Serologic testing remains crucial for Zika virus diagnosis. We found that urea wash in a Zika virus nonstructural protein 1 IgG ELISA distinguishes secondary dengue virus infection from Zika virus infection with previous dengue . This test will aid serodiagnosis, serosurveillance, and monitoring of Zika complications in dengue-endemic regions. The rapid spread of Zika virus and its association with fetal microcephaly and other birth defects  present a pressing need for sensitive and specific diagnostic tests , cause substantial disease in humans. Because of cross-reactivity of antienvelope antibody to Zika virus and other flaviviruses, positive or equivocal IgM results based on envelope protein require further testing with plaque-reduction neutralization tests . Convalescent-phase samples from patients with confirmed Zika virus infection who were either DENV-naive (designated as pZIKV panel) or previously exposed to DENV (designated as DENV-ZIKV panel) were from a cohort study in Nicaragua . Convalehttps://www.graphpad.com/scientific-software/prism). The expression and purification of Zika virus NS1 protein (strain HPF2013) have been described  of urea wash resulted in significantly lower rODs in the sDENV panel than in the probable DENV-ZIKV and DENV-ZIKV panels , panel D>0.28  to 80% (5–6 years) and for ZIKV-NS1 IgG ELISAs from 83.3% to 40%, respectively (>0.28 , panel C>0.28 , panel C>0.28 , panel DAlthough neutralization tests can confirm pZIKV infection, they remain difficult to interpret for patients who have previously experienced flavivirus infections, including sDENV and DENV-ZIKV infections. A recent study reported reduced cross-neutralization against Zika virus among samples from patients with sDENV infection >6 months after symptom onset; however, 23% still cross-neutralized Zika virus  Figure 1This study has limitations. First, we tested only convalescent- and post–convalescent-phase samples. Second, the sample size in each panel was small; future studies with larger samples, including acute-phase and more sequential samples, are needed to validate these observations. Third, because our previous study showed cross-reactivity of anti-DENV NS1 antibodies within the DENV serocomplex  Table 2.Our method of combined ELISAs plus 6 mol/L urea wash in Zika virus-NS1 IgG ELISA is simple, cost-effective, and applicable for use at field sites. This method could be used for routine serologic testing for Zika virus in dengue-endemic regions and for serosurveillance and Zika pregnancy studies to clarify epidemiology, transmission, and complications (Supplemental results for study of using urea wash ELISA to distinguish Zika and dengue virus infections.


The organization of health care for breast cancer (BC) constitutes a public health challenge to ensure quality of care, while also controlling expenditure. Few studies have assessed the global care pathway of early BC patients, including a description of direct medical costs and their determinants. The aims of this multicenter prospective study were to describe care pathways of BC patients in a geographic territory and to calculate the global direct costs of early stage BC during the first year following diagnosis.in situ for each patient, using a prospective direct observation method.OPTISOINS01 was a multicenter, prospective, observational study including early BC patients from diagnosis to one-year follow-up. Direct medical costs  and direct non-medical costs (transportation and sick leave costs) were calculated by using a cost-of-illness analysis based on a bottom-up approach. Resources consumed were recorded Data from 604 patients were analyzed. Median direct medical costs of 1 year of management after diagnosis in operable BC patients were €12,250. Factors independently associated with higher direct medical costs were: diagnosis on the basis of clinical signs, invasive cancer, lymph node involvement and conventional hospitalization for surgery. Median sick leave costs were €8,841 per patient and per year. Chemotherapy was an independent determinant of sick leave costs . Forty percent (n = 242) of patients declared additional personal expenditure of €614/patient/year. No drivers of these costs were identified.Initial stage of disease and the treatments administered were the main drivers of direct medical costs. Direct non-medical costs essentially consisted of sick leave costs, accounting for one-half of direct medical costs for working patients. Out-of-pocket expenditure had a limited impact on the household. Breast cancer (BC) is the most common cancer in women in France with more than 54,000 new cases and 12,000 deaths per year –6. Most The costs of BC are also underestimated. However, knowing the costs makes it possible to adapt health policies, allocate health budgets, adjust social assistance and evaluate the contribution of innovations. Most published health economics, cost-effectiveness or cost-utility studies have focused on a specific drug , surgicaThe aims of this multicenter prospective study were to describe care pathways of BC patients in a geographic territory and to calculate the global direct costs of early stage BC during the first year following