor-like kinase; S, serine; SB, syntenic block; SQL, structured query language; TAIR, the Arabidopsis information resource; TM, transmembrane; Un, unassigned; Y, tryptophan.


We surveyed morphologic alterations in calves in Belgium that were naturally infected in utero by Schmallenberg virus (SBV) and born with deformities during January–March 2012. SBV-specific RNA was distributed unevenly in different tissues. Natural intrauterine SBV infection of calves might cause serious damage to the central nervous system and muscles. During summer and fall 2011, a nonspecific febrile syndrome characterized by hyperthermia and decreased milk production was reported in adult dairy cows from farms in the Netherlands and Germany . During January–March 2012, the consequences of SBV infection on bovine fetuses were not yet known, which prompted the staff to look systematically for the new virus in all deformed calves and in calves that died spontaneously without obvious cause. Among the 67 animals in these categories, SBV genetic material was detected in 15 calves by reverse transcription quantitative PCR, and IgG specific for SBV nucleoprotein was systematically highlighted in their serum by ELISA. In addition, all attempts to retrieve the genetic material of bluetongue virus 8 and bovine viral diarrhea virus from the tissues of these 15 seropositive calves failed. None of these calves carried the mutation responsible for noninfectious arthrogryposis in local livestock. These 15 calves, in which both SBV RNA and antibodies against SBV were detected, are the subject of this study.Detailed information about the methods used to examine the calves is available in + 15 kg vs. 49 kg + 4 kg, p<0.05). The body mass deficit, severity of deformities in whole-body conformation, and amount of skeletal muscle were obviously correlated . Tendons spanning fixed joints were shorter than expected, and corresponding muscles displayed decreased mass and altered color. Often the animal’s head was distorted, having a horse-like or pig-like shape, brachygnathism, prognathism, and/or diverging sagittal axes .We observed overall permanent deviations of the axial skeleton in all 3 planes  Figure 2We systematically observed major alterations after we opened the skull and spinal canal  Figure 4Microscopic examination of the spinal cord revealed a significant decrease in neuron numbers, the magnitude of which correlated positively with the severity of whole-body deformities . Muscle The viral RNA was always present in the CNS and sometimes in the lungs and colon Table 2. >120–150 days after conception), and we deduce that damages inflicted by the virus consequently have no or little effect on its further development. Conversely, the infection probably spreads more easily and lasts much longer if the virus contaminates an immunologically immature fetus. Because transplacental infection is possible only when the first placentome is present (30 days after conception in cattle), the window during which the infection of a bovine fetus might lead to a porencephaly/hydranencephaly-micromyelia-arthrogryposis syndrome ranges from 30 to 150 days after conception. The degree of overall body deformity correlated with a progressively greater reduction in the size of the spinal cord  and with fewer spinal neurons, suggesting that the lack of movement leading to arthrogryposis results directly from the spinal cord lesions, leading to denervation atrophy of skeletal muscle. This primary role for the spinal cord lesion is further supported by the tendency of forelimbs and hind limbs to be affected bilaterally because muscle involvement might be expected to lead to more randomly distributed lesions.Our findings show that natural in utero infection of the bovine fetus by SBV may result in serious damage to the CNS and muscles. Mechanistic hypotheses that could explain these alterations are discussed in When SBV virus infects the bovine fetus during the risk window mentioned above and causes neuromuscular defects, its genetic material remains detectable at term—thus 4 months later—at a minimum. The physical form of this persisting virus and the way it persists in the face of the seroconversion are unknown. The hypothesis of the existence of sites of persistence must be addressed, for example, in the CNS (≈90% of cases were virus positive at term) or lungs (≈30%). In practice, a priority is to establish whether SBV persists in calves infected in utero but born asymptomatic.Materials and methods.Gross pathology and histology.Definition of whole-body deformity (WBD) scores in Schmallenberg virus (SBV)–infected newborn calves, Belgium, 2012; deformities of the axial skeleton ; deformities of the head ; micromyelia ; occipital ratios (mean ± SD) in control and SBV-positive calves ; and end-stage muscles in a typical SBV-infected calf.Distribution of microscopic lesions and virus-specific RNA in the skeletal mucles of 15 Schmallenberg virus–infected newborn calves ; distribution of microscopic lesions and virus-specific RNA in the central nervous system ; and distribution of microscopic lesions and virus-specific RNA in thoraco-abdominal organs.Diagnostic considerations.Mechanistic hypotheses underlying central nervous system and muscle changes.


Bovine Leukemia Virus (BLV) is distributed worldwide and causes important economic losses on dairy farms. Currently, there are no effective vaccines or antivirals against BLV. Egg yolk antibodies (IgY) has many advantages over mammalian IgG. Despite the higher yields, they are non-invasively extracted from egg yolk, do not cross react against mammalian antigens or activate the mammalian complement system. In this work we evaluate the reactivity of Igy antibodies against Bovine Leukemia Virus p24 core protein and against the whole virus particle. Hens were immunized by intramuscular inoculation with purified p24 or the virus particle until the development of high antibody-titers. Total IgY was purified from egg yolks by ammonium sulfate precipitation. The purified egg yolk antibodies strongly reacted with BLV particles from a persistently infected cell line, with supernatants from ex vivo cultures of PBMCs from natural infected animals and also with purified p24 by both ELISA and Western blot. These data suggest that chicken IgY may be a suitable platform to produce large amounts of anti-BLV antibodies for diagnostic systems. Furthermore, the use of IgY for passive immunization against BLV infection should also be explored in order to develop new strategies to control the disease in cattle.


Cucumber mosaic virus (CMV) in its natural host Arabidopsis thaliana. To do so, we serially passaged three CMV strains under strict vertical and strict horizontal transmission, alternating both modes of transmission. We quantified seed  transmission rate, virus accumulation, effect on plant growth and virulence of evolved and non-evolved viruses in the original plants and in plants derived after five passages of vertical transmission. Our results indicated that vertical passaging led to adaptation of the virus to greater vertical transmission, which was associated with reductions of virus accumulation and virulence. On the other hand, horizontal serial passages did not significantly modify virus accumulation and virulence. The observed increases in CMV seed transmission, and reductions in virus accumulation and virulence in vertically passaged viruses were due also to reciprocal host adaptation during vertical passages, which additionally reduced virulence and multiplication of vertically passaged viruses. This result is consistent with plant-virus co-evolution. Host adaptation to vertically passaged viruses was traded-off against reduced resistance to the non-evolved viruses. Thus, we provide evidence of the key role that the interplay between mode of transmission and host-parasite co-evolution has in determining the evolution of virulence.For the last three decades, evolutionary biologists have sought to understand which factors modulate the evolution of parasite virulence. Although theory has identified several of these modulators, their effect has seldom been analysed experimentally. We investigated the role of two such major factors—the mode of transmission, and host adaptation in response to parasite evolution—in the evolution of virulence of the plant virus  Cucumber mosaic virus (CMV) and its natural host Arabidopsis thaliana, we studied the role of two such factors: the mode of transmission, and host adaptation in response to parasite evolution. We serially passaged CMV under strict vertical and strict horizontal transmission, and a combination of both. Subsequently, we analysed differences in CMV seed  transmission rate, accumulation and virulence between evolved and non-evolved viruses. We also compared whether these differences varied in original plants and in plants evolved during vertical passaging. Vertical passaging increased CMV seed transmission, and reduced accumulation and virulence, while horizontal passaging had no effect. Changes during vertical passaging were determined also by reciprocal host adaptation, which additionally reduced virulence and accumulation of vertically transmitted viruses. Hence, we provide evidence that the interplay between the transmission mode and host-parasite co-evolution is central in determining virulence evolution.Virulence is a key property of parasites, and is linked to the emergence of new diseases