Urophonius brachycentrus and U. achalensis) and one that does not (Zabius fuscus). Here we performed the first morphological description of the natural plugs of the two Urophonius species. We described complex three zoned structure anchored to the female genital atrium and based on this information we placed implants in the genitalia  of virgin females of the three species and measured the immune encapsulation response to this foreign body. We found a greater and heterogeneous response in different zones of the implants in the plug producing species. To corroborate the specificity of this immune response, we compared the local genital reaction with the triggered response at a systemic level by inserting implants into the female body cavity of U. brachycentrus and Zabius fuscus. We found that the systemic response did not differ between species and that only in the plug producing species the local response in the genitalia was higher than the systemic one. We also compared the total hemocyte load before and after the genital implantation to see if this parameter was compromised by the immunological challenge. We confirmed that in Urophonius species the presence of a strange body in the genitalia caused a decrease in the hemocyte load. Besides, we find correlations between the body weight and the immunological parameters, as well as between different immunological parameters with each other. Complementarily, we characterized the hemocytes of the three scorpion species for the first time. This comparative study can help to provide a wider framework of the immunological characteristics of the species, their differences and their relationship with the particular postcopulatory mechanism such as the genital plugs.Immune defense is a key feature in the life history of organisms, expensive to maintain, highly regulated by individuals and exposed to physiological and evolutionary trade-offs. In chelicerates, relatively scarce are the studies that relate postcopulatory mechanisms and immune response parameters. This work makes an approximation to the female’s immunological consequences produced after the placement of a foreign body in the genitalia of three scorpions species, two species that normally receive genital plugs during mating ( Arthropods have a relatively simpler immune system than vertebrates since they lack acquired immunity  are produced and secreted to the hemolymph . In arthGryllus texensis  where mated females have higher parasite resistance than virgins, or Allonemobius socius  wher eas mating frequency increases, hemocyte load, lytic activity, and encapsulation ability [Tenebrio molitor with a reduction in phenoloxidase activity after mating [Several studies have found changes in the immune system after mating . For exa ability –18. Conv ability  as foundr mating . Also, mr mating , 23 or cr mating . In the r mating –26.Among the many reproductive strategies that organisms exhibit, there are some that involve males’ adaptations favored by sperm competition to increase their reproductive success –29. MaleIn chelicerates, some studies investigate hemocyte ultrastructure –65 and tUrophonius brachycentrus  and U. achalensis  [Zabius fuscus (Thorell 1877), a buthid species with no genital plug [Urophonius has a ‘mixed’ plug with a combination of detachable portions of the spermatophore and glandular substances [Urophonius brachycentrus and U. achalensis transfer an ‘initial plug’ formed by two hemi-mating plugs (one per hemispermatophore) that join in the formation of the spermatophore during sperm transfer. This ‘initial plug’ presents a translucent coloration when it has just been transferred to the female and shows a progressive darkening during the reproductive season (see below the results of this study). Certain changes in the size and coloration of the plugs of Urophonius, and other species [In this work, females of three scorpion species are compared. l, 1974) , 78 two tal plug . Urophonbstances . It is k species , 39, 43, species –82.Urophonius species to begin by approaching a foreign body which the females face naturally. In second place, we evaluated and compared the systemic and local cellular immune response of the species placing implants in the body cavity and in the reproductive tract respectively. We expect not to observe interspecific differences in the systemic cellular immune reaction, but in the local response to a foreign body with a higher local immune response  in the plug producing species compared with the non-plug species. In turn, we expect to observe differences between the systemic and local immune responses in Urophonius, and not to observe these differences in Z. fuscus that does not have a genital plug. Thirdly, we surveyed the types of hemocytes present in the hemolymph and total hemocyte load (THL) of the females of each species. We observed if there were changes in the THL before and after the placement of the genital implants, in search of possible connections between the triggering of the local and systemic immune response. Finally, we looked for relations between the immunological parameters (encapsulation areas / coloration as a proxy of melanization and THL) and between the parameters and body weight of individuals, since heavier females could have greater THL and be more competent to face an immune challenge such as the genital implant.To elucidate the immunological mechanisms triggers by the female against a foreign body in the reproductive tract, we first described the genital plugs and their positioning within the female genital atrium in both Urophonius brachycentrus and U. achalensis present winter surface activity [Zabius fuscus individuals are active in summer (November to March) [U. brachycentrus: 31°23'30.7"S 64°43'42.1"W; U achalensis: 31°21'52.9"S 64°46'33.6"W; Z. fuscus: 31°22'42.5"S 64°35'28.7"W). Although Zabius fuscus (Fam. Buthidae) is phylogenetically distant from the two species of Bothriuridae, it was chosen for this study because as far as we know, there are no bothriurid species that do not have a genital plug [Tenebrio molitor  or adults of Shelfordella tartara . We maintained the specimens at constant temperatures . Voucher specimens were deposited in the collection of Laboratorio de Biología Reproductiva y Evolución, Universidad Nacional de Córdoba, Argentina.Two sister species of the family Bothriuridae were studied. activity  and wereo March) , and inso March) , 18. Indtal plug , 85–87. U. brachycentrus n = 20; U. achalensis n = 20). The specimens were sacrificed in a freezer at -20°C for fifteen minutes and then dissected under a stereoscopic microscope (Nikon SMZ 1500). After the dissection, we removed the genital plug from the female’s atrium with straight tweezers. The dissected specimens and genital plugs were photographed with a digital camera  coupled to the stereoscopic microscope. Some genital plugs (n = 10 per species) were kept in a 1 mL microcentrifuge tube exposed to the air and were photographed every week for a month. In this way, we observed if there were changes in the coloration or morphology of the genital plugs outside the female . We also evaluated changes in the coloration and morphology of the plugs inside the females throughout the reproductive season (n = 10 per species) by examining the external portion of the plugs below the genital operculum.We performed dissections to observe the positioning of the plug within inseminated females (Z. fuscus n = 8 and U. brachcyentrus n = 11)  . After this period, the implants were carefully removed, and the tissue remnants were cleaned. Each implant was photographed from two perspectives (front and back), rotating 180° [The foreign body consisted of a piece of sterile monofilament (3 mm x 0.1 mm)  (from now on ‘implant’) . To elic n = 11) . The spe n = 10) . For thiing 180°  with a dUrophonius species studied also have three zones   See   See . The zonWe completely excised the second left leg of individuals (between tarsus and tibia) to allow a considerable drop of hemolymph to flow from the wound. Immediately, we took a sample of 0.75 μl of hemolymph with a glass capillary from the wound . This sa2) and the average grayscale value of each type of encapsulation (coloration). The zone of the implant , the type of immune response elicited , the species , and the individual’ body weight were the fixed effects. The body weight of the individuals was measured before and after the placement of the implants and since there were no significant differences between both instances  the average weight value for statistical analyses was considered. In the quantification of hemocytes, the variable response was the THL, and the fixed factors were the species, the stage of quantification  and the body weight of the individuals. We also evaluated the possible interactions between the fixed factors analyzed. We included the individuals’ identity in all the models as a random effect. If the random effect variance was small, the effect of the random variable was discarded. Normality and homogeneity of variances of the variables were assessed graphically and analytically. If the assumptions were not met, the variable according to the best distribution was modeled. The coloration of ME and the THL presented a normal distribution and were analyzed with the package lme4 [We analyze the data with generalized linear mixed models (GLMM). In the analysis of the implants encapsulations, the variables response were the ME and NME areas  with translucent coloration and thin, fragile consistency. As the reproductive season progressed, the plug darkened and acquired a sclerotized consistency (June to August). We observed a decrease in the size of the distal zone of the plug towards the end of the season before parturition (November to December). Conversely, no changes were observed in the coloration or morphology of the plugs extracted from the females and exposed to the air.In both species, the genital plug adjusted exactly to the female’s atrium and blocked the lumen and the genital aperture . The gen ‘wings’ . In contaperture . Next toructures . Finallyructures . In U. bDifferent characteristics and magnitudes of encapsulation response were observed, depending on the species, the type of immune response elicited, and the zone of the implant . OccasioZ. fuscus, we did not found a statistical effect of the type of immune response elicited in the areas of encapsulation  , although there were no differences between the zones of the implant for the NME . We also did not observe differences in the coloration of ME and NME encapsulations between the types of immune response elicited  . No relationship was found between the immunological parameters analyzed and the individuals' body weight .We observe cellular response in implants of both sites of implantation , showing: NME encapsulations with transparent excrescences in all zones of the implant, and ME encapsulations located more restricted to certain portions of the implants  . In Z. f= 0.637) . We foun= 0.803) , nor bet2 = 32.655, p<0.0005; NME: Df = 2, χ2 = 11.672, p = 0.003)  . There were no significant differences for the NME coloration between zones of the implant  and between the type of immune response elicited . We did not fond a relation of the analyzed immunological parameters and the body weight of the individuals .A significant statistical interaction was found between the type of immune response elicited and the zone of the implant for the encapsulation areas  . The imp= 0.003) . The imp= 0.013) , becauseU. brachycentrus) and not-plug producing species (Z. fuscus) in area of encapsulations  and ME coloration  . The NME encapsulations were clearer in all zones of the implant in Z. fuscus. We did not observe significant differences between the zones of the implants in any of the immunological parameters of both species .The encapsulation of implants