diagnosis.OPTISOINS01 was a multicenter, prospective, observational study conducted in patients from a determined regional health territory around Paris. This study was approved by the French National ethics committee (CCTIRS Authorization No. 14.602 and CNIL DR-2014-167) for the research conducted at all participating hospitals. This study was registered with ClinicalTrials.gov (Identifier: NCT02813317). The design of this study has been previously published . Most patients presented invasive BC  and 23% (n = 142) had axillary lymph node involvement. Less than 40% of patients received adjuvant chemotherapy . Nearly one-half of patients were working at the time of diagnosis . Patient characteristics and treatments administered are presented in The majority of patients  were managed in a CCC, 14% in a GH (n = 83) and 12% in a TH (n = 12%). Median distance between residence and center was 12 km. Patients travelled longer distances to attend a CCC (18 km) than a GH or TH (6 km).Among the 379 patients between the ages of 50 and 74 years (target population of systematic screening in France), 290 (77%) were diagnosed following a screening examination and 89 (23%) were diagnosed on the basis of clinical signs.More than 80% of group 1 patients were managed by outpatient surgery . In the logistic regression model, 2 factors were associated with outpatient surgery in this group: being operated in a CCC  and living in a couple .Resources consumed included medical consultations, imaging examinations for diagnosis , biopsies, staging examinations , pre-treatment examinations, supportive care , surgery, hospitalizations, radiation therapy sessions, chemotherapy courses; for each phase of diagnosis .Median direct medical costs of 1 year of management from diagnosis of operable BC were €12,250 . In multivariate analysis, 4 factors were independently associated with higher 1-year 44direct medical costs: diagnosis on the basis of clinical signs, invasive cancer, lymph node involvement and conventional hospitalization for surgery .Sick leave costs were calculated for the 156 working patients included in the study for whom salary and duration of sick leave during the first year after diagnosis were available. Median sick leave costs were €8,841 per patient and per year . In multivariate analysis (multiple linear regression), only chemotherapy was found to be an independent determinant of sick leave costs, which were increased nearly threefold in the case of adjuvant chemotherapy .One hundred and ninety-five patients declared transportation costs. The median cost of transportation for these patients was €902 . No significant difference in terms of transportation costs was observed between the 3 groups, and the only determinant identified on multivariate analysis was a distance between the patient’s residence and hospital greater than 16 km  of patients declared additional personal expenditure, including medical  and non-medical costs , with a median cost of €614 per patient and per year . A majority of the patients (n = 362), did not have out-of -pocket expenditure. Costs were higher for group 2 and 3 patients  compared to group 1 patients . In multivariate analysis, no additional factor was found to be associated with personal expenditure in this population.This multicenter prospective study describes BC pathways in a given health territory and the direct medical and non-medical costs during the first year after diagnosis. BC pathways mostly depend on 2 factors: initial stage of disease, which determines treatment and the resources consumed and the type of center. For example, more than 80% of group 1 patients undergoing breast-conserving surgery without adjuvant chemotherapy were operated on an outpatient basis. Undergoing surgery in a CCC was associated with outpatient surgery. Moreover, most patients of this study were managed in a CCC and we also observed that patients were willing to travel many miles to receive treatment in a CCC. All public centers involved in the management of BC in the area studied participated in this study; patient recruitment therefore reflects the reality of the health care territory. We have also previously shown that compliance with selected EUSOMA (European Society of Mastology) quality criteria was observed more frequently in CCC . The typProgramme de médicalisation des systèmes d’information (PMSI) [medical information systems programme] were classified into 19 care pathway groups, corresponding to particular morbidity profiles, as evaluated by main and associated diagnostic codes: invasive or in situ cancer, palliative care, aplasia, inflammatory breast disease. The average cost of a care pathway was €9,600. The greatest cost variability was explained by the invasive cancer code, associated with chemotherapy followed by palliative care, reconstructive surgery and aplastic anemia. The largest group in terms of numbers (62% of patients) had the lowest pathway cost. The second study proposed a macroeconomic method to calculate the overall cost of primary treatment of non-metastatic breast cancer using aggregated data from hospital activity, the national cost scale, and incidence data for a given year [Median direct medical