and to the reduction of ecosystem biodiversity. Consequently, scientists have devoted a great effort to build theoretical models that predict which factors may modulate virulence evolution. However, whether (and how) these factors affect virulence evolution has been seldom analysed experimentally. Using the plant virus  Understanding which factors determine the evolution of virulence–the negative effect of parasites on host fitness Experimental analyses have shown that multiplication rates and/or transmission rates are positively correlated with virulence for most parasites of humans, animals and plants when transmitted horizontally, i.e., between host individuals that are not parent and offspring Despite the abundance of theory, and the numerous examples of important parasites with vertical or mixed modes of transmission, the largest fraction of experimental analyses of the effect of transmission mode on the evolution of virulence has been done under strict horizontal transmission. Studies of parasite evolution under vertical transmission have mostly reported a negative correlation between virulence and rate of vertical transmission, supporting the ‘continuum hypothesis’ Arabidopsis thaliana plants to virus infection is achieved through developmental reprogramming, so that resources are reallocated from the vegetative growth to reproduction Most theoretical and experimental analyses of virulence evolution overlook the important fact that virulence and transmissibility are not just parasite traits Arabidopsis thaliana L. Heynh. (Brassicaceae) - Cucumber mosaic virus . A. thaliana  has been developed as model organism for molecular and genetic analyses of a wide range of plant traits, and also is increasingly used in analyses of host-parasite co-evolution Arabidopsis facilitates performing serial passage experiments of vertical transmission, and the large numbers of seeds produced by a plant Arabidopsis, the efficiency of CMV seed transmission ranges between 2 and 8% [unpublished data].Here we analyse experimentally the role of the mode of transmission, vertical or horizontal, on the evolution of virulence, while also accounting for the effect of host-parasite co-evolution in virulence-related traits. For this, we used the plant-virus system Arabidopsis genotype for five generations under three transmission modes: strict vertical transmission, strict horizontal transmission, and alternation of vertical and horizontal transmission. We monitored the vertical transmission rate through seeds, virus accumulation and virulence after each passage. After the last passage, we compared virus accumulation and virulence of the evolved and the non-evolved virus lineages, both in the original plant stock, and in the progeny of plants derived from the fifth passage of vertical transmission. Results indicate that vertical passaging led to adaptation of the virus to greater vertical transmission, which was associated with reductions of virus accumulation and virulence. Increases in seed transmission and reductions in virus accumulation and virulence were determined also by reciprocal host adaptation during vertical passages, which additionally reduced virulence and multiplication of vertically evolved viruses. Host adaptation to vertically passaged viruses was traded-off against reduced resistance to the non-evolved viruses.In this work, we serially passaged three CMV strains in one Arabidopsis under strict vertical, strict horizontal or alternated vertical and horizontal transmission (i/SWmSW) , were determined in each of five passages of vertical transmission , virus accumulation , and virulence . Thus, lineage was not considered as a factor for further analyses.We studied the evolution of three strains of CMV; Fny-CMV , De72-CMV , and of LS-CMV  smission , Table 14,72F = 48.72; P<1×10−5), or independently . Indeed, a significant positive linear correlation between seed transmission rate and number of vertical passage was found either when the three strains were considered together  or individually . Slopes and intercepts of these regression lines did not differ significantly between CMV strains , indicating that seed transmission increased at the same rate for the three viral strains  or separately   and independently . The slope and intercept of the De72-CMV regression were different from those of the Fny-CMV and LS-CMV regressions, so that virus accumulation decreased more slowly in the former than in the latter two strains   , Table 1P≤0.010) .4,72F≥6.53; P≤0.002)  or separately . Slopes and intercepts of the regression lines for each viral strain were similar .Virulence decreased as the number of passages increased for all viral strains together and individually (P≤0.002) , Table 1r = −0.32; P = 0.005) and separately     . In addiP≤0.006) . FinallyP≤0.034) . OverallRW) and inflorescences (IW), respectively. We previously reported that tolerance of Arabidopsis to CMV is attained by reallocating resources from vegetative to reproductive structures RW and IW ratios . We also quantified the effect on SW (virulence) and virus accumulation as described above. Vertical transmission rate in plants from the ‘original’ seed stock was quantified only for vertically evolved viruses. The five virus lineages for each combination of CMV strain and mode of transmission did not differ for any of the parameters estimated  , as well as the initial non-evolved strains, were inoculated in plants from the ‘original’ seed stock. In these plants, we determined the effect of virus infection on plant vegetative and reproductive growth, through the weight of the rosettes (P≥0.077) . Thus, l3,135F≥7.53;P≤1×10−5). In general, evolved viruses of the three strains showed lower virus accumulation than non-evolved viruses (P≤0.024), with the exception of horizontally evolved Fny-CMV (P = 0.378) (P = 0.154), and in both cases was significantly lower than for horizontally evolved viruses (P≤0.001). In LS-CMV, the virus accumulation of vertically evolved viruses was lower than that of horizontally and alternately evolved viruses (P<1×10−4), which were not significantly different (P = 0.898) (P≥0.200)  . In addi= 0.898) . The modP≥0.200) .RW ratio) of any CMV strain . The effect of virus infection on reproductive growth (IW ratio) . In Fny-CMV and LS-CMV, the IW ratio was significantly higher in vertically and alternately evolved viruses than in horizontally evolved and non-evolved viruses (P≤0.051) , which were not significantly different (P>0.645) . In both strains, virulence was lower when viruses were vertically and alternately evolved than when they were horizontally evolved or non-evolved (P≤0.030). No differences in virulence were observed between evolved and non-evolved De72-CMV viruses regardless of transmission mode .The mode of transmission did not significantly affect the effect of infection on vegetative growth   varied sP≤0.051) . In De72P>0.645) . The eff1,42F≥4.42; P≤0.043), while no differences were observed for De72-CMV   .Thus, CMV evolution under strict vertical transmission results in decreased virus accumulation and virulence. Vertical transmission also results in a reduced effect of infection on plant growth, particularly of the plant reproductive structures. These effects of passaging are higher in Fny-CMV and LS-CMV than in De72-CMV.RW and IW) and on fecundity (SW) were analysed as in ‘original’ stock plants, but percentage of CMV seed transmission was not determined. For each combination of CMV strain and mode of transmission, the different virus lineages did not differ in any of the parameters estimated   , and the3,200F≥3.59; P≤0.015). Fny-CMV and LS-CMV evolved viruses accumulated at lower levels than non-evolved viruses (P<1×10−4), while the opposite was observed in De72-CMV (P≤0.055) (P = 0.001), with intermediate accumulation values of horizontally evolved viruses (P≤0.005) (P = 0.446), but was significantly less in horizontally evolved viruses (P<1×10−4). The mode of transmission did not affect the accumulation of De72-CMV (P≥0.216) (Virus accumulation in evolved plants differed among treatments (P≤0.055) . The effP≤0.005) . In LS-CP≥0.216) .RW and IW were observed among the different modes of transmission , and followed similar patterns in the three virus strains (RW and IW ratios than the non-evolved viruses (P<1×10−4), with intermediate values for horizontally and alternately evolved viruses , which were not significantly different among themselves (P≥0.068) , with vertically evolved viruses always being less virulent than viruses in the other three treatments (P<1×10−4). In addition, virulence was significantly lower in alternately evolved viruses than in the other two treatments (P≤0.052) (Differences in the effect of CMV infection in  strains . In Fny- viruses . In De72P≥0.068) . In all P≤0.052) .1,333F≥450.11; P<1×10−5), and for each strain individually . Accordingly, virulence was always lower in vertically infected plants .Finally, we explored if there were differences in virus accumulation and virulence of vertically evolved viruses in plants derived from the fifth vertical passage with respect to whether they were infected by vertical transmission (via seed) or horizontal transmission . To do so, we compared the data above for plants mechanically inoculated  with resIn summary, CMV evolution under vertical transmission decreases virus accumulation, virulence and effect of infection on the growth of plants derived from