placed in the body cavity were not different between plug producing species (= 0.542) . We founU. brachycentrus, U. achalensis) and not-plug producing species (Z. fuscus) and between the zones of the implants     . ME valu<0.0005) . Urophon<0.0005) . Zabius We identified different types of hemocyte cells in the hemolymph of the females in the species studied . Hemocyt2 = 35.364, p<0.0005) . Body weight had a positive effect on THL .We found a significant interaction between the species and the stage of quantification  . Zabius We noted that the immunological parameters of the local immune response, in general, were highly correlated with one another . A positU. brachycentrus and U. achalensis, and that allowed us to perceive a complex three zoned structure anchored to the female genital atrium. We also observed changes in the coloration of plugs over time, probably attributable to a female’s role. Given the need for experimental approaches to answer questions about the function or origin of genital plugs [Urophonius species the presence of a strange body in the genitalia caused a decrease in the hemocyte load. These results could suggest a possible female role in the plugs’ formation in Urophonius and may help to provide a wider framework of the different physiological consequences related to this post-copulatory mechanism.Here we report the cellular immune response at a local and systemic level in females of three scorpion species. The study of the local immune response of encapsulation in the genitalia acquires relevance because females of two evaluated species face the placement of a natural genital plug  by the male. We have described for the first time the genital plug morphology of the al plugs –33, we pDrosophila nasuta, where the male transfers a substance within the ejaculate that would activate the phenoloxidase pathway leading to the formation of a large, opaque mass in the female’s uterus [Urophonius species the implants placed in the body cavity (systemic immune response elicited) were less encapsulated and more homogeneous than the genital implants . This would suggest a difference in the magnitude and specificity between the local and systemic immune response in these species. Conversely, Z. fuscus, the species without a genital plug, presented the same immunological parameters in the local and systemic cellular response. In general, the species did not differ in their systemic cellular immune responses, although implants with clearer NME encapsulations were observed in Z. fuscus. This does not necessarily mean a lower immune response at the systemic level in Z. fuscus, but perhaps interspecific differences in the effectiveness or time necessary for the melanization of implants. However, the interspecific differences in the local immune response in the female genital tract were very remarkable, which would suggest that the difference could be due to different agents or substances to which the genitalia is exposed . In scorpions, the cuticle thickness and histological complexity of the female’s genital atrium have been related to mechanical damage caused by the capsular eversion of the spermatophore or by the introduction of genital plugs [Z. fuscus presents simple spermatophores and a thin-walled genital atrium, whereas plug producing bothriurid species have more complex spermatophores and the genital atrium with folded epithelium and thick cuticular walls [U. brachycentrus, there have been seen regions in the atrium’s apical zone which contain epithelial cells with microvilli and pores connected to ducts [Z. fuscus. A change in the THL after an immunological challenge has been previously described in species of insects and crabs [The immune system of the female genital tract epithelium has evolved by making contact with sperm, with male's ejaculate substances and with potentially infectious agents , 100–103s uterus –106. Heral plugs , 38, 76.nd crabs –113. ThiU. achalensis and U. brachycentrus. In all cases, the plugs blocked completely the lumen of the atrium, the genital aperture, and, in some cases one of the spermathecae’ ducts. Both the female and the male could be involved in the plug’s formation. The double conformation of the plug indicates a male’s contribution in that, the ‘initial plug’ transferred by the male is formed from the ‘hemi-plugs’ in each hemispermatophore when fused in the spermatophore (involving portions of the ejaculate and glandular products). The changes of the plug over time in morphology, size and coloration could indicate a female’s role. Our results have allowed us to rule out the hypothesis that these changes of the genital plugs were due to air contact or O2 influence. The Urophonius’ genital plugs present three different zones. Females of U. achalensis showed darker melanotic encapsulation in the ‘distal’ zone, and females of U. brachycentrus in the ‘middle’ zone. The ‘proximal’ zone of the genital plug does not present dark coloration, and coincidentally, this was the area in the genital implants with smaller melanotic encapsulations and clearer coloration. It was also found that the ‘distal’ and ‘proximal’ zones of the genital implants presented the larger non-melanotic encapsulations areas. The projections formed in the ‘distal’ zone of the genital implants extending below the genital operculum were similar in shape, size, and consistency to those found in the genital plugs. These results and the specificity of the local immune response of the plug producing species add evidence to the possible female’s role in the formation of portions of the plug.We described inseminated females’ genital plugs of Urophonius achalensis, U. brachycentrus and, Z. fuscus. Two main types of cells were found: plasmatocytes (PLs) and granulocytes (GRs), in agreement with the findings of existing works on the subject [Zabius fuscus had the highest values of THL compared those of plug producing species. The causes of this difference could be related to the evolutionary history of each species and the sexual and ecological context in which they have evolved [We have described, for the first time, the types of hemocytes found in the female’s hemolymph in  subject , 114–117 subject ,114–117  subject , which c subject –122 but  subject ). There  subject . Even th subject , 114–115 evolved –126. Alt evolved –84. In a evolved .per se (aggregation of layers of hemocytes) [Z. fuscus females) presented smaller areas of melanotic encapsulations on the genital implant and clearer encapsulations. A higher THL value would indicate a higher concentration of free hemocytes in hemolymph, and potentially a lower number of hemocytes in the genital area, resulting in less encapsulated and melanizated genital implants. It has also been reported that individuals with large numbers of hemocytes have a lower proportion of phagocytic hemocytes [Since multiple immunological parameters can be costly to maintain  trade-ofmocytes)  and the mocytes) . There wmocytes) , and sommocytes) ,129–130.mocytes) ,131 sincmocytes) ,132. Heamocytes)  but see mocytes) ). Howeveemocytes .Urophonius. The description of the plug morphology and the cellular encapsulation of the genital implants have allowed us to observe complex structures and physiological processes. The encapsulation and melanization patterns on the genital implants may indicate greater and more specific response in females of species that have a genital plug. Future studies would evaluate the mechanisms underlying the observed immune responses. Sperm and other ejaculate accessory substances accompany the natural plug, and its formation will depend on multiple highly regulated factors and as a result of the interaction between the male and the female. In addition to this, the plugging occurs in the context of mating and multiple behavioral variables of mate evaluation, sex stimulation, or mating resistance cannot be ruled out, which can determine to some extent the characteristics of the plug. Therefore, next work will focus on the comparison of the immune response in females in the context of mating and taking into account the natural formation of genital plugs. Future and more detailed experiments will confirm the female role in the formation of the natural genital plugs of this species and the evolutionary interests underlying these post-copulatory mechanisms [Z. fuscus. Further studies would include analysis of changes in THL comparing virgin and inseminated females  to elucidate whether the observed results with genital implants actually reflect what happens when females are plugged. In addition, it is still necessary to corroborate whether the THL decrease could be due to recirculation of hemocytes with labeling methods or phagocytosis assay using Indian ink or fluorescent labelled bacteria. A first morphological description of the hemocytes has been made in these species, and in the future we plan to make more detailed descriptions, functional characterizations, inclusion of novel techniques such as genetic markers or antibodies, as well as other microscopy techniques such as scanning or electronic transmission, for the purpose of a precise classification, quantification and elucidation of the action mechanisms of these cells [These results help to elucidate some of the immune mechanisms triggered by the female against a foreign body in the reproductive tract. The study of the local immune response of encapsulation in the female genitalia is important to understand the plugging phenomenon in the two species of chanisms , 60–61. se cells , 136–139S1 TableThe melanotic (ME) and non-melanotic (NME) encapsulation response was measured in all three zones of implants placed in the body cavity (eliciting systemic immune response) and in the female genitalia . The total hemocyte load (THL) before and after genital implantation, and the decrease in hemocyte concentration between both stages are presented. a, b, and c indicate the grouping and separation between stages and zones of the implant (p <0.05). Capital letters  indicate the grouping and separation between species (p <0.05) .(DOCX)Click here for additional data file.S1 FigUrophonius achalensis. (B) Protruding distal portion of the genital implant positioned within the female genital atrium of Urophonius achalensis. (C) Implant inserted in the body cavity (in the membrane between the fifth and sixth segment of the mesosome) of Z. fuscus. (D) Genital plug extracted of an inseminated female of Urophonius brachycentrus. (E) Implant (nylon monofilament) before being placed on a female. (F) Scheme of a genital plug (Urophonius) and its positioning within the female genital atrium. (G) Scheme of a genital implant and its positioning within the female genital atrium. Abbreviations: ag, genital atrium; al, lumen of the genital atrium; bci, implant inserted in the body cavity (eliciting systemic immune reaction); bp, basal piece; Dz, distal zone; ga, genital aperture; gi, implant inserted in the genitalia ; gp, genital plug; ls, lumen of the seminal receptacle; ME, melanotic encapsulation; Mz, middle zone; NME, non-melanotic encapsulation; og, genital operculum; p, pectine; Pz, proximal zone; sr, seminal receptacle. Scale bars: 1 mm.(A) ‘Distal’ zone of genital plug below the female genital operculum of (TIF)Click here for additional data file.S2 FigZabius fuscus female. (B) Implant inserted in the body cavity of Urophonius brachycentrus female. (C) Implant inserted in the genitalia of Z. fuscus female. (D) Implant inserted in the genitalia of U. achalensis female. Abbreviations: bci, implant inserted in the body cavity (eliciting systemic immune reaction); Dz, distal zone; gi, implant inserted in the genitalia ; ME, melanotic encapsulation; Mz, middle zone; NME, non-melanotic encapsulation; Pz, proximal zone. Scale bars: 0.5 mm.(A) Implant inserted in the body cavity of (TIF)Click here for additional data file.