costs of 1 year of management from diagnosis of operable BC were €12,250, from a National Health Insurance perspective. The cost of breast cancer in Europe has been estimated to be €15 billion . In Franven year . The aveven year : mean toThese estimates are close to the direct medical costs estimated in the present study. However, these studies did not determine direct non-medical costs, such as transportation or sick leave costs. Nevertheless, median sick leave costs were €8,841/year/working patient, corresponding to the majority of absenteeism  in the global cost of BC, and accounting for more than half of annual direct medical costs. In the light of these results, health authorities should develop individual support for working patients in order to decrease the duration of sick leave and improve return to work. Transportation costs, as well as out-of-pocket costs, were declared by a small proportion of patients and represented a minor share of global costs. No significant difference, especially in terms of sociodemographic characteristics, was observed between patients according to whether or not they reported out-of-pocket expenditures. Out-of-pocket expenditure obviously depends on the country of care. In France, cancer patients receive 100% cover of all breast cancer-related health care costs from French National Health Insurance. Out-of-pocket costs therefore essentially consist of complementary paramedical therapies , or additional prosthetic material . Similar results were reported in Canada, with a median out-of-pocket expenditure of US$1002 during the year after diagnosis . The burA variability of direct costs is also observed between countries. Most studies in the US have described higher direct medical costs. A study based on a private insurance database reported a total health care cost of US$42,401 per patient for the first year . In anotBC costs mainly depend on treatment and BC pathways. Optimizing clinical pathways could therefore help to control costs. Moreover, implementation of organized clinical pathways helps to improve quality of care . In the These global cost data can be useful to all health actors, from health authorities to private insurers and caregivers. Even if social care systems differ between countries, comparing costs can be informative. The costs from one developed country to another were not substantially the same, regardless of the payer. For government health policy, knowing the distribution of the costs of a disease makes it possible to better allocate budgets. For private insurers, an inventory of such expenditures is very useful to adjust the financial support. In addition, a good evaluation of the costs of a disease is a necessary basis to evaluate the cost effectiveness of new drugs and other innovations.S1 Table(XLSX)Click here for additional data file.


We study the dynamics of semiflexible hyperbranched macromolecules having only dendritic units and no linear spacers, while the structure of these macromolecules is modeled through T-fractals. We construct a full set of eigenmodes of the dynamical matrix, which couples the set of Langevin equations. Based on the ensuing relaxation spectra, we analyze the mechanical relaxation moduli. The fractal character of the macromolecules reveals itself in the storage and loss moduli in the intermediate region of frequencies through scaling, whereas at higher frequencies, we observe the locally-dendritic structure that is more pronounced for higher stiffness. Macromolecular systems with a high amount of branching units continue to attract high attention ,4,5,6,7.Let us now briefly introduce the fractal system, the so-called “T-fractal”, on which we focus here. Belonging to the class of exactly decimable fractals, T-fractals enjoy a constant theoretical attention ,24,25,26The theoretical description of a polymer’s dynamics requires a suitable mathematical model. In order to find the relationship between the dynamics of a macromolecule and its topology, one can use in a first approach the model of generalized Gaussian structures (GGS) , which oThe outline of the paper is as follows: In this section, the model of semiflexible treelike polymers (STP) is briefly recalled; details of the STP model can be found in .In the STP-model, polymer structures are described by beads, located at the positions In Equation  the inciT the temperature and Considering the easiest case, the so-called GGS-model  that extThe mean squared length of the bonds is fixed a and b, directly connected over a bead i, a and b; otherwise, the minus sign is obtained. The common stiffness parameter related to a and b is denoted by For adjacent bonds a and c  of . HoweverNeither the volume of the monomers nor restrictions on the bond angles are considered in the GGS-model. A first approximation taking into account these restrictions leads to a correlation of successive bonds. Introducing semiflexibility in the GGS-model by imposing geometrical restrictions for the bonds’ orientations results in a generalized potential:VSTP({da}In the limit quations  and 3)  qi→0, W i-1; see . This reom below . A detaifound in .