the fifth vertical passage. Differences among modes of transmission were larger in these plants than in those from the ‘original’ stock for most traits, which suggests that plants changed during the vertical passages by increasing their resistance to virus infection. Whether these changes co-evolved with those observed in the viruses is analysed in the next section.Arabidopsis plants with better performance under virus infection, the effect of infection with evolved and non-evolved strains would differ between ‘original’ stock plants and plants derived from the fifth vertical passage. To test this hypothesis, virus accumulation, RW and IW ratios and virulence in ‘original’ stock plants were compared with plants derived from the fifth vertical passage , and infected plants had higher RW and IW ratios, and lower virulence  in plants derived from the fifth vertical passage than in ‘original’ stock plants . The exception was that no differences in RW and IW ratios were observed between LS-CMV-infected plants of the ‘original’ stock and those derived from the fifth vertical passage  , but virus accumulation was similar in both types of plants . Non-evolved viruses were marginally more virulent in plants derived from the fifth vertical passage than in ‘original’ stock plants  .Fny-CMV and LS-CMV vertically evolved viruses accumulated to lower levels (k plants . The oppP≥0.198) . For De7P≤0.090) . VerticaIW and were more virulent in plants derived from the fifth vertical passage compared with ‘original’ stock plants . In De72-CMV virus accumulation was higher in plants derived from the fifth vertical passage than in the ‘original’ stock plants .Fny-CMV and LS-CMV strains horizontally evolved  caused l1,100F≥4.40; P≤0.040), but virus accumulation of De72-CMV was higher in ‘original’ stock plants than in plants derived from the fifth vertical passage . No significant differences were found in the rest of comparisons  by vertically transmitted viruses, often at the cost of increased virus accumulation and/or virulence of non-evolved viruses. These results support the hypothesis of plant-virus co-evolution during vertical transmission passages.Arabidopsis thaliana, a system with several traits suitable for our objectives: i) CMV is a pathogen of Arabidopsis that is found at high incidence in Arabidopsis wild populations ArabidopsisArabidopsis has a short generation time, which allows serial passages of vertical transmission to be performed in a reasonable time frame; and iv) recovery of Arabidopsis plants to CMV infection has not been described. From the perspective of viral fitness, host recovery is equivalent to host death in which the virus can no longer replicate or be transmitted. Therefore, recovery would affect the transmission rate, potentially blurring the virulence-transmission trade-off Arabidopsis under strict vertical or strict horizontal transmission, and quantified virulence and traits related to viral fitness in the evolved and non-evolved viruses. We used the effect of virus infection on plant fecundity as a measure of virulence. Virulence encompasses the negative effect of a parasite on host longevity and fecundity ArabidopsisMost experimental analyses of virulence evolution are based on the hypothesis that virulence, which is correlated with parasite multiplication, is determined by trade-offs with parasite transmission rate Barley stripe mosaic virus (BSMV) in barley As predicted by theory, our results show a key role of the mode of transmission in the evolution of virulence. The comparison of non-evolved viruses with those evolved under strict vertical transmission indicated that the latter increased their rate of vertical transmission, and that adaptation to this transmission mode was associated with the reduction of virus multiplication and virulence. In contrast, evolution under strict horizontal transmission did not result in changes of virus multiplication or virulence. We did not quantify the rate of vertical transmission of horizontally evolved lineages. However, the absence of evolution in virus multiplication and in virulence of horizontally passaged viruses − two traits that are correlated with vertical transmission rate in our system − may be suggestive of a lack of change in vertical transmission rate. Thus, our results support predictions of the models of virulence evolution based on the trade-off hypothesis Arabidopsis, a negative correlation between virus multiplication and transmission rate would be explained: 1) if lower virus titer would result in a less efficient triggering of plant defenses that prevent seed infection; and/or 2) if serial passages of vertical transmission selects for virus variants with mutations that facilitate direct or indirect access for CMV to the seed even at low multiplication levels.Perhaps one of the most striking results of this work is the observed negative correlation between virus multiplication and transmission rate. In other plant-parasite systems higher parasite load is associated with higher percentage of infected seeds Arabidopsis genotypes, however, as genotype-specific host tolerance to virus infection uncoupled both traits Arabidopsis genotype used in the present work (Cen-1) was rated as a low-tolerance genotype Virulence and within-host multiplication were positively correlated in viruses evolved under strict vertical transmission in this study, consistent with the central assumption of the trade-off hypothesis RW and IW). Vertically transmitted parasites may select for modifications of host life-history traits that enhance host reproductive success and parasite transmission. For instance, insects have increased survival when infected by bacteria such as Rickettsia or WolbachiaRW and IW ratios of Arabidopsis infected with vertically evolved viruses compared with non-evolved viruses might be interpreted similarly, as Arabidopsis fecundity is positively correlated with both the vegetative and the reproductive growth of the plant The reduction of CMV virulence associated with increased rates of vertical transmission is also associated with smaller effects of infection in both the vegetative and reproductive efforts of the host . De72-CMV was obtained from a field-infected plant of Diplotaxis erucoides (Brassicaceae), a host closely related to ArabidopsisNicotiana tabacum) plants for virus multiplication. CMV virions from tobacco leaves were purified as described in Three virus strains were used: Fny-CMV and De72-CMV belonging to subgroup I of CMV isolates, and LS-CMV, belonging to subgroup II. Fny-CMV and LS-CMV are well characterized and were derived from biologically active cDNA clones Arabidopsis accessions as it combined a higher efficiency of CMV vertical transmission − ranging between 2–8% depending on the virus isolate [unpublished data] −, with a relatively short life cycle Accession Cen-1  was selected from a panel of eighteen The three CMV isolates were serially passaged in Cen-1 plants five times by strict vertical transmission, strict horizontal transmission, or alternating both modes of transmission .2HPO4 when rosettes had 4–5 leaves  generously provided by Dr. Carlos Alonso-Blanco  were mechanically inoculated with purified CMV RNA of the three CMV strains (100 ng/ml) in 0.1 M NaTo analyse virus evolution under strict horizontal transmission, sap extracts from the fifteen Cen-1 plants inoculated to generate the vertically transmitted lineages were used to mechanically inoculate fifteen uninfected Cen-1 plants from the ‘original’ Cen-1 seed stock; these fifteen plants represented the first passage of horizontal transmission in five independent lineages for each virus strain. Sap extract from each of these fifteen infected plants was used to inoculate ten plants at stages 1.04–1.05 In parallel, five viral lineages per CMV strain were evolved alternating vertical and horizontal transmission to generate five alternately transmitted lineages. To do so, 100 seeds from each of the fifteen plants used to generate the vertically transmitted CMV lineages were grown, and CMV infection was detected, as described above. Sap extracts from one infected plant per lineage were used to inoculate ten Cen-1 plants from the ‘original’ seed stock. The procedure for this treatment to this point is identical to that used for the strict horizontal treatment. However, seeds from these horizontally infected plants were harvested at complete senescence. One hundred seeds per plant were grown, virus infection was detected, and one plant per lineage was randomly chosen to generate the new vertically transmitted generation. This process was repeated until the third horizontal passage was completed, for a total of three vertical passages and three horizontal passages .Multiplication and virulence of non-evolved and evolved CMV strains after vertical, horizontal and alternated passages, referred to as vertically evolved, horizontally evolved and alternately evolved viruses, were analysed in plants from the ‘original’ seed stock and in plants derived from the fifth vertical transmission passage. Sap extracts from CMV-infected plants of the fifth vertical and horizontal passages were used to inoculate ten Cen-1 plants from the ‘original’ seed stock and ten plants from seeds from the fifth passage of vertical transmission per virus lineage. Only five plants were inoculated for each of the alternately evolved viruses. In the fourth vertical transmission passage, no