As such itis a very valuable source on information regarding bird migration.Recently, the Dutch bird migration atlas has been launched in the Netherlands might have died due to HPAI A/H5N8 virus strains during autumn–winter2016–2017 . However, AI virus strains have been circulating and diversifying in wildbird populations for at least the last 100 years  have continued tocause outbreaks in poultry , Eurasian pochard (A. ferina), mallard(Anas platyrhynchos), common teal (A. crecca), Eurasianwigeon (A. penelope), and gadwall (A. strepera). Birdswere 8–11 months of age at time of inoculation and captive-bred. Prior to simultaneoustracheal and esophageal inoculation with HPAI A/H5N1 virus some animals had positive orsuspected positive antinucleoprotein antibody titres. Inoculation with the 2005 (A/H5N1)virus caused clinical signs at 3–4 days following inoculation in both diving duck species,which were more severe in tufted ducks than in Eurasian pochards. Viral excretion washighest in Eurasian pochards and mallards. Severely affected birds died or were euthanizedin a moribund state at 4 days post inoculation  versus 6.9 (2.8–17.1) days] or cloacaroutes [2.5 (1.0–6.2) and 6.6 (2.7–16.3) days]  Cygnusolor)] on 9 December 2017  positive for HPAIafter the first detection of HPAI H5N8 . Similarly, first detection inthe Netherlands of HPAI A/H5N6 was on a commercial farm with meat ducks on 7 December, 2017,whereas the virus was first detected in wild birds [mute swan , VetsuisseFaculty, University of Bern, Bern, Switzerlandjohannes.vanderkolk@vetsuisse.unibe.chJohannes H. van der Kolk, Editor-in-Chief


Arising from F.H. Coutinho et al. Nature Communications 10.1038/ncomms15955 (2017).1 reported metagenomics-derived evidence in support of the ‘Piggyback-the-Winner’ (PtW) hypothesis that lysogeny prevalence increases at high microbial abundances. Coutinho et al.1 did not directly estimate lysogenic prevalence, but instead, found that the ratio of virus-to-microbial host abundances decreased as microbial cell abundances increased. This pattern represents potential  evidence in support of PtW. Here, we show that the bulk of these reported abundance relationships are likely spurious. Instead, we find absence of evidence for positive, sublinear correlations between virus and microbial abundances as estimated in dozens of putative virus-microbe pairs identified by Coutinho et al.1 The absence of correlations between virus and microbial abundances is a counter-indicator for PtW. Altogether, our re-analysis suggests the need for caution in using correlation-based inference to identify viral strategies from metagenomics-derived abundance relationships.Coutinho et al.1, who developed a metagenomics-based approach to characterize the diversity, ecology, host-associations, and strategies of marine phage. In doing so, they introduced a “new method for host prediction based on co-occurrence associations”, in which “virus–virus abundance associations were used for host affiliation”1. As a result, Coutinho et al.1 claimed that observed abundance relationships amongst phage and bacterial hosts in a range of marine habitats are consistent with the recently introduced mechanism of PtW2.To begin, consider the work of Coutinho et al.2, Wigington et al.3, and Parikka et al.4), with similar patterns found in predator–prey relationships5.The hypothesis underlying PtW is that viruses have increased lysogenic prevalence (and decreased lytic activity) with increasing microbial abundances. This hypothesis is meant to provide a mechanistic basis for empirical findings that total virus abundances increase with total microbial abundances even as the number of viruses per microbe decreases as microbial abundances increase. This pattern is found across marine, freshwater and other environmental systems  increases in virus abundances with microbial abundances may arise from multiple governing mechanisms. These mechanisms include PtW, whose underlying mathematical model predicts that viral abundances increase with increasing microbial abundances, albeit sublinearly . Addditional mechanisms that could explain sublinear increases include variation in life history traits in antagonistic virus–microbe dynamics7 or trade-offs in Kill-the-Winner models8. As a consequence, the value of these patterns as exclusive indicators of any particular mechanism is disputed . Nonetheless, the possibility of using metagenomics-based methods to infer virus–host pairs and their abundance relationships could provide insights into viral strategies and their consequences in marine systems.Sublinear . Upon initial inspection, many reported slopes in Coutinho et al.1 appear to be close to −1 .This observation forms the basis for the present analysis. If slopes of see Fig. . A lack y and x using a permutation test. The permutation test takes each host–virus pair and then permutes the virus abundances randomly, while maintaining the same host abundances. After each permutation, we recalculated the correlation between virus and host abundances across metagenomes. Such permutations should, in principle, eliminate latent correlations between the two variables. Yet, by chance, we might find some correlations even after permutation. Hence, this test enables a quantitative answer to the question: how often should there be slopes with at least as large a magnitude as observed in the data in the event that there were no underlying relationship between virus and host abundances? We can apply similar permutation methods for nonparametric slopes. In practice, we implemented a two-tailed randomized permutation test to generate a sampling distribution of expected slopes with mean 0.To investigate this further, we evaluated the statistical relationship between p = 0.05 level  total comparisons. Hence, the threshold of p = 0.05 is permissive. Whether using p = 0.05 or p = 0.01 for product–moment or rank-based correlations, our results suggest that nearly all of the relationships reported in Coutinho et al.1, do not show evidence of a relationship between virus and host abundance and therefore do not provide even indirect support for PtW  and 4 phylum datasets out of 16) have significant correlations at the see Fig. . A stric6 have si8 and 4 p1 do not have robust evidence in support of PtW. To the contrary, nearly the entire dataset of putative relationships between viruses and hosts do not have evidence that virus abundances are related to host abundances. It is this lack of a relationship  that explains why the observation that the ratio of viruses to hosts decreases with increasing host abundances  was incorrectly interpreted in Coutinho et al.1. Such a decrease can occur in the absence of a relationship between virus abundances and host abundances. This has been termed spurious self-correlation12.Given this reanalysis, we conclude that the abundance relationships derived from marine metagenomics datasets in Coutinho et al.13. The absence of a relationship may also indicate that the method for host prediction developed by Coutinho et al.1 fails to identify genuine interactions. In that event, the absence of a correlation may reflect the absence of an underlying interaction between putative virus–host pairs. However, correlation does not imply causation, just as the absence of correlation does not imply the absence of causation. Multiple studies have reported that correlation in microbe–microbe dynamics and virus–microbe dynamics may be a poor indicator of interaction16.Our reanalysis raises a number of questions. The absence of significant abundance relationships between viruses and hosts may be a consequence of analyzing correlations based on relative abundances, which can lead to spurious findings1 to identify virus–host pairs. If the methods of Coutinho et al.1 accurately identify virus–host pairs, then our re-examination suggests that abundance relationships between viruses and microbes may differ starkly when considered amongst lineages1 vs. when given total abundance data4. If robust, the absence of a significant relationship between virus and microbial abundances given lineage-specific interactions may result from other eco-evolutionary drivers, e.g., “cryptic dynamics” in which rapid evolution masks latent abundance relationships17.We recognize that correlation-based inference of virus–host interactions was one of multiple methods used by Coutinho et al.1 are functionally relevant. However, we can conclude that the abundance relationships inferred from metagenomes do not provide robust, indirect support for PtW (or other mechanistic hypotheses) that predict sublinear increases in viral abundances with microbial abundances. Moving forward, we hope that the combined use of new in situ technologies and principled, in silico analyses can help advance the identification of likely virus–host interactions, viral strategies, and population-level consequences of viral infection.In summary, we cannot say definitively whether the identified virus–microbes associations in Coutinho et al.1 were partitioned based on whether interactions were identified at the genus or phylum scale. In the original dataset, there were 17 and 93 virus–host datasets at the phylum scale and genus scale respectively. Each dataset has 39 distinct sampling sites. However, those sites with zero virus abundance or zero host abundance were excluded. In addition, we did not include any datasets for which there were not at least five sample sites with both non-zero levels of microbes and viruses. As a result, the datasets analyzed included 16 of 17 original virus–host pairs at the phylum level and 48 of 93 original virus–host pairs at the genus level.The datasets of Coutinho et al.4 randomized samples of size ni (the number of sample sites for virus–host pair i). In each sample, we permuted the virus abundances without replacement while holding the host abundances fixed. Distributions of measured correlations were compared to the original samples using product–moment correlation (main text and Supplementary Information) and Spearman rank correlation (see Supplementary Information).We implemented permutation tests to assess measured correlations between virus and host abundances. For each host dataset we generated 10Supplementary Information


Using a state of the art nano-electrochemical technique, we show that a single virus ‘tagged’ with silver nanoparticles can be rapidly detected in real time at the single virus level. Using a state of the art nano-electrochemical technique, we show that a single virus ‘tagged’ with silver nanoparticles can be rapidly detected in real time at the single virus level. A solution containing a low concentration of influenza virus is exposed to silver nanoparticles which are adsorbed onto the virus surface, as revealed by UV-Vis spectroscopy and transmission electron microscopy. With sufficient potential applied to a carbon electrode introduced into the solution, current spikes are observed which correspond to the oxidation of the nanoparticles decorating the virus. The frequency of the current spikes and their magnitude are linearly proportional to the virus concentration and to the surface coverage of the nanoparticles, respectively. Differences observed from single bacterium detection are discussed and a comparison with existing detection methods is made, with emphasis on the favourability of the proposed technique towards the realization of point of care test devices. The emerging need for rapid, reliable and cost effective methods of virus detection has been the subject of intensive investigation in the last decade.i.e., concentration of the bacteria or virus in the blood or urine). Therefore, there is a practical need for a simple, cost effective, point of care test device that can selectively detect in real time the existence of bacteria or a virus, and the quantification of the species' concentration is desirable. In this work we present a proof of concept towards this goal and show that by using commercially available electrochemical tools, the detection of individual viruses can be rapidly achieved. The method presented here exploits the interaction of viruses such as influenza with silver nanoparticles. This phenomena is extremely well investigated from the antiviral perspective,In practice, the evaluation of a patient’s diagnosis is generally performed rapidly by an authorized person, which is usually heavily reliant on the patient's symptoms and the prevalence of a specific common disease/infection within the community at the time and location. This diagnosis by itself in many cases