ζ denotes the friction coefficient of a bead. The stochastic Gaussian force acting on the i-th bead α and β denote the three spatial directions z.Within the framework of the STP-model, the following set of Langevin equations describes the dynamics of the polymer:The system of Langevin equations  requiresombining  and 3),,VSTP is i  elements i. In the following, i or q for all beads of functionality three.Referring to one picked bead shown in , the anahown in =GN is the number of beads of a T-fractal that for generation G reads:As we discussed in quations –8) of S of SASTPμ), those related to nearest neighboring (NN) beads (ν) and those to the next-nearest neighboring (NNN) ones (ρ). All of these elements depend on the stiffness parameter q, which reflects the bonds’ orientations; see Below, we discuss all possible non-vanishing entries of Each diagonal element corresponds to a bead. Therefore, the diagonal elements depend on the beads’ topological position within the T-fractal. The topology of T-fractals results in five different diagonal elements, as shown in Terminal beads with functionality  For T-fractals of generation An internal bead with two terminal and one internal NN is described by the diagonal element:The diagonal element If an internal bead has two internal and one terminal NN, it is described by the diagonal element:Besides the diagonal elements, there are two types of non-vanishing NN elements following from the analysis of NN pairs of beads, as depicted in If one of the two considered beads is a terminal bead, we obtain the NN element:Otherwise, two internal beads in NN position result in the matrix element:According to Equation  of SectiG. Among them, the first G groups are based on the branches G represent the dynamics of dendrons of the generations 1 to G, and the In order to analyze the eigenmodes of the T-fractals, we concentrate on the homogeneous form of the set of Langevin equations introduced in Equation , grows elity f=3  and latelity f=3 ,48; see lity f=3  for flexlity f=3 ,40 illusndrimers , the gron-th group (The Equation  vanish.i and j) connected by a common NN . Each of these terminal beads represents a i and j results in the relations:l and m, connected by a path containing more than two bonds, vanishes. Hence, in case of immobile beads, it is necessary to discuss only the equations of motion of bead k and its immobile NN. In both cases, the sum in Equation i and j) quations  reduces Equation  leads toquations  and (20)uations  of the two branches perform an opposing movement, whereby the amplitudes k and its immobile NN vanish, so that the equations of motion of these two beads become trivial. Thus, considering the second group, the set of Equations . Let n-th group, we have:G-th group, there are only two linearly independent realizations of two oppositely-moving Furthermore, according to the iterative construction of a T-fractal, described in we have:Dn(G)=3G-Since the central bead does not have any symmetric counterpart, the eigenmodes coming from the The number of independent variables Equation , in combEquation , gives ttion τk=The analysis of the eigenvectors corresponding to eigenvalues less than or equal to q. Thus, the scaling exponent is robust under the introduction of local constraints. This behavior is in line with the scaling of the spectral density The analysis of the eigenvalue spectrum plotted in double logarithmic scales b shows t-fractal  ds=log〈Rg〈Rg{λi}, Equation , one obtq, respectively. The comparison of The knowledge of the eigenvalue spectra allows the calculation of many dynamical characteristics . Here, wq leads to a broadening of the Obviously, the increasing value of the stiffness parameter ndrimers  and lessndrimers . We notendrimers ,43, wherndrimers . These tq and proportional to On the other hand, one observes a scaling behavior in the range of middle frequencies; such a feature is typical for fractals and not for dendrimers. For the purpose of a more precise analysis, the derivation:ctations ,54,55 [GIn this paper, we have studied the dynamics of hyperbranched, dendritic macromolecules modeled through T-fractals. The symmetry of the T-fractal structure enabled us to construct a full set of eigenmodes and to analyze it in detail. Moreover, the set has reduced the computational efforts by having much smaller reduced dynamical matrices. The analysis of the corresponding eigenvalue spectra has shown a significant broadening of the spectra with increasing stiffness. Thus, the relaxation of the large-scale eigenmodes feels the increase of size of the macromolecule with growing stiffness and becomes slower; meanwhile, the relaxation of the small scale eigenmodes becomes faster due to the locally-constrained motion. These features become relevant for the mechanical relaxation moduli, which show a broadening with increasing stiffness. Moreover, while for high frequencies, the moduli reflect the local dendritic nature of the T-fractals (this behavior is more pronounced by the loss modulus for higher stiffness), a broad range of intermediate frequencies reveals through a scaling the fractal character of the macromolecules (which is less influenced by stiffness), a feature that is rather typical for hyperbranched polymers .


The Avian Influenza A