infected seeds were detected for one of the De72-CMV lineages; therefore, only four replicates were analysed for this treatment. Plants derived from the fifth passage of vertical transmission were chosen randomly to represent plants passaged with each of the three CMV strains, and inoculated with evolved lineages of the corresponding strain. In addition, ten plants of the ‘original’ Cen-1 stock and ten plants from one randomly chosen uninfected replicate after five vertical passages per each of the three strains were inoculated with sap from Cen-1 plants infected with non-evolved Fny-CMV, De72-CMV and LS-CMV. Mock-inoculated plants, with ten replicates per seed stock, were included as a control. Virus multiplication, and effect of virus infection on rosette and inflorescence growth, and on seed production were determined in each plant as described below.32P-labeled RNA probes obtained by transcription from cDNA clones representing the 3′ non-coding region of the three genomic RNAs, which is highly similar within a CMV isolate. For Fny-CMV and De72-CMV, a probe representing nucleotides 1933 to 2215 of Fny-CMV RNA3 (GeneBank Acc. No. D10538) was used, and for LS-CMV the probe represented nucleotides 1861 to 2193 of LS-CMV RNA3 (Acc. No AF127976). Internal CMV standards for subgroup I (Fny-CMV or De72-CMV), and subgroup II (LS-CMV) were included as a two-fold dilution series of purified RNA (0.5 to 0.001 µg) in nucleic acid extracts from mock-inoculated Arabidopsis plants. RNA extracts from infected plants were blotted at different dilutions to ensure that hybridization signal was on the linear portion of the RNA concentration-hybridization signal curve. All hybridizations were done at 65°C overnight in 6× SSC, 5× Denhardt's mixture, 0.1% sodium dodecyl sulphate, and yeast tRNA at 50 mg/ml +2 store phosphor screens to the labelled samples, and CMV multiplication was quantified by using Image-Quant 5.2  CMV multiplication was quantified as virus RNA accumulation. Total nucleic acid extracts from four leaf discs (0.01 g fresh weight) collected from four different rosette and inflorescence leaves were obtained using TRI-reagent . RNA quantification was done by dot-blot hybridization with SW) as a measure of host fitness. Thus, virulence was estimated as one minus the ratio of the total seed weight of infected (iSW) to total seed weight of mock-inoculated (mSW) plants, 1−(i/SWmSW). We also measured plant dry weight at complete senescence after drying at 65°C until constant weight, as a measure of tolerance (see RW) and inflorescence weight including seeds (IW) were measured separately. To quantify the effect of CMV infection on RW and IW, the value of each infected plant was divided by the mean value of the mock-inoculated plants .Virulence is defined as the negative effect of infection on host fitness ance see . RosetteIn each vertical passage, the rate of seed transmission was determined as the percentage of infected individuals out of the 100 germinated plants per lineage. Two leaves of twenty-day-old plants were harvested and pooled in groups of ten individuals, and total nucleic acid extracts of these pools were obtained. We pooled leaves among plants because of the low percentage of plants infected [unpublished data]. The presence of CMV in each pool was detected by dot-blot hybridization with the same probes used for quantification of virus accumulation. As negative controls, total nucleic acid extracts from pools of ten twenty-day-old non-infected plants were used. Samples with hybridization signal more than two-fold higher the negative controls were considered as positive. Two leaves were harvested from each plant within each positive pool for detection of CMV individually by dot-blot hybridization as described above.In the ‘original’ stock plants infected with the vertically evolved CMV strains, five infected plants per lineage were randomly chosen and 100 seeds per plant were grown as described above. Five-day-old seedlings were harvested in pools of ten individuals and the presence of CMV in each pool was detected essentially as described above. Because we pooled seedlings for this analysis, seed transmission rate was estimated assuming that the number of seedlings infected follows a Poisson distribution vs. passaged), and mode of transmission were considered as fixed effect factors. Significance of differences among classes within each factor was determined by Least Significant Difference (LSD) analyses. Linear associations between virus multiplication, seed transmission rate and virulence during serial passages was analysed by bivariate tests and using Pearson's correlation test Data on virus multiplication, seed transmission rate, and rosette, inflorescence and seed weights, and their various transformations, including virulence, were homoscedastic and were analysed using full factorial General Linear Models (GLM). Virus strain/lineage, type of plant Click here for additional data file.Table S2Estimates of virus accumulation, effect of infection in vegetative and reproductive growth, and virulence for each lineage in Cen-1 plants derived from the fifth vertical transmission passage.(DOCX)Click here for additional data file.


After a quasi-stable state of approximately 20 s to 2 min, depending on the species and hypertonic osmotic strength difference of disaccharides, there was a second phase of viral shrinkage. At the highest osmotic strengths, this led to an undulating light scattering profile that appeared to be related to perturbation of the viral envelope resulting in loss of virus activity, as determined by in vitro hemagglutination measurements and in vivo immunogenicity studies in mice. Addition of carboxymethyl cellulose effectively prevented vaccine activity loss in vitro and in vivo, believed to be due to increasing the viscosity of concentrated sugar solution and thereby reducing osmotic stress during coating of microneedles. These results suggest that hyperosmotic solutions can cause biphasic shrinkage of whole inactivated influenza virus which can damage vaccine activity at high osmotic strength and that addition of a viscosity enhancer to the vaccine coating solution can prevent osmotically driven damage and thereby enable preparation of stable microneedle coating formulations for vaccination.Enveloped virus vaccines can be damaged by high osmotic strength solutions, such as those used to protect the vaccine antigen during drying, which contain high concentrations of sugars. We therefore studied shrinkage and activity loss of whole inactivated influenza virus in hyperosmotic solutions and used those findings to improve vaccine coating of microneedle patches for influenza vaccination. Using stopped-flow light scattering analysis, we found that the virus underwent an initial shrinkage on the order of 10% by volume within 5 s upon exposure to a hyperosmotic stress difference of 217 milliosmolarity. During this shrinkage, the virus envelope had very low osmotic water permeability (1 – 6×10 However, there has been little mechanistic study done on the initial activity loss of the vaccine during the MN preparation process [This study focused on virus particle stability in hyperosmotic conditions is motivated by the need to stabilize vaccines during preparation of microneedle (MN) patches. Previous studies have shown that MN patches coated with influenza and other vaccines provide effective skin vaccination that generates protective immune responses that are at least as potent as conventional intramuscular or subcutaneous vaccination –3. Advan process . Among mEnveloped biological systems are subjected to osmotic stress during drying processes and in high osmotic strength solutions. Osmotic pressure, arising from osmolarity differences across a semipermeable lipid membrane, induces swelling or shrinkage of biological systems as a result of water/osmolyte transportation . The resMycobacterium smegmatis, Bacillus Calmette—Guérin (BCG), and influenza vaccines were not damaged during drying due to the absence of osmolytes  in the process [Previous work has shown that spray-dried  process ,16. Unfo process ,18. Howe process . Despite process –22. HoweE. coli when subjected to hypertonic osmotic conditions [Studies on the morphological changes of vaccines in the presence of osmotic gradients are essential to understand the role of osmotic stress in vaccine stability. Osmotic pressure most likely induces morphological changes such as shrinkage, fission, swelling, and fusion of viral membranes, as seen in other enveloped organisms. For instance, events such as the formation of daughter cells in unilamellar liposomes  were alsnditions . In the nditions –27. FromAlthough there has been much research on the effects of osmotic pressure on the growth and viability of viruses  analysis at different osmotic gradients. Results were correlated with the infectivity and hemagglutination (HA) activity of the live and inactivated viruses, respectively. Based on the assumption that osmotic pressure-induced morphological change is a key factor affecting vaccine stability, vaccine-coating formulations were modified by increasing viscosity to delay the viral shrinkage rate and to minimize viral membrane perturbation. To validate this idea, the This research shows that enveloped influenza vaccine in hypertonic solutions experiences step-wise morphological changes: a rapid initial shrinkage together with membrane perturbations following a secondary shrinkage. This shrinkage was found to be related to vaccine activity loss. Our data suggest that osmotic pressure-induced vaccine destabilization in both liquid and dry states can be prevented by adding a viscosity enhancer to the vaccine formulation. This research is expected to contribute to improving our understanding of the behavior of enveloped viruses under osmotic stress as well as the development of immunogenic influenza vaccine-coated MNs.All animal care and procedures presented in this work were approved by the Emory University Institutional Animal Care and Use Committee (IACUC) review board and conducted in accordance with the guidelines of the Emory University IACUC. Emory IACUC operates under the federal Animal Welfare Law (administered by the USDA) and regulations of the Department of Health and Human Services.A/PR/8/1934 influenza virus was prepared using hen’s eggs and purified as described previously . InactivOsmotic leakage properties of the inactivated influenza virus were analyzed by SFLS. The osmotic behavior of the virus was characterized by recording light scattering after a rapid mixing of inactivated influenza virus stock in DPBS with an equal volume of a separate solution of trehalose-dihydrate, sucrose, or NaCl  in sterile Dulbecco’s phosphate-buffered saline .