causes an unnecessary use of antibiotics. Moreover, the dosages of antibiotics or antiviral drugs given are uncorrelated with the actual intensity of the infection, as the magnitude of the infection is not quantified . Later, we verify that the virus covered with silver nanoparticles is electroactive, and we conclude with a realization of single virus electrochemical detection E. coli solution (blue dashed curve).E. coli containing solution and influenza virus containing solution indicates that in both cases a similar red shift in the plasmonic peak is observed, pointing to a similar adsorption and stabilization mechanism. It should be noted however, that the absorption signals in these cases are indistinguishable, highlighting that the selective detection of the virus and bacteria cannot be achieved solely through such an optical method.First, optical and microscopic characterizations of the synthesized silver nanoparticles were made (see methods section for silver nanoparticles synthesis). As can be seen in via nanoparticle tracking analysis  simply by exploiting its adsorption mannerism together with the inherent electrochemical activity of the silver nanoparticles labels. It is worth mentioning that a solution containing E. coli and silver nanoparticles could not be tracked in vitro and only after modifying the electrode (by drop casting), could the electroactive properties of the hybrid bacteria/nanoparticles be resolved.The sticking ability of the influenza virus may be ascribed to exposed proteins that enable adsorption on the electrode surface.via chronoamperometric measurements. In brief, by this so called nano-impacts technique, random individual collisions of nanometre sized particles can be seen as current spikes or steps in the chronoamperogram, providing that the electrode is held under a sufficient potential to drive an electrochemical reaction such as the oxidation (or reduction) of the nanoparticles. The area under each individual current spike describes the amount of charge oxidised (or reduced) during each collision. By this method, the detection of various individual nanoparticles,vs. SCE) and the random spikes reflect the oxidation of nanoparticles on the ‘tagged’ virus randomly colliding with the electrode. i.e. the molarity ratio is changed), a similar spike frequency is observed but with a different average current magnitude arising from individual spikes. This observation can be explained by a higher coverage of the nanoparticles on the unsaturated virus surface. Hence, upon an individual collision of a virus with the biased electrode, a higher charge is seen when a higher concentration of silver nanoparticles is used, reflecting a higher number of silver nanoparticles oxidized at each individual collision. The charge magnitude reflects the number of silver nanoparticles that are being oxidized upon virus/electrode impact. In the same manner, comparison of After showing that the virus can be electrochemically tracked by simply exposing it to silver nanoparticles, the electrochemical properties of the ‘tagged’ virus were then extended to realise detection at the single virus level. This was performed through the use of the nano-impacts technique.Both the average charge observed in each spike  and the Via this method, in less than an hour a specific infection can be detected.One of the leading techniques for rapid viral and bacterial detection is real time reverse transcription polymerase chain reaction (RT-PCR).43From this perspective, an unadorned approach is required. A simple diagnostic tool that can answer this question with a minimal number of components, that can be implemented at the point of care, is at least as important as the ability to selectively detect different species. Moreover, the technique presented here is performed by a nonspecific modification of the nanoparticles and can in principle provide greater selectivity towards sub-species by modifying the electrochemically active nanoparticles with appropriate antibodies or other recognition elements.Very recently, ‘blocking collisions’ of proteins, DNA and cytomegalovirusin vitro detection of single E. coli ‘tagged’ with silver nanoparticles produced a maximum charge of 9 pC . Using the same approach for the C Fig. S5. The survia the use of modified nanoparticles that can bind only to a specific organism. These kinds of modifications have been established already for various species such as streptavidin modified magnetic nanoparticles for the detection of the hepatitis B virus,E. coli detection and separation,49etc.Here, we show that nanoparticles themselves can serve as a detected element, as an integrated part of the virus species upon interaction. By this approach, two important goals are achieved: (a) the electrochemical signal detected is enhanced, and (b) commercially available microelectrodes are sufficient for detection purposes. We have shown here by UV-Vis characterization that upon the interaction of silver nanoparticles with the bacteria or virus, the plasmonic peak is maintained, even at high ionic strength. However, by UV-Vis spectroscopy the two species are indistinguishable . ExploitLastly, commercially available microfluidic electrochemical sensors are now reaching the sub nA noise level and a further decrease in the noise level is more of a low demand issue rather than an electronic obstacle. Consequently, automated microfluidic electrochemical sensors combined with the proposed technique may be designed for point of care testing of viral and bacterial infections as a standard diagnostic tool.In summary, we have applied here a state of the art electrochemical technique used mostly for the detection and characterization of a low concentration of nanoparticles in solution, and broadened its application to demonstrate fast, cost effective and easy to use detection of the influenza virus in solution.3) was supplied by Fisons Scientific Equipment, Loughborough, UK. Trisodium citrate  was ordered from BDH Laboratory Supplies, Poole, UK. Silver nitrate  and potassium chloride were obtained from Sigma-Aldrich, Dorset, UK. Sodium borohydride  was supplied by Fisher Scientific, Loughborough, UK. The influenza A virus (H1N1), A/PR/8/34, was purchased from LGC ATCC (ATCC-VR-95) and experiments were done in a biosafety level 2 authorized environment. All solutions were made with ultrapure water from Millipore with a resistivity of not less than 18.2 MΩ cm at 298 K.Silver nanoparticles of 9.4 ± 3.6 nm were synthesized according to the literatureThe characterization of the silver nanoparticle suspension with and without the influenza virus was performed by UV-Vis spectroscopy . For all measurements a quartz cell with a 1 cm optical path was used.http://www.OriginLab.com) and SignalCounter software developed by Dario Omanovic .For the electrochemical measurements of the ‘sticky’ virus, a 7 μm diameter carbon fibre wire (Goodfellow Cambridge Ltd.), of approximately 1 mm in length was used as a working electrode, as reported earlier.via the scanning transmission electron microscopy (STEM) mode. Here the electron probe was limited to a diameter of c.a. 1 nm and scattered electrons were collected with a high-angle annular dark field (HAADF) detector with inner angle of 50 mrad, giving highly Z-contrast imaging.Transmission electron microscopy was performed using a JEOL 3000F instrument operated at 300 kV. Sample preparation consisted of modifying holey carbon grids by drop casting suspensions of the nanoparticles, viruses or nanoparticle-modified viruses and allowing them to dry. It should be noted that no stain was used during this imaging. Energy dispersive X-ray mapping was performed using an Oxford Instruments Si(Li) detector running the INCA processor, Supplementary informationClick here for additional data file.


Metagenomic next-generation sequencing (mNGS) is a novel approach to identify pathogens undetected by conventional methods. Herein, we report a case in which mNGS was used to identify JC virus from the cerebrospinal fluid sample of an HIV positive patient with progressive multifocal leukoencephalopathy (PML). Human immunodeficiency virus (HIV)-associated central nervous system (CNS) infections could be caused by any type of microbe  . Diagnos3; Viral Load = 2.66 × 105 copies/mL) and transferred to our hospital. MRI of the brain showed multiple lesions more marked in the white matter around bilateral ventriculus cerebri lateralis  performed from the CSF sample for CMV, HSV, and EBV identification was negative. We failed to find another public or private lab to perform the JC virus PCR test in Tianjin, China. Thus, for a quick diagnosis, we had no choice but to send the CSF for NGS analysis to an independent clinical lab . An unbiased metagenomic sequencing of CSF revealed a high load of human polyomavirus 2 . Based Human polyomavirus 2 genome (Human polyomavirus 2 genome (Human polyomavirus 2 (GenBank ID: AB103411). The phylogenetic tree presenting our sequence similarities with other Human polyomavirus (Human polyomavirus 2 with a target fragment was carried out using the primers F-GGTTTAGGCCAGTTGCTGACTT and R-GTCTCCCCATACCAACATTAGCTT. The PCR products (https://blast.ncbi.nlm.nih.gov/Blast.cgi). A 129 bp PCR product was assembled and found to be 100% identical to a reference Human polyomavirus 2 sequence. Consequently, all these results indicated that the patient was infected with Human polyomavirus 2.The patient's CSF specimen was analyzed by metagenomic sequencing. DNA was extracted from 200 μL of CSF specimen using a TIANamp Micro DNA Kit DP316. The sequencing library was constructed via NEBNext Ultra II DNA library Prep Kit. The library concentration and quality were checked using Qubit and agarose gel electrophoresis. The library was sequenced by Illumina Miniseq system. The raw data were generated by Illumina sequencing machine. High-quality data were generated after filtering out adapter, low-quality, low-complexity, and shorter reads. Then, the human sequences were excluded by mapping reads to the human reference genome via SNAP tool. The remaining data were aligned to the microbial genome database, which is more than 20000 genome. Finally, we got the microbial compositions of the sample. Thus, we noted 34 sequencing reads uniquely aligned to 2 genome , and the2 genome . We, theomavirus  is shownproducts  were seqPML is a demyelinating disease of the CNS caused by reactivation of latent JC virus that typically infects oligodendrocytes and astrocytes , 7, and Sometimes when clinical suspicion remains and yet non-invasive neuroimagings are equivocal; laboratory findings mostly enable the correct diagnosis. Unfortunately, routine technology has limited power. Under diagnosis due to a shortage of imaging or diagnostic methods are common in developing countries including China . QuantitHerein, we described the metagenomic sequencing analysis as a diagnostic tool to establish the presence of JCV infection in the CSF sample and finally confirmed the etiology of the brain lesions. In this patient, conventional diagnostic studies had failed to define causative pathogens. Our patient represents a good proof that mNGS studies are useful and perhaps provide a better characterization of the JC virus genome for diagnosis purpose.To our knowledge, there are no reports of mNGS used for PML detection from CSF. There is only one published PML case diagnosed by mNGS from a brain biopsy. RNA reads aligned to JCV and the result was confirmed by immunohistochemistry and neuropathology . In our We did not verified the sensitivity and specificity of mNGS technique for JC virus detection. But, We evaluated the sensitivity and specificity of mNGS for virus detection through spike-in experiments with other virus. The sensitivity was > 95% and the specificity was > 99%. The limit of virus detection for mNGS was estimated at 10 copies/mL. We have also detected JC virus by mNGS in several other specimens of immunocompromised patients. In our case, we detected 34 JC virus reads with mNGS. And all these sequences only matched to the genome of JC virus (identity > 98%). But the alignment with other genome sequences was very poor (identity < 90%), indicating that the JC virus sequences we detected had a good specificity.Enterobacteriaceae or Cutibaterium acnes, but so far Mycobacterium tuberculosis or JC virus are not found as contaminants in reagents or part of normal microbiota (Sometimes mNGS findings were considered indeterminate as suspected agents, frequently seen as contaminants for their pathogenic role, could not be testified