–1 (injection volume 66 μL) at 4°C. The excitation wavelength was set at 546 nm using a 150 watt Xe light source and monochromator (f/3.5 grating).Osmolarities of all solutions were measured using a vapor pressure osmometer  with reference to Wescor osmolarity standards. Several osmolarities were tested to compare the effect of osmotic gradient on water permeability of the virus and to monitor its shrinking behavior. This was performed with a stopped-flow spectrometer  at a flow rate of 7 mL sPf) was calculated using the equation,k = rate constant, V0 = initial virus volume, A = initial virus surface area, Vw = molar volume of water, and ΔCos = osmolarity difference across the viral envelope [−16 cm3 and a virus surface area of 3.46 × 10−10 cm2 in iso-osmotic condition were determined in this way and used for calculations. Values of k were obtained from the single exponential curve fitting of the light scattering spectra using Bio-Kine 32 V4.46 software (Bio-Logic USA).The osmotic water permeability coefficient of viruses . Measurements were made at six temperatures  maintained using a recirculating water bath.Temperature-dependent water permeability coefficient of the virus membrane was measured at a hyperosmotic difference of ΔCUnder the assumption that the relationship between SFLS light scattering and volume is the same for inactivated influenza virus and liposomes, the time-course of viral volume change was calculated following a method reported previously . Briefly–1) of the live or inactivated influenza virus in DPBS was mixed with an equal volume of trehalose solution to apply a hyperosmotic difference of ΔCos = 217, 420, 682, and 1351 mOsm in the same manner as for osmotic shrinkage experiments. That is, an osmotic strength difference of 217, 420, 682, or 1351 ΔmOsm was achieved by mixing virus stock with a 450, 800, 1200, and 2000 mM trehalose solution in DPBS, respectively. Virus-trehalose mixtures were incubated at 4°C in a sealed container to prevent drying-induced activity changes. They were subsequently diluted with DPBS after 10 s, 1 min, 5 min, 10 min, and 30 min to remove further application of the osmotic pressure. Over the course of incubation time, activity of the virus at each osmotic gradient was assessed by measuring HA activity. For HA titer measurements, a serial dilution of the sample was mixed with a 0.7% suspension of chicken red blood cells . HA activity of the samples at each condition was calculated relative to that of virus or inactivated virus in iso-osmotic condition . All experiments were performed at 4°C to eliminate the temperature effect on the remaining HA activity.To study the effects of osmotic pressure on virus activity, 2 μg (1 μg μLos = 682 mOsm), 0.5% w/v CMC, and inactivated influenza virus in DPBS was tested by measuring HA titer. HA titer measurements were performed on inactivated influenza virus (1 μg) in trehalose solution  with 0.5% w/v CMC after incubation for 30 min.To assess the effects of viscosity on the stability of the virus against osmotic stress, carboxymethyl cellulose sodium salt  was added to the trehalose solution as a viscosity enhancer. Over time, the HA activity change of a mixture composed of trehalose (ΔCos = 682 mOsm) and trehalose (ΔCos = 682 mOsm) plus 0.5% w/v CMC. Samples were incubated for 30 min at 4°C. Live influenza virus in iso-osmotic condition was used as a control. Viral titers were determined by counting the number of plaques formed on MDCK cells.To investigate the effects of osmotic pressure on the stability of the live virus, viral infectivity was assayed by reading viral plaque forming units (PFU) from live influenza virus both with and without viscosity enhancer. Live influenza virus was mixed with trehalose only (ΔCos = 682 mOsm) solution; and trehalose (ΔCos = 682 mOsm) plus 0.5% w/v CMC solution. It should be noted that the addition of CMC in the formulation had no detectible effect on the osmolarity of the solutions. One microgram of the live or inactivated influenza virus in these formulations was coated on a Ti plate at room temperature. The Ti plate was cleaned using acetone, methanol, and isopropanol (Sigma Aldrich), followed by plasma cleaning  prior to use. The effects of viscosity enhancers on the stability of inactivated influenza virus were further tested using vaccine coating formulations composed of trehalose plus xanthan gum (Sigma Aldrich) or sodium alginate (Sigma Aldrich). For equal comparison, the concentrations of xanthan gum and sodium alginate were adjusted to have the same viscosity  as the trehalose plus 0.5% CMC formulation . Coatings were air-dried at ambient conditions for 24 h and their remaining activity was assessed by HA titer measurement. HA titers of live and inactivated influenza virus in iso-osmotic condition at 4°C were used as controls of virus coatings. For HA titer measurement, dried coating samples were reconstituted in 200 μL of DPBS overnight at 4°C. Viscosity of the coating formulations was measured using glass capillary viscometers following the manufacturer’s protocol . Viscosity was determined in triplicate.To investigate the effects of viscosity on viral stability during drying, remaining HA activity of both live and inactivated viruses was tested using virus-embedded coatings on a titanium (Ti) plate, which is the same material and surface on which microneedle coatings are performed. Two different coating formulations were used for stability tests: trehalose  solution; and virus coating with trehalose (ΔCos = 682 mOsm) plus 0.5% w/v CMC solution. MN arrays of five in-plane microneedles were fabricated by lithographic masking followed by wet etching of Ti sheets (needle dimension: 750 μm in length and 200 μm in width), as described previously [To further evaluate the effects of viscosity on osmotic stress-induced viral instability, eviously . MNs wereviously  and air-os = 682 mOsm) solution coated onto microneedles and then reconstituted, virus in trehalose (ΔCos = 682 mOsm) solution containing 0.5% w/v CMC coated onto microneedles and then reconstituted. Protein concentrations were calculated using the bicinchoninic acid assay  with bovine serum albumin as a standard. To avoid interference from the coating materials, the concentration of coating excipients was maintained to be the same between the reconstituted virus solution from MNs and bovine serum albumin standard samples.Female inbred BALB/c mice  6–8 weeks of age were immunized once with inactivated influenza virus. Dried virus-coated MNs were reconstituted in DPBS for intramuscular immunization. Four groups of mice (6 mice per group) were immunized with 0.2 μg of the protein via intramuscular route: naïve (negative control), virus in iso-osmotic buffer solution at 4°C (positive control), virus in trehalose (ΔCSerum samples were collected on the second week following immunization. Influenza virus-specific IgG antibody were measured using enzyme-linked immunosorbent assay (ELISA) plates coated with inactivated influenza virus and by goat anti-mouse IgG-specific secondary antibodies  . Opticalt-test and general linear model (ANOVA), and P value of less than 0.05 was considered to be significant. In some cases, mean values were compared to validate the results.All parameters were recorded for individuals within all groups. Multiple conditions were compared using Student's os = 0 mOsm is 105 ± 16 nm, see Fig A in To understand the effects of osmotic stress on influenza virus stability, we first determined viral membrane permeability by measuring changes in virus size as a function of osmotic strength. The osmotic permeability of the viral membrane was characterized using SFLS by monitoring the change in light scattering intensity after rapidly mixing of inactivated influenza virus , no detectable level of SFLS intensity variation was observed from trehalose, sucrose, or NaCl solutions, indicating no change in virus size. In contrast, when exposed to outwardly directed, hyperosmotic gradients, viruses showed an abrupt increase in light scattering intensity  followed by a slow saturation over time. Thus, the hyperosmotic gradient is thought to have triggered an efflux of water molecules out of the virus particles leading to a viral shrinkage with a rate dependant on the level of the applied hyperosmotic gradient.As shown in k increased in proportion to the osmotic strength imposed on the virus , indicating the existence of a smaller osmotic difference across the viral membrane in the presence of NaCl than for sugar molecules. This can be explained by leakage of the viral membrane to small ions like Na+ and Cl−. As shown in Pf of the virus measured from all osmolytes ranged from 1×10−4 cm s–1 to 6×10−4 cm s–1 under the test conditions employed, which is similar to that of a lipid bilayer membrane [Further examination of these data shows that the delay to saturation decreased as the osmotic differences across the viral membrane increased B. The she virus C. It shmembrane .os = 304 mOsm with trehalose across the viral membrane. Thermal fluctuations and instability in the virus envelope led to a noisy spectroscopic response upon hypertonic osmotic shock that showed greater variation at higher temperature  vs. 1/T plot, a Arrhenius activation energy of 15.0 kcal mol–1 was calculated (–1), as opposed to the much lower activation energy required for water transport through aqueous channel proteins  [The kinetics of osmotic water transport through the viral membrane was investigated at six different temperatures between 4°C and 40°C by applying a hyperosmotic difference of ΔCperature A. Moreoperature . For exalculated B. This porin Z) ,44. Thisos = 217 mOsm of trehalose. It is of interest to note that the SFLS curve exhibited a biphasic intensity increase over time. A steep increase in scattering intensity in the first few seconds  decreased with the increase of osmotic stress  comprising the envelope of the virus cannot adjust themselves to accommodate the stress during the secondary shrinkage phase, which results in the loss of membrane integrity and/or irreversible morphological change. This is discussed further below. In contrast, below this threshold osmotic gradient, secondary shrinkage can proceed without generation of significant or permanent membrane deformation. This explains why no notable level of intensity undulation was observed at ΔCos = 28, 217, 304, and 420 mOsm osmotic strength after the secondary shrinkage. Therefore, we expect that the threshold stress above which viruses lose membrane integrity would be between ΔCos = 562 and 682 mOsm.We carried out similar studies over a range of osmotic gradients generated by trehalose C, sucroc stress D. It is2nd. Thus, an osmotic difference of ΔCos = 2388 mOsm by NaCl (t2nd = 84 s) generated a similar level of effective osmotic difference to that of roughly 217 mOsm of trehalose (t2nd = 86 s).These results, however, were not the same for a similar range of osmotic gradients caused by NaCl, which showed no such biphasic intensity increase  in To evaluate the correlation between morphological change and viral activity, the functional activity of hemagglutinin in live and inactivated influenza virus was investigated at four osmotic conditions using trehalose. Functional hemagglutinin activity of the virus was assessed via HA assay as a function of incubation time in high osmotic strength solutions .os = 682 mOsm, with an HA activity loss of about 10–20% after 30 min. The live virus reacted in a similar way, with a slight activity loss after the first minute followed by an increase in activity loss to about 10–20% after 5 min in response to osmotic differences of ΔCos = 682 mOsm and 1351 mOsm, as shown in Fig F in os = 682 mOsm  and CMC (0.5% w/v). As shown in the plot, the virus did not show any significant activity loss, which contrasts with the results in the trehalose-only solution at the same concentration (os = 1351 mOsm) (ntration . Additio51 mOsm) B.os = 682 mOsm) and trehalose (ΔCos = 682 mOsm) plus CMC (0.5% w/v) solutions were assayed and compared to a control . As expected from the experiment of live virus hemagglutinin activity loss in hypertonic solutions , a delay in the secondary shrinkage (1.8 fold increase of t2nd), and no irregular scattering after t2nd compared with the trehalose-only solution (data not shown). These experiments indicate that the addition of a viscosity enhancer produces a dampening effect on the effective osmotic stress applied to the virus, thereby stabilizing viral membranes. The significance of this finding is that viscosity enhancers can play a critical role in stabilizing enveloped virus/vaccine under osmotic stress conditions.In the case of live influenza virus, the effect of osmotic stress on its infectivity was investigated via plaque assay. For this purpose, viral titers of live influenza virus in both trehalose-only  plus CMC (0.5% w/v) solution maintained most of their original activity even after drying , only 56% and 45% of the remaining HA activity were measured with the trehalose-only (ΔCos = 682 mOsm) solution from the inactivated and live viruses, respectively .Next, the stabilizing effects of a viscosity enhancer on the influenza virus were tested during drying. It is necessary to remember that during the drying process, osmotic stress increases with the progress of dehydration. After one day drying at ambient conditions, functional HA activity change was measured both in the presence and absence of CMC using live and inactivated influenza virus (os = 682 mOsm) plus xanthan gum (0.075% w/v) and trehalose (ΔCos = 682 mOsm) plus sodium alginate (0.3% w/v). As shown in t-test, P < 0.001). These results further support the hypothesis that a viscosity enhancer can stabilize enveloped virus against osmotic stress by suppressing membrane perturbation-induced defunctionalization of antigenic proteins.The stabilizing effect of the viscosity increase was further investigated using the trehalose solutions with different viscosity enhancers, but with the same viscosity as the trehalose (ΔCos = 682 mOsm) plus CMC (0.5% w/v): i.e., trehalose  formulation  in os = 682 mOsm) plus CMC (0.5% w/v) formulation  in In vivo experiments were performed using four different groups of mice: naïve (negative control), virus in iso-osmotic solution (positive control), solution reconstituted from dried vaccine coating on MN , and solution reconstituted from dried vaccine coating on MN . Vaccine from MN coatings was reconstituted and given by injection rather than being administered directly using the MNs in order to remove the effects of the intradermal route of administration using solid MNs and thereby provide a more direct comparison among liquid formulations injected intramuscularly.To confirm previous To evaluate the immunogenicity of these four groups, virus-specific IgG antibody levels were determined by ELISA from the sera of mice two weeks after vaccination with 0.2 μg of viral proteins. As shown in Vaccine-coated MNs have been proposed as a promising vaccine administration method that simplifies vaccination and has the potential to thereby increase vaccination coverage. This led us to investigate destabilizing factors on influenza vaccine-coated MNs and their associated mechanisms. Similar to other enveloped organisms, osmotic pressure is an important factor affecting the stability of influenza viral vaccines. Unlike bacteria or other cells, there is an inherent lack of systematic study on viral osmotic behavior. This makes it difficult to predict the stability of viral vaccines under various environmental conditions, and to identify solutions to stability problems. Therefore, this study deals with conditions under which enveloped virus becomes damaged under osmotic stress and proposes a strategy to avoid osmotic pressure-induced stability loss when making influenza vaccine-coated MNs.os = 304 , 314 mOsm (sucrose), and 326 mOsm (NaCl), osmotic water permeability was measured to be 3.4 ± 0.4×10−4 , 2.7 ± 0.6×10−4 (sucrose), and 2.2 ± 0.5×10−4 (NaCl) cm/s, with a high activation energy . These measurements are similar to those previously found for pure liposomes without any osmoregulatory proteins, indicating that only pathway for water transport is through the membrane lipids [Inactivated influenza virus was used as a model system to investigate osmotic stability of enveloped viruses. Our first goal was to characterize osmotic water leakage of the viral envelope via SFLS analysis using trehalose, sucrose, and NaCl as osmolytes. Under a hypertonic osmotic gradient, at the initial shrinkage phase , the virus’ shrinking behavior was similar to other enveloped biological systems, as determined by scattered light intensity as a measure of viral volume change. At an osmotic difference of ΔCe lipids .