by cultures or other techniques , 16. Thecrobiota . AccordiThe turnaround time for mNGS in our case was 4 days from clinical specimen receipt. With this technique, a timely etiologic diagnosis could be attained. Furthermore, it could help to narrow antimicrobial therapy and consequently reduce the treatment cost and drug-related toxicities. mNGS studies of CSF provide possibility for great improvements in our ability to detect a broad scope of CNS infection agents, with potential benefits in sensitivity, speed, and cost. It might supplement the currently used diagnostic methods for the determination of CNS disorders. With suitable case selection, proper sample managing, and result interpretation; mNGS could emerge as a promising methodology for infectious disease diagnostics. It really is expected that over another couple of years, the cost and time-to-result of metagenomics will certainly reduce, and with this, it is foreseen that it will be possible to offer this technique as the first-line diagnostic check.The studies involving human participants were reviewed and approved by Tianjin Second People's Hospital Research Ethics Committee. The patients/participants provided their written informed consent to participate in this study. Written consent for publication was obtained from the patient's family.ZW, HuX, ZY, and PM were involved in the clinical management of this patient. HaX and YG performed the mNGS and human polyomavirus 2-specific PCR. HuX, HaX, SZ, YG, and PM wrote the article. All authors read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Spirulina  is one of the cheapest sources of protein and essential vitamins. However, bitterness and bad flavor of spirulina protein may limit its use in food products. In this study, spirulina was encapsulated using ionic gelation to facilitate protein delivery. The objective was to study the effects of different types of gelation methods on particle size, texture, morphology, and crude protein content of the beads. Spirulina protein was encapsulated in alginate using both internal and external gelation methods and varying concentrations of sodium alginate and calcium chloride. A total of six different treatments were evaluated, and characterization of the beads included crude protein content, hardness measured using a texture analyzer, and thickness and width of the beads. The morphology was also studied using a scanning electron microscope (SEM). Results from the texture analysis show that the hardness of the external gelation beads is three times that of the internal gelation beads, and an increase in the alginate percentage in both gelation methods increased the firmness of the beads. The crude protein content was significantly higher with the beads formed using external gelation than with internal gelation. The SEM micrographs also show that the surface morphology of the beads produced with internal gelation has a more heterogeneous structure. Overall, the beads formed with external gelation were superior to those formed with internal gelation methods. Results from this study suggest that alginate is suitable for encapsulation of spirulina protein and these hydrogel beads could be used to enhance the protein delivery and facilitate the design of foods with alternative sources of protein. An increasing world population and depleting natural resources have created a need to develop a sustainable and cost-effective protein source. Today, protein malnutrition is a problem worldwide, and the World Health Organization reports that protein malnutrition is one of the largest contributors to child mortality . EspeciaSpirulina platensis) and for people in Chad (Spirulina maxima)[Microbial cells have the potential to provide an alternative source of protein around the world. The protein extracted from different microbial sources is known as “Single Cell Protein” (SCP). Primary sources of Single Cell Protein are bacteria, moulds, yeasts, and algae . SCP hasa maxima).Among algae proteins, spirulina is considered to be a powerhouse of nutrients and it is one of the cheapest sources of protein and essential vitamins . It has Incorporation of bioactive proteins and peptides into food products is a challenging task since they are sensitive to chemical or biochemical degradation and susceptible to aggregation. These proteins also possess a potential to cause off-flavors like bitterness or astringency in the food products . In geneEncapsulation using ionic gelation can be used to mask the bitter taste of spirulina. Ionic gelation is a chemical reaction between sodium alginate and calcium chloride, where the sodium ions are replaced by the calcium ions to form a gel-like structure. This unique property of the alginate makes it a suitable material for encapsulation of bioactive compounds and protein , 13. TheAlginates can be cross-linked by external or internal gelation methods. In the external gelation or diffusion method, the cations diffuse from the external medium into the interior of an alginate phase to form the hydrogel beads. The bioactive compound to be encapsulated is mixed with the alginate solution, and then the solution is extruded dropwise into an aqueous solution with cross-linking cations  to form gelation . For intIn this study, spirulina-alginate beads were prepared using both external and internal gelation methods with the extrusion technique. The effects of sodium alginate concentration, calcium chloride concentration and the type of gelation on the protein retention, morphological characteristics, and size and texture of the beads were studied. The goal was to identify the most suitable formulation of spirulina-alginate beads to be incorporated into a food product.Spirulina powder  was purchased from Nuts.com, NJ, USA. Sodium alginate (W201502) was purchased from Sigma- Aldrich, USA and calcium chloride was purchased from Modernist Pantry, York, ME, USA. Sodium dodecylbenzene sulfonate (289957) was purchased from Sigma- Aldrich, USA, and Polysorbate 80/ Sorbitan monooleate was purchased from Vantage, Gurnee, IL, USA. Maltodextrin (DE = 18) was purchased from Myprotein, USA.For preparation of beads using the external gelation process, the plain alginate solution was prepared by dissolving sodium alginate (1% w/w or 7% w/w) and Polysorbate-80 surfactant (1%w/w) in distilled water stirring at 65°C for 20 minutes. The spirulina powder was then mixed with the previously prepared alginate solution to obtain 15 % w/w concentration at 65°C for 20 minutes until it formed a homogeneous solution. The cross-linking solution (10% or 15% w/w) was prepared by dissolving calcium chloride powder in distilled water. The spirulina-alginate solution was drawn into a 3ml syringe with a 26 G needle and dropped manually into the cross-linking solution to form the alginate beads. As shown in For the internal gelation process, the plain alginate solution was prepared by dissolving sodium alginate (0.5% w/w or 1.5% w/w) and SDS (0.5% w/w) in distilled water stirring at 65°C for 20 minutes. The cross-linking solution was prepared by dissolving calcium chloride (2 % w/w) in distilled water. Maltodextrin (10% w/w) was used to adjust the viscosity of calcium chloride solution and ensure that the alginate beads were of uniform shape. Spirulina powder was mixed with calcium chloride solution to reach 15% w/w concentration. The spirulina-calcium chloride solution was drawn into a 3ml syringe with a 26 G needle and dropped manually into the alginate solution to form the beads. The formed beads were filtered using a strainer, rinsed using distilled water, and stored under refrigeration.External gelation: 1% sodium alginate and 10% calcium chlorideExternal gelation: 1% sodium alginate and 15% calcium chlorideExternal gelation: 7% sodium alginate and 10% calcium chlorideExternal gelation: 7% sodium alginate and 15% calcium chlorideInternal gelation: 0.50% sodium alginate and 2% calcium chlorideInternal gelation: 1.5% sodium alginate and 2% calcium chloridePreliminary trials were conducted to examine the effect of the concentrations of sodium alginate and calcium chloride on gel formation. More than 30 different formulations were developed using different concentrations of sodium alginate and calcium chloride. All preliminary formulations were evaluated for crude protein and hardness of the beads. With external gelation, the beads were not formed when the concentration of calcium chloride was below 10% or the concentration of sodium alginate was below 1%. With internal gelation, the beads were not formed when the concentration of calcium chloride was above 2%. The range of concentrations of sodium alginate and calcium chloride selected for internal and external gelation were determined from the preliminary trials. A total of six different treatments were evaluated, including the following:Samples of 10 spirulina-alginate beads obtained from each formulation and type of gelation were taken at random and measured with a digital caliper  to measure their width and length.A pair of beads from each formulation and type of gelation method was viewed under a scanning electron microscope  to determine both external and cross-sectional morphology. The beads were attached to stubs using adhesive tape and coated with gold. Finally, the beads were examined using an acceleration voltage of 10 kV at 25x and 50x magnification.The texture of the spirulina-alginate bead was analyzed using a texture analyzer (TA-XT 2i), and the compression testing was performed using a cylindrical probe. The samples were examined at a test speed of 0.5 mm/s, over a varied distance adjusted based on the dimensions of the samples in order to achieve complete compression. The maximum force (N) needed for compression represents the maximum resistance of the bead to compression of the probe, which indirectly gives an indication of the hardness of the samples. In order to obtain representative results of the hardness of the beads, experiments were performed in triplicate (with ten samples per experiment) and expressed as mean ± SDThe Dumas method  for estimation of crude protein is based on combustion of the whole sample in an oxygen- enriched environment at 950°C in order to ensure complete combustion. All samples were analyzed for crude protein content using the Dumas method in triplicate. Samples (10 g) from each formulation were dried at 102°C for 18 hours and homogenized. The homogenized samples were analyzed for percent protein using a Leco combustion instrument .The encapsulation efficiency (%) of the alginate beads was determined by dividing the amount of spirulina remaining in the beads by the initial amount of spirulina added to each formulation. The amount of spirulina remaining in each formulation was determined based on the protein content of the beads and the total protein content of spirulina.α =0.05. The research study was designed as a completely randomized design. The ANOVA procedure was used to evaluate any significant differences between the gelation methods in terms of bead dimensions, protein content, and hardness of the beads. A generalized linear model was used with different factors being the dependent variables and treatments being the independent variables. Tukey's Studentized Range Test was used to detect the significantly different treatments using Spirulina beads were prepared using both external and internal gelation methods, and varying levels of sodium alginate and calcium chloride. Differences in the formation methods resulted in different size beads. Due to the formation method, the bead shapes were not truly spherical but were more teardrop shaped, so two different dimensions were measured, termed thickness and length. For incorporation into food products, the smallest possible beads would be ideal, because they are easier to ‘hide' in existing food products . Bead si2+ ions from the shell to the core  or length (mm) of the beads. External gelation beads exhibited a more uniform, homogeneous morphology compared to internal gelation beads. The hardness of the beads prepared by external gelation was significantly higher than the hardness of the beads prepared by internal gelation. The external gelation beads also possessed significantly higher protein content than the internal gelation beads and therefore exhibited much higher encapsulation efficiency than the internal gelation beads. Findings from this study suggest that alginate is suitable for encapsulation of spirulina protein and in the future these beads could be used for incorporation of alternative protein sources into food products.