+ and Cl− ions through the viral membrane. Theses SFLS results observed over a range of experimental conditions and seen in both live and inactivated virus indicate that this biphasic pattern represents a characteristic osmotic shrinking behavior of influenza virus in response to hyperosmotic gradients. Furthermore, from the relationship between osmotic gradient and t2nd, we hypothesize that t2nd can be used to estimate the effective osmotic gradient applied to the virus, regardless of osmolytes.The most significant finding from the long-term measurements by SFLS with trehalose and sucrose is that the virus experienced biphasic osmotic shrinking behavior under hypertonic osmotic conditions. As demonstrated by the biphasic increase of scattered light intensity, a gradual secondary shrinkage over the course of tens of seconds to minutes was observed to follow a rapid primary shrinkage. For the NaCl osmolyte solution, a much higher level of osmolarity was required to observe the secondary shrinkage than sucrose/trehalose, believed to be due to leakage of small NaTo further understand this osmotic shrinking behavior, we should consider the viral structure. An influenza virus contains two outer layers: a lipid bilayer (~ 3.8 nm thick) and a matrix protein (M1) layer (~ 7.2 nm thick) ,48. The We hypothesize that the presence of biphasic morphological changes could be attributed to this double-shell structure. Upon exposure to hyperosmotic shock, an efflux of water molecules from the virus causes the viral envelope to shrink, which is expected to increase lateral packing density of both lipid molecules in the lipid bilayer and protein molecules in supporting the M1 layer. These layers will compress easily to a certain degree, as reflected by the initial rapid change in viral volume followed by a quasi-steady state indicated by the plateau seen in the SFLS spectra. This resistance to further shrinkage can be explained mainly by the resistance of the M1 protein layer to further compression. In a previous study by Ivanovska et al., bacteriophage capsids have been found to exhibit bimodal elastic properties . We are os = 682 mOsm for trehalose and 737 mOsm for sucrose) as shown in When the compressive stress exceeds a tolerance level during and/or after the secondary shrinkage, membrane integrity is lost, and further deformation of the virus is expected to occur in order to relieve the stress being applied to it. The membrane perturbation/morphological change would result in highly undulating light scattering intensity profile, which explains SFLS spectra of the influenza virus during strong osmotic shock .As a way to prevent osmotic stress-induced viral activity loss, we tested the effect of viscosity on viral stability. The data showed that time-dependant osmotic shrinkage was significantly inhibited by the presence of a viscosity enhancer. In other words, the viscosity enhancer played a critical role in reducing osmotic pressure-mediated effects on the virus. As a result, the effective osmotic gradients which would cause the virus to become destabilized under low-viscosity conditions were reduced to below destabilizing levels. For that reason, no loss in viral activity  was observed even at ΔCAnother effect of viscosity enhancer is on the stabilization of viral vaccines during drying. It is well known that osmotic pressure increases as water content decreases through dehydration. The resulting increased stress is a major destabilization factor for biomolecules . Therefore, controlling osmotic stress-induced destabilization is of special interest to scientists working in the area of designing drug formulations  and devising drying methods. As seen in the liquid state, we similarly observed a stabilizing effect on both live and inactivated influenza viruses in the post-drying, solid state when a viscosity enhancer was added see . Importain vitro findings about the effects of osmotic stress on viral stability could be generalized from the liquid state to the more important solid state. Although our research was performed only on influenza virus, we propose that the general idea of osmotic pressure-induced viral deactivation and the stabilizing effect of a viscosity enhancer can be used in the future development of both solid and liquid drug formulations for other enveloped vaccines, as exemplified by influenza vaccine-coated MNs.As a result, solid MNs coated with influenza virus using the trehalose-plus-viscosity enhancer formulation were shown to maintain their original antigenic immunogenicity even after 24 h of drying. This indicates that our in vitro and in vivo experiments. Overall, our results suggest that enveloped influenza virus undergoes biphasic shrinkage under hyperosmotic conditions that can lead to loss of virus immunological activity. The addition of a viscosity enhancer (CMC) in a hyperosmotic sugar-based vaccine formulation was shown to inhibit this osmotically driven damage and therefore is a useful excipient in dried-vaccine coating formulations on MNs.In this work, SFLS analysis has been employed in monitoring the time-dependent structural stability of an enveloped influenza virus under various osmotic test conditions and in developing MN vaccine formulations. We found that disaccharide-induced osmotic stress generated biphasic morphological change of the influenza virus. The onset time for the secondary shrinkage/morphological change and the degree of undulation in scattered light intensity provided quantitative/qualitative information about the effective osmotic stress applied to the virus and the structural integrity of the virus envelope, respectively. The presence of a viscosity enhancer in the solution was shown to protect virus from osmotic damage both in liquid and in dried states, by means of S1 FileFig A Transmission electron microscopy image of inactivated influenza virus. As shown in the micrograph, viruses are spherical in shape and spike-shaped glycoproteins are observed on the surface of the viral envelope. A mean diameter of the virus was measured to be 105 ± 16 nm from the analysis of such micrographs. Fig B Time course of SFLS analysis of inactivated influenza virus exposed to hypertonic solutions. Virus suspensions (0.2 μg μL−1) were abruptly exposed to hyperosmotic solutions of (i) sucrose and (ii) NaCl in a stopped-flow apparatus and the resulting changes in scattered light intensity were monitored at 546 nm as a function of time for 12 s. All measurements were made at 4°C. Data are presented as the mean ± standard deviation (SD) for n > 30. Hypertonic osmotic differences (ΔCos) are indicated within the plots of (i) and (ii). Fig C Temperature dependence of the osmotic water permeability coefficients of inactivated influenza virus derived from data in n = 15–30.) Fig D Long-term course of SFLS behavior of inactivated influenza virus in response to hyperosmotic gradients by (i) sucrose and (ii) NaCl solution. Hypertonic osmotic differences (ΔCos) are indicated at the right side of each curve. Fig E Long-term course of SFLS analysis of inactivated influenza virus in response to a hyperosmotic difference of ΔCos = 239 mOsm by NaCl solution. A biphasic intensity increase was not observed from the virus with NaCl solution at this condition. This can be explained by the leakage of the viral envelope to Na+, Cl− ions, as indicated by a gradual intensity decrease following the first phase of rapid intensity increase. Fig F HA activity change as a function of incubation time in hypertonic solutions. The effect of osmotic pressure on the activity of live influenza virus was investigated by measuring HA activity change at four osmotic strength differences  using trehalose with the increase of incubation time . HA activity change was calculated with respect to HA titer of the virus in iso-osmotic solution. All measurements were performed at 4°C.  Fig G Viscosity of the trehalose (ΔCos = 682 mOsm) solution and the trehalose (ΔCos = 682 mOsm) plus CMC (0.5% w/v) solution.  Fig H Dried vaccine-coated MNs with inactivated influenza virus in formulations of (i) trehalose (ΔCos = 682 mOsm) only and (ii) trehalose (682 mOsm) plus viscosity enhancer CMC (0.5% w/v). Influenza vaccine-coated MNs were air-dried for one day at ambient conditions and reconstituted in DPBS for vaccination of mice.(PDF)Click here for additional data file.S1 Dataset(XLSX)Click here for additional data file.


Understanding the costs to implement SBI is important for providers in planning resource needs, and for decision makers considering widespread implementation of SBI. Unfortunately, little is known about the initial costs to start an SBI program.The objective is to estimate the start-up costs of two models of SBI delivery to adolescents in primary health care settings: BI delivered by a behavioral health specialist  and BI delivered by a primary care provider .SBI was implemented in a multi-site, cluster randomized trial (N = 7) guided by Proctor's model of implementation. The economic costs of starting SBI were calculated using an activity-based costing methodology. Data collection instruments were developed to collect staff time spent in identified SBI activities and non-labor resources. Start-up activities included: 1) administrative activities, such as changes to existing electronic medical record systems and planning meetings; 2) staff training; and 3) technical assistance.The average total cost for initial implementation of SBI was $5,017 and $3,838 for the specialist and generalist models, respectively. Planning activities had the greatest impact on costs for both models . This was followed by contracted services for training and technical assistance . The average cost of staff time spent in training was similar across the two models (approximately $770). Overall, more resources were devoted to planning activities in specialist sites, making this model of delivery slightly more costly than the generalist model, largely due to its increased complexity.The initial resource investment for providers to implement SBI should not be ignored as these costs may present obstacles toward implementation. The level of resources depend on the delivery model and its integration in current practice.


In Japan, epizootic arboviral infections have severely impacted the livestock industry for a long period. Akabane, Aino, Chuzan, bovine ephemeral fever and Ibaraki viruses have repeatedly caused epizootic abnormal births and febrile illness in the cattle population. In addition, Peaton, Sathuperi, Shamonda and D’Aguilar viruses and epizootic hemorrhagic virus serotype 7 have recently emerged in Japan and are also considered to be involved in abnormal births in cattle. The above-mentioned viruses are hypothesized to circulate in tropical and subtropical Asia year round and to be introduced to temperate East Asia by long-distance aerial dispersal of infected vectors. To watch for arbovirus incursion and assess the possibility of its early warning, monitoring for arboviruses was conducted in the Yaeyama Islands, located at the most southwestern area of Japan, between 1994 and 2014.Blood sampling was conducted once a year, in the autumn, in 40 to 60 healthy cattle from the Yaeyama Islands. Blood samples were tested for arboviruses. A total of 33 arboviruses including Akabane, Peaton, Chuzan, D’ Aguilar, Bunyip Creek, Batai and epizootic hemorrhagic viruses were isolated from bovine blood samples. Serological surveillance for the bovine arboviruses associated with cattle diseases in young cattle  clearly showed their frequent incursion into the Yaeyama Islands. In some cases, the arbovirus incursions could be detected in the Yaeyama Islands prior to their spread to mainland Japan.We showed that long-term surveillance in the Yaeyama Islands could estimate the activity of bovine arboviruses in neighboring regions and may provide a useful early warning for likely arbovirus infections in Japan. The findings in this study could contribute to the planning of prevention and control for bovine arbovirus infections in Japan and cooperative efforts among neighboring countries in East Asia.The online version of this article (doi:10.1186/s12917-016-0747-z) contains supplementary material, which is available to authorized users. Culicoides biting midges. Nearly 500 arboviruses have been recorded so far and some of them can seriously harm animal health [Arthropod-borne viruses (arboviruses) are transmitted by hematophagous arthropod vectors, such as mosquitoes, ticks and l health . Arbovirl health , 3, and l health –7. The rl health , 9.Orthobunyavirus in the family Bunyaviridae is principally associated with recurring epizootics of abnormal births [Orbivirus in the family Reoviridae is also known to be an etiological agent of congenital abnormalities in cattle [Orthobunyavirus were confirmed in Japan in the past 16 years [Orbivirus is a member of Palyam virus (PALV) group and has repeatedly been isolated in Japan since 1987 [Ephemerovirus in the family Rhabdoviridae causes a febrile illness in cattle and water buffalo, and is associated with reduction of milk production in dairy cattle and loss of condition in beef cattle [Orbivirus, and difficulty swallowing is its main manifestation [Culicoides biting midges [In Japan, epizootic abortion, stillbirth, premature birth and congenital malformations in cattle caused by arboviruses have severely impacted the livestock industry for a long period . Akabanel births –12. It wn cattle , 14. In n cattle , 16. Fur16 years –19. Alth16 years , 21. As nce 1987 , 23. Itsf cattle . The lasf cattle , 26. Ibaestation . The conestation –30. The g midges  while mog midges . Becauseg midges , 32, 33.Culicoides- and mosquito-borne arboviruses are considered to be introduced from lower latitudes in Asia to Japan with the infected vectors carried by airstreams [The ideal climate for vector insects in the tropical zone permits arbovirus circulation through most of the year. Therefore, the lower-latitude regions are considered a potential source of arboviruses in the temperate zone, where vector insects are usually absent during the colder months. rstreams , 34. Altrstreams –37, detarstreams , 26, 38.The Yaeyama Islands comprise an archipelago located in the most southwestern part of Japan  from Ishigaki Island to Yonaguni Island  and hamster lung (HmLu-1) cells as described previously . Briefly2O2 in TBS.Cytopathic agents in the cultured cells were characterized by a dot immunobinding assay (DIA) as described previously , 39. BriViral RNA was extracted from the supernatant of cell cultures showing CPE with the High Pure Viral RNA Kit . Group-specific RT-PCRs targeting the S RNA segment of orthobunyaviruses, segment 3 of PALV group viruses and segment 7 of EHDV were performed using the Titan One tube RT-PCR Kit (Roche Diagnostics) in accordance with previous studies , 41. SpeGenome RNA of an orbivirus that was unidentified by the above-mentioned methods was extracted from the infected cells using the TRIZOL LS Reagent . Amplification of the cDNA of segment 2 was performed by Full-Length Amplification of cDNAs (FLAC) as described previously . In brieThe PCR products were purified with the QIAquick PCR Purification Kit  and directly sequenced with the BigDye Terminator Cycle sequencing Kit v3.1 (Life Technologies) on the ABI 3100-Avanti Genetic Analyzer (Life Technologies). The nucleotide (nt) sequences were edited by DNASIS Pro Ver. 3.0 , and a sequence similarity search was conducted with the basic local alignment search tool (BLAST). Pairwise nt sequence identities were calculated with GENETYX software ver. 10 .2 for 1 h. Then, 100 μl of the suspension of HmLu-1 cells in GIT medium  was added into each well and incubated at 37 °C under 5 % CO2 for 7 days. The antibody titer was calculated as the reciprocal of the highest serum dilution inhibiting the CPE. Samples were deemed positive if they had neutralizing antibodies to the viruses in at least a dilution of 1/8.Virus neutralization tests with AKAV OBE-1, AINOV JaNAr28, PEAV KSB-1/P/06, SATV KSB-2/C/08, SHAV KSB-6/C/02, BEFV YHL, CHUV C31, DAGV KSB-29/E/01, IBAV No.2 and EHDV-7 KSB-14/E/97 were performed. After heat inactivation at 56 °C for 30 min, bovine sera were serially diluted twofold in serum-free Eagle’s MEM containing 10 μg/ml gentamicin sulfate from 1:2 to 1:64 in the 96-well microplates. Fifty microliters of serum dilution was mixed with an equal volume of virus inoculum containing 100 × the 50 % tissue culture infective doses and incubated at 37 °C under 5 % COOrthobuyavirus [A total of 33 arbovirus isolates were obtained from bovine blood samples during the study period Table . Some ofuyavirus .Table 2AAll arboviruses that were investigated in this study were observed as present in cattle blood in the Yaeyama Islands between 1995 and 2014  of BEFV were also detected without clinical cases in 1995, 1997, 1998, 2000 and 2002, suggesting that the incursion and small-scale circulation of BEFV often occurs in the Yaeyama Islands. This also reflects the pattern of frequent epizootics of BEFV in neighboring regions, such as Taiwan and mainland China, in recent years , 51–53.The resurgence of IBAV in the mainland was confirmed in 2013 after a 26-year absence . HoweverThe pathogenicities of PEAV, SATV, and SHAV in cattle remain uncertain. However, colostrum-free calves with congenital abnormalities sometimes carry neutralizing antibodies to these viruses, suggesting their teratogenicity –19. AlthAttempts at virus isolation proved the incursions of BATV, BCV and an EHDV isolate to the Yaeyama Islands. Serological surveillance in 1994 and 1995 also revealed the transient incursion of BATV in 1994 (data not shown). It was reported that BATV occasionally causes a febrile illness in humans and ruminants . It was Annual seroprevalence patterns differed among the islands. Several factors could contribute to this result. The sampling size and cattle population on Ishigaki Island are significantly larger than those on other islands, probably resulting in the higher frequency of the virus and detection of neutralizing antibodies. Because the distance between Ishigaki Island and surrounding islands including Iriomote, Taketomi, Kohama and Kuroshima Islands is not so far , wind dispersal of infected vectors likely occurs between the islands. However, Yonaguni Island is approximately 65–110 km from the other islands in the archipelago. This geographical feature might cause independent epizootics there. In addition, Yonaguni Island is the part of Japan closest to Taiwan and mainland China, and thus more sensitive detection of arbovirus activities in adjacent regions could be conducted in some cases. Although the source of arboviruses remains unknown, long-distance migration of the infected insect vectors by air flows is the most probable route by which bovine arboviruses are introduced to the Yaeyama Islands. Atmospheric dispersal modeling would be useful to estimate plausible incursion events and possible source sites of infected vectors.It is of interest that fewer clinical cases have been reported in the Yaeyama Islands, despite the frequent incursion of ruminant-pathogenic arboviruses. Most cows kept for reproduction in the islands probably gain humoral immunity to the viruses by vaccination or natural infection before their pregnancy. Therefore, the abnormal births might be prevented in most cases if the teratogenic arboviruses were introduced to the islands. Also, constant and high seroprevalence in the cattle population might limit acute and massive epizootics of arboviruses.Culicoides biting midges are still active in the Yaeyama Islands between October and December [Because viremia of bovine arboviruses is generally short, the occasion of isolation is highly limited from sentinel cattle . TherefoDecember  and thusToo often ruminant arbovirus diseases are ignored and neglected in tropical countries because they have not yet impacted the livestock industry on the surface. However, these diseases might have caused continuous, but unrecognized losses in these countries. Further investigation regarding the ruminant arboviruses will be necessary to assess their impact on the livestock industry in the regions where they persist. Moreover, environmental change through global warming will enhance the virus spread from the tropical to the temperate zone . MonitorWe showed that long-term monitoring in the Yaeyama Islands could estimate the activity of bovine arboviruses in neighboring regions and may provide a useful early warning of risk of arbovirus infections in Japan. The findings in this study could contribute to planning of prevention and control for bovine arbovirus infections in Japan and also to beneficial information-sharing on monitoring efforts among neighboring countries. Enhancement of collaborative work in this region should be essential to reduce the livestock loss caused by arbovirus infections.AINOV, Aino virus; AKAV, Akabane virus; BATV, Batai virus; BCV, Bunyip Creek virus; BEFV, bovine ephemeral fever virus; CHUV, Chuzan virus; CPE, cytopathic effects; DAGV, D’Aguilar virus; DIA, dot immunobinding assay; EHDV, epizootic hemorrhagic disease virus; FLAC, Full-length Amplification of cDNAs; IBAV, Ibaraki virus; MEM, minimum essential medium; PALV, Palyam virus; PEAV, Peaton virus; SATV, Sathuperi virus; SBV, Schmallenberg virus; SHAV, Shamonda virus; VNT, virus neutralization test