Pax6 gene, and was able to determine locus conformations across cell types with varying levels of transcriptional activity and explain different mechanisms of enhancer use. Minimal a priori knowledge of epigenetic marks is sufficient to recapitulate complex genomic loci in 3D and enable predictions of chromatin folding paths.Chromatin folded into 3D macromolecular structures is often analyzed by chromosome conformation capture (3C) and fluorescence in situ hybridization (FISH) techniques, but these frequently provide contradictory results. Chromatin can be modeled as a simple polymer composed of a connected chain of units. By embedding data for epigenetic marks (H3K27ac), chromatin accessibility (assay for transposase-accessible chromatin using sequencing [ATAC-seq]), and structural anchors (CCCTC-binding factor [CTCF]), we developed a highly predictive heteromorphic polymer (HiP-HoP) model, where the chromatin fiber varied along its length; combined with diffusing protein bridges and loop extrusion, this model predicted the 3D organization of genomic loci at a population and single-cell level. The model was validated at several gene loci, including the complex   •HiP-HoP: highly predictive heteromorphic polymer model to analyze chromatin structure•Polymer simulations use widely available epigenetic and protein binding data as input•Validate HiP-HoP model at complex loci using 3D FISH and Capture-C•Simulations uncover striking conformational variability in chromatin fiber folding Buckle et al. develop a highly predictive heteromorphic polymer (HiP-HoP) model that uses epigenetic and protein binding data to predict 3D chromatin organizations, validated by FISH and Capture-C experiments. These simulations show striking conformation variability in fiber folding within a cell population and can explain different mechanisms of enhancer use. Pax6 genomic locus at high resolution, which we probed experimentally at different levels using fluorescence in situ hybridization (FISH) imaging and Capture-C. Pax6 is surrounded by constitutively expressed genes and multiple enhancers, providing a paradigm for complex genetic interactions -cohesin loops, as well as the inherent structure of the underlying fiber. Chromatin is far from a bland homomorphic fiber; rather, it is a structurally heterogeneous material that is frequently disrupted at transcriptional hotspots , and is ractions .Pax6, globin, and SOX2 loci. Unlike other widely used “inverse modeling” approaches for predicting 3D chromatin folding, such as the recent polymer-physics-based approach PRISMR (any fitting to preexisting chromosome conformation capture carbon copy (5C) or Hi-C data, making it applicable to a wider variety of experimental situations, for instance to investigate the 3D conformation of rare or hard-to-obtain tissues and cell types.Our previous models use a simple bead-and-spring polymer to represent the chromatin fiber and as such assume that this has a uniform structure. We speculated that certain histone modifications would be indicative of disrupted chromatin with decreased linear fiber compaction; to this end, we developed a predictive heteromorphic polymer (HiP-HoP) model. Simulations of these “heteromorphic” chromatin fibers gave a much better recapitulation of locus conformation at transcriptionally active regions of the genome, providing a universal model for chromatin fiber folding that could potentially be applied to map 3D structures genome-wide in the future; as examples, here we studied the h PRISMR , or prevh PRISMR , the prePax6 locus using three different immortalized cell lines that expressed Pax6 at different levels   . The hisctively) A, usuallll lines A, but adPax6 locus. This model, where a chromosome region is represented as a bead-and-spring polymer (with each bead representing 1 kb), combines two views on what drives chromatin conformation . Both probe pairs showed a non-monotonic variation in separation as a function of Pax6 activity, both in vivo and in simulations. Separations were reduced in Pax6-ON compared to Pax6-OFF cells but were larger in Pax6-HIGH cells than in Pax6-ON cells. To quantify how well the simulations predicted the data, a measure called the K-score, ranging from zero to one (from no agreement to perfect overlap of separation distributions), was defined . Our previous studies  showed tThe model described above agreed with population based 3C-style data but could not accurately predict trends observed in single-cell FISH. We reasoned that because our model assumed a homomorphic fiber, variation between cell types could only arise from differences in the TF binding or CTCF locations as loop extruder anchor sites. However, it is known that chromatin fibers can adopt alternate configurations , and recPax6 . We focus here on the case of Pax6-HIGH cells. First, a qualitative inspection of simulated conformations see , and S5  classes A. The di classes B and 5C;ar apart D. These Beads that overlap peaks of H3K27ac are shown in yellow; beads at the Pax6 promoters, the URR, and the DRR are shown in red, blue, and green, respectively. All other beads are gray. In the top left, the same snapshot is shown, but now all beads are invisible except those at the URR, Pax6, and the DRR. The 3D structure has been orientated such that the triangle made by the URR-Pax6-DRR beads is in the plane of the image. The order in which the structures are shown is such that the conformation that most closely matches the current one is shown next .Beads that overlap peaks of H3K27ac are shown in yellow; beads at the Pax6 promoters, the URR, and the DRR are shown in red, blue, and green, respectively. All other beads are gray. In the top left, the same snapshot is shown, but now all beads are invisible except those at the URR, Pax6, and the DRR. The 3D structure has been orientated such that the triangle made by the URR-Pax6-DRR beads is in the plane of the image. The order in which the structures are shown is such that the conformation that most closely matches the current one is shown next .Beads that overlap peaks of H3K27ac are shown in yellow; beads at the Pax6 promoters, the URR, and the DRR are shown in red, blue, and green, respectively. All other beads are gray. In the top left, the same snapshot is shown, but now all beads are invisible except those at the URR, Pax6, and the DRR. The 3D structure has been orientated such that the triangle made by the URR-Pax6-DRR beads is in the plane of the image. The order in which the structures are shown is such that the conformation that most closely matches the current one is shown next .Pax6 promoters , or all three were in contact. Interestingly, within the 200 structures for each cell type, none had interactions between the two enhancers without them also interacting with Pax6, and the DRR never interacted with Pax6 in isolation. Consistent with the observations detailed above, in Pax6-OFF cells, the majority of conformations had no Pax6-enhancer interactions; in Pax6-ON cells, a large proportion of conformations had Pax6 interacting with both enhancers; and in Pax6-HIGH cells, Pax6 more often only interacted with the URR   H, we spend LDHA) A and thend LDHA) E from ound LDHA) . Pax6-ONnd LDHA) I, analogPax6 folding, but since our predictive heteromorphic polymer simulation approach only requires four different datasets as input , it is applicable to a large number of active chromatin loci in different cell lines (associated with different levels of locus activity). To test how well the HiP-HoP model performs at other loci, we studied the folding of the α and β-globin loci in mouse erythroid cells, which involve simpler genomic interactions with respect to Pax6. As expected, our simulated structures for globin compared favorably with previously published Capture-C and FISH data . Additionally, in Pax6-HIGH cells, microscopy and HiP-HoP simulations showed that the chromatin fiber at the DRR must undergo a dramatic decondensation, which is associated with a 50% increase in the mean separation between this enhancer site and the Pax6 promoter. Two possible explanations for these results are that the downstream enhancer is not involved in upregulation of Pax6 in these cells or that there is an enhancer activity that does not require physical proximity to the promoter but is instead associated with chromatin decompaction , there is a large degree of cell-to-cell variation in locus conformation, and this probably reflects how cells require different transcription levels and heterogeneity in various cellular situations, regulated by alternate enhancers. This is different to the case of the globin loci where both loci were found to adopt one of a small number of preferred configurations , the same model will be applicable to active loci generally. Indeed, we have shown that these predictive heteromorphic polymer simulations can be successfully applied to loci in different cell types and organisms, such as α and β-globin or nick.gilbert@ed.ac.uk).Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Nick Gilbert  were isolated from a mouse insulinoma , Pax6-ONH3K27ac , CTCF , and Rad21 (Abcam Ab992) antibodies where used for ChIP with two biological replicates per antibody condition. Two T75 flasks of 80% confluent cells were used for each ChIP, representing two biological replicates per antibody condition and this was performed using a cross-linked ChIP protocol adapted from . All cenEach IP was performed with 25 μl of Protein G Dyna Beads (ThermoFisher), pre-washed with 2x 1 mL PBS/BSA, and incubated in 200 μl PBS/BSA with 10 μl of capture antibody (CTCF antibody (D31H2 XP Rabbit mAb #3418), Rad21 antibody (Abcam Ab992), H3K27ac  or control Rabbit IgG (I5006 Sigma)) for 3 hr, rotating at 4°C. One T75 worth of cross-linked and sonicated chromatin was used per ChIP, diluted in sonication buffer and pre-blocked using 10 μl of native beads for 30 min rotating at 4°C. Blocking beads were removed and added to pre-prepared bead/antibody complex, for incubation overnight rotating at 4°C. 10% of chromatin was stored as input. CTCF and Rad21 Beads were washed sequentially at 4°C in 1 mL each of ChIP wash, 1x ChIP wash 1, 3x ChIP wash 2, 1x ChIP wash 3, beads were transferred to a new tube, and washed 2x ChIP wash 4, 10 min 4°C . H3K27acChIP Lysis Buffer: 50 mM HEPES pH 6.8, 140 mM NaCl, 10% (v/v) glycerol, 0.5% (v/v) IPEGAL, 0.25% (v/v) Triton X, 1mM EDTA, 0.25 mM PMSFPre ChIP Wash: 200 mM NaCl, 10 mM Tris pH 8, 1 mM EDTA, 0.5 mM EGTA, 0.25 mM PMSFSonication Buffer: 100 mM NaCl, 0.1% (v/v) Na deoxycholate, 0.5% N-lauroylsarcosine 10 mM Tris pH8, 1 mM EDTA, 0.5 mM EGTA, 0.25 mM PMSFChIP Wash 1: 25 mM Tris pH 8, 150 mM NaCl, 1% Triton X-100, 0.1% SDS, 2 mM EDTA, 0.25 mM PMSFChIP Wash Buffer 2 : 25 mM Tris pH 8, 500 mM NaCl, 1% Triton X-100, 0.1% SDS, 2 mM EDTA, 0.25 mM PMSFChIP Wash Buffer 3 (detergent): 10 mM Tris pH8, 250 mM LiCl, 1% Triton X-100, 1% IPEGAL, 1% sodium deoxycholate, 2 mM EDTA, 0.25 mM PMSFElution Buffer: 50 mM Tris pH 8, 10 mM EDTA, 1% SDSRIPA Buffer B: 50 mM HEPES (pH 7.6), 1 mM EDTA, 0.7% Na deoxycholate (v/v), 1% (v/v) Nonidet P-40, and 0.5 M LiClChIP Wash 4: TE, 50 mM NaClPax6  were performed. Agilent 180K custom tiling arrays were used for Rad21 ChIP-Chip samples (4 Mb Pax6), labeling was performed with CYTAG CGH labeling kit (Enzo) and processed at the VU microarray facility, Amsterdam. For ChIP-ChIP Ringo (Bioconductor) was used for pre-processing, normalization, combining replicates and peak calling of ChIP-chip data , Rad21 and Inputs had a second amplification  purified  and labeled . ChIP samples (Cy5) and Input samples (Cy3) were hybridized using a NimbleGen Hybridization and Sample Tracking Control Kit (Roche Cat. 05993776001) according to the manufacturer’s instructions. Slides were washed  and scanned at 2 μm resolution on a MS 200 Microarray Scanner (Nimblegen). Images were processed using NimbleScan (version 2.5). Two replicates of ChIP hybridization along with input to custom genomic microarrays (Nimblegen 720K) tiling a 66 Mb region around hip data .STORM software  was used2, 1.5 mM CaCl2, 0.2 mM PMSF, 1 mM DTT). Nuclei where checked for purity, and counted. Protocol for transposition reaction and PCR based on Pax6 region and genome-wide peak calling was performed using MACS version 2.1.1, with a q-value cut off pf 0.05.ATAC-seq was performed in duplicate for Pax6-HIGH, ON and OFF cells. Cells were cultured and harvested to provide a single cell suspension. To make nuclei cells were pelleted at 1400 rpm and resuspend in 2ml cold NBA buffer  and mixed with 2 mL of cold NBB buffer  for 2 min at 4°C, and centrifuged at 2000 rpm before washing in 2 mL NBR 4°C, centrifuged again and resuspended in 1ml NBR . Briefly, total RNA was extracted using QIAGEN RNeasy mini kit (QIAGEN) and ribosomal RNA depleted using RiboMinus Eukaryote Kit for RNA-Seq (Life Technology); libraries were made with NEBNext mRNA Library Prep Master Mix Set (NEB) and sequenced on an Illumina Hi-Seq 2000 SE 50. Reads where aligned to the mm9 genome using TopHat v2 and processed with Samtools v1.6  with 1.6% Triton X-100, and digested with 3 sequential additions of 750 units DpnII enzyme at 37°C with shaking 1200rpm over 24 hr. Samples were heat inactivated at 65°C for 20 min, and 3 samples from each replicate combined into 7 mL with 1 × T4 DNA Ligase Buffer (NEB), with 1% Triton X-100, and 12,000 units of T4 DNA ligase at 16°C overnight. Samples were treated with Proteinase K at 65°C overnight and RNase A/T1 (ThermoFisher) at 37°C for 1 hr, before a standard Phenol/Chloroform, Chloroform extraction and ethanol precipitation was performed. Complete digestion and ligation was assessed by gel electrophoresis.NG Capture-C was performed on Pax6-HIGH, ON and OFF cells , with thPax6 locus elements: CTCF and/or Cohesin binding sites, known and predicted enhancer clusters, and the multiple promoters of three genes in the locus Pax6, Elp4 and Rcn1 (a full list of targeted restriction enzyme fragments is given below). Capture oligos were designed to each end of the targeted DpnII fragments (Purified 3C DNA from each sample was sonicated to 200-400 bp with a Soniprep 150 probe sonicator at 4°C and purified with a standard Ampure XP Bead protocol (Beckman Coulter) using a 1/1.5 DNA to bead ratio. Two Illumina sequencing libraries were prepared per capture pool replicate, with 6 μg of starting DNA in each, and generated using NEBNext DNA Library Prep Kit (NEB), with samples indexed with unique barcodes using NEBNext Multiplex Oligos for Illumina (NEB). Two separate capture pools were designed to the following ragments , and eacNG Capture-C sequence capture was performed using SeqCap EZ HE-Oligo Kit A or B (dependent on the multiplex barcode) and SeqCap EZ Accessory Kit (Nimblegen) , using ein silico digested,” and broken into shorter fragments at DpnII sites. Thus, each paired end read was converted into a set of DpnII fragments, which were called a read group; each fragment was then aligned separately to the mm9 reference genome as single end reads using Bowtie .Capture-C data were analyzed according to methods developed by g Bowtie . The resDpnII fragment, and every other mappable DpnII fragment in the genome. We normalized such that the total number of interactions for each target was 100,000,000 reads genome-wide; this implies the assumption that each target should have the same interaction “visibility.” To obtain the interaction profiles shown in figures, we applied a sliding binning window, which collects the data into 3-kbp bins which contain data from a 6-kbp window (The above scheme gives an interaction frequency between each target Pax6 promoters interact with the upstream regulatory region (URR)”?, as shown in Pax6 , and counted the number of normalized reads falling within a window of 10 kbp around the probe at the URR (probe CTCF6).To assess “How much do the In Target name pool restriction fragmentPax6_P1 1 chr2:105515339-105515903Pax6_P0 1 chr2:105508737-105509119Pax6_Palpha 1 chr2:105521487-105522094CTCFp11 1 chr2:105511309-1055123037CE12_CTCF 1 chr2:105527664-105528138Elp4_pro 1 chr2:105744078-105745820CTCF5 2 chr2:105639621-105640392CTCF6 2 chr2:105456258-105457375CTCF4 2 chr2:105748530-105748848CTCF7 2 chr2:105363080-105364092CTCF10B 2 chr2:105173505-105175699Rcn1_pro 2 chr2:105238728-105239162e200_Enh 2 chr2:105284625-105285412List of the targeted restriction enzyme fragments, names, which pool of oligos they belong to, and genomic position (mm9 genome build).−1 RNase A (Invitrogen) at 37°C for 60 min. Slides were then rinsed briefly with 2 × SSC, dehydrated through an ethanol series and air-dried. Slides were warmed by incubation in a 70°C oven for 5 min before denaturation for 40 min in 70% formamide in 2 × SSC, pH 7.5, at 80°C. Slides were then transferred to 70% ethanol on ice, dehydrated through an ethanol series and air-dried before overnight hybridization at 37°C with probes. Fosmid probes (BacPac resources) were labeled in green-500-dUTP (ENZO life sciences), digoxigenin-11-UTP (Roche) or biotin-16-dUTP (Roche). 100 ng of each labeled probe was hybridized with 5 μg salmon sperm and 20 μg human Cot1 DNA. Slides were washed four times for 3 min in 2 × SSC at 45°C and four times for 3 min in 0.1 × SSC at 60°C before being transferred to 4 × SSC with 0.1% Tween 20 at room temperature. Probes used in this study are listed in the table below. Digoxigenin-labeled probes were detected by using one layer of rhodamine-conjugated sheep anti-digoxigenin and a second layer of Texas red–conjugated anti-sheep (Vector Laboratories). Biotin-labeled probes were detected by using one layer of Cy5-conjugated streptavidin followed by a layer of biotin-conjugated anti-avidin and a second layer of Cy5-conjugated streptavidin (Vector Laboratories). Slides were counterstained with 0.5 μg ml−1 DAPI and mounted.Cells were grown overnight on glass slides. Slides were rinsed with PBS and fixed in 4% paraformaldehyde for 10 min , rinsed Four-color stained 3D slides were imaged using a Photometrics Coolsnap HQ2 CCD camera , on a Zeiss Axioskop II MOT fluorescence microscope with Plan-neofluar or Plan apochromat objectives, a Lumen 200W metal halide light source  and Chroma #89000ET single excitation and emission filters  with the excitation and emission filters installed in Prior motorised filter wheels. A piezoelectrically driven objective mount  was used to control movement in the z dimension. Hardware control, image capture and analysis were performed using Volocity . Image stacks (0.2 μm slices) were collected from at least 70 randomly selected nuclei for each experiment. Images were deconvolved using a calculated PSF in Volocity  and the distances between probes was measured using Volocity. Image color balance was adjusted to improve data visualization in the manuscript.The three-color probes allow simultaneous measurement of three distances in a single cell. This information was used to give a measure of the size of the locus, As well as a size of the locus, this triplet of separations can be used to give a measure of the cell-to-cell variability of the conformation of the locus. The three separations can be thought of as a vector representing a point in a three-dimensional “conformation” space. The set of imaged cells gave a set of points in the conformation space E, and thName Fosmid ID genome position LabelRcn1 WIBR1-0075F18 chr2:105218785-105254871 green-500-dUTPPax6 WIBR1-0322P22 chr2:105508853-105550057 digoxigenin-11-dUTPElp4 WIBR1-1660D19 chr2:105726249-105764673 biotin-16-dUTPURR WIBR1-1230I10 chr2:105430492-105469674 digoxigenin-11-dUTPPax6 WIBR1-0322P22 chr2:105508853-105550057 biotin-16-dUTPDRR WIBR1-2859L14 chr2:105612707-105655961 green-500-dUTPCoarse grained molecular dynamics simulations were performed, in which collections of molecules are represented by “beads,” which interact with phenomenological force fields and move according to Newton’s laws. A chain of beads connected by springs represent a region of the chromosome, and individual beads represent complexes of transcription factors or other DNA binding proteins (TFs). We also include loop extruding factors, which are represented by additional springs between non-adjacent chromatin chain beads. We use the multi-purpose molecular dynamics package LAMMPS  .The position of the For the chromatin fiber the otential .Each TF is represented by a single bead and the WCA potential is used to give a steric interaction between these. Chromatin beads are labeled as binding or not-binding according to the input data (see below). For the interaction between proteins and the chromatin beads labeled as binding, we use a shifted, truncated Lennard-Jones potentialailed in , this noLoop extruding factors (LEs) are represented by additional springs between non-adjacent beads along the chromatin chain. Binding of extruding factors occurs at a rate The polymer is initialized as a random walk, and the dynamics are first evolved in the absence of TF interactions and extruders in order to generate an equilibrium coil conformation. Interactions with the TFs and extruders are then switched on, and the dynamics are evolved until a new steady-state conformation is obtained.So far the system has been described in units As regards timescales, we first note that the previously defined simulation units define a natural time unit Other important parameters are: the TF switching rate, chosen as We note that simulated Capture-C results depend rather weakly on the TF and LE parameters, but the simulated FISH results are more sensitive. Previous work on the loop extrusion model  also shoFor the simulations with the heteromorphic polymer presented in essivity   we assigned the anchor bead as having forward, reverse or both orientations. In order to model cell-to-cell variation of CTCF binding we used the relative height of each peak to determine an occupation score – in each repeat simulation, each anchor bead was chosen to be present or not with a probability depending on this score. During LE dynamics, extrusion was halted when the direction of the anchor bead was opposite to the direction of motion – in this way persistent LE loops were only formed between convergent pairs of anchor beads.In In order to obtain a fiber which varies in thickness along its length, we use the H3K27ac data to identify de-compacted regions, and do not include the DnpII restriction enzyme fragment are sampled stochastically from a large population of cells. From our 13 targets and three cell types, on average each target had 107.2 normalized reads (reads per 100 million) from interactions within the chr2:105,000,000-106,000,000 region. To generate similar profiles from simulations we take a set of “snapshots” of the locus conformation  over all targets in all cell types is the same in simulations and experiments. Importantly all profiles are scaled in the same way to preserve target-to-target and cell type variation.To plot simulated Capture-C profiles alongside experimental data we scaled the simulated interaction count such that the total number of interactions within the Each FISH probe covers a region of the genome as indicated in In order to map simulation length units to physical ones, we take six probe pair distributions from each of the three cell types . We use the two-sample Kolmogorov-Smirnov statistic to find the “distance” between each simulated distribution and the corresponding experimental distribution; we then sum these distances. We then numerically find the value of Pax6 locus, we performed a hierarchical clustering analysis. To do this we took a set of snapshots of the simulated locus conformations , and a distance metric based on the full polymer conformation gave informative clusters of conformations . The metric was defined asWe previously showed that for the Pax6 showed less clear grouping of conformations than was observed in our previous work on globin loci. That is to say, while groups were observed , there was not much correlation with the overall configuration of the locus . Also, different models can show clear improvements in some respects  and 1 (when there is no overlap). The overall K-score is the average of these normalized distances subtracted from 1 . The K-score can therefore be interpreted as the average degree of overlap between the simulated and experimental probe pair separation distributions, The standard deviation of the individual Kolmogorov-Smirnov statistic values gives a measure of the variation of agreement across probe pairs and cells. In order to further elucidate the scale of K-score values, we generated a random simulated FISH dataset ; this yielded a K-score of 0.59 : The processed simulation and experimental data used to generate all figures. The full set of 200 simulated locus configurations for each cell type used to generate plots and simulation snap-shot images for The accession numbers for the data reported in this paper are GEO:


West Nile Virus (WNV) is an arthropod-borne flavivirus whose zoonotic cycle includes both mosquitoes and birds as amplifiers and humans and horses as dead-end hosts. In recent years WNV has been spreading globally and is currently endemic in Africa, The Middle East, India, Australia, central and southern Europe, and the Americas. Integrated surveillance schemes and environmental data aim to detect viral circulation and reduce the risk of infection for the human population emphasizing the critical role for One Health principles in public health. Approximately 20% of WNV infected patients develop West Nile Fever while in less than 1%, infection results in West Nile Neurological Disease. Currently, the diagnosis of WNV infection is primarily based on serology, since molecular identification of WNV RNA is unreliable due to the short viremia. The recent emergence of Zika virus epidemic in America and Asia has added another layer of complexity to WNV diagnosis due to significant cross-reactivity between several members of the Flaviviridae family such as Zika, dengue, Usutu, and West Nile viruses. Diagnosis is especially challenging in persons living in regions with flavivirus co-circulation as well as in travelers from WNV endemic countries traveling to Zika or dengue infected areas or vise-versa. Here, we review the recent studies implementing WNV surveillance of mosquitoes and birds within the One Health initiative. Furthermore, we discuss the utility of novel molecular methods, alongside traditional molecular and serological methods, in WNV diagnosis and epidemiological research. Culex genus which can potentially transmit the virus to every vertebrate on which they feed (West Nile Virus (WNV) is a member of the family Flaviviridae from the genus Flavivirus which also contain other viruses pathogenic to humans such as Zika, dengue, yellow fever, Usutu, and Japanese encephalitis . WNV is hey feed . While mhey feed . Mammalshey feed .West Nile Virus infection in humans is mostly asymptomatic, however, in approximately 20% of the cases WNV infection induces a mild disease with influenza like symptoms termed West Nile Fever (WNF), while in less than 1% of cases, mainly in elderly and immunocompromised people, infection results in a severe neuroinvasive disease (WNND) which may lead to death . Horses During the last 20 years WNV has spread geographically and is now common in countries in Africa, Europe, the Middle East, North America, and East Asia where it can cause disease outbreaks . While tTable 1).The One Health concept recognizes that the health of humans, animals, and environment are all connected and that only a collaborative inter-disciplinary approach can effectively achieve optimal health outcomes . WNV is Following the first outbreak of WNV in horses in Italy in 1998  the ItalWest Nile Virus has emerged in Greece in 2010 and caused outbreaks for three consecutive years in both humans and horses . FollowiThe number of WNV cases detected in horses and humans in Spain is significantly smaller than Italy and Greece and so far only two outbreaks with two and three human patients have been recorded in Andalusia, in 2010 and 2016, respectively . NeverthWest Nile Virus has been recognized as endemic in Israel since its establishment in 1948. However, despite sporadic cases and a few small outbreaks , WNV wasMuch like its neighbor, Greece, WNV outbreaks emerged in 2010 and 2011 with 47 and 5 human cases, respectively . Since tWest Nile Virus was first identified in Austria in birds in 2008  and humaWest Nile Virus had not been detected in North America before 1999, when a large WNV outbreak occurred in New York City . PhylogeFollowing WNV introduction to the United States, the virus has spread to Canada and was responsible for major outbreaks in 2002 and 2012 in Ontario and Quebec and in 2003 and 2007 in the Prairie Provinces . In ordeAt this point it is hard to assess the effects that One Health initiatives have on controlling WNV disease in each country. In Italy, an unbiased quantitative evaluation protocol has been developed to examine the implementation of the One Health approach in order to “fine tune” the system . HoweverWith the development of molecular biology methods in the 1980s, laboratory diagnosis of viral infections have developed from traditional viral isolation in cell culture which can take days to weeks  to molecFigure 1). Recently, several studies have demonstrated that WNV is retained in the kidneys and can therefore be detected in urine samples for a longer period of time than plasma, CSF or serum . Several commercial and in house molecular diagnostic tests with varying sensitivities are available which are different primarily in their amplification target of the viral genome.In recent years, the detection of viral genome in bodily fluids by quantitative Real-Time Polymerase Chain Reaction (qRT-PCR) has become the routine diagnosis method for many viral infections due to its industry standard format, high levels of repeatability and reproducibility, high sensitivity and specificity, fast turnaround time and ease of use . Unfortuor serum , 2015. Ior serum  it can por serum . We haveTransmission of WNV during blood and organ donations has been recorded in the United States  and EuroFigure 1). Numerous commercial kits for WNV antibody detection are available which are primarily based on detection of specific antibodies against the Envelope (E) protein of WNV using either enzyme linked immunosorbent assays (ELISAs) or immunofluorescence tests (IFAs). The advantages of these qualitative assays are that they are very easy to use in a laboratory setting, the results are mostly reproducible and part of their protocol can be automated. However, since WNV IgM can persist in serum for months and even years after infection  and Immunoglobulin G (IgG) antibodies can usually be detected by day 4 and day 8 after onset of symptoms, respectively  and thernfection , 2015 annfection  and is cnfection . There aThe Zika epidemic, which emerged in 2015 in Brazil , poses aSerological methods aided by detection of viral nucleic acid by qRT-PCR will, most probably, continue to be the primary and preferred diagnostic methods for WNV. Nevertheless, the establishment of next generation sequencing (NGS) methods in recent years, which are also called deep or high-throughput sequencing, led to the increased use of NGS to complement diagnosis and monitoring of infectious diseases caused by both bacteria  and viruRecently, a new and novel method for the detection of flavivirus RNAs including Zika, dengue and WNV from body fluids has emerged. Using CRISPR-cas13 technology, a fluorescent protein was used to track and detect flavivirus RNAs present in patients samples generating a field-deployable viral diagnostic platform with high performance and minimal equipment or sample processing requirements . This plThe emergence and geographical spread of WNV in recent years has forced endemic countries to initiate integrated surveillance programs to identify, predict, and reduce WNV circulation. Future programs should be implemented to combine all data and assess the effectiveness of these surveillance programs to reduce the burden of WNV infections. In addition, due to the influx of other flaviviruses, such as Zika virus, and their global expansion, more specific, yet sensitive assays for WNV diagnosis should be established to be able to distinguish between the different diseases and ultimately pave the way for the development of a WNV vaccine.YL reviewed the design, researched the literature, and wrote and edited the manuscript. DS researched the literature and edited and provided critical review of the manuscript. EB researched the literature and wrote and edited the manuscript. EM reviewed the design, researched the literature, and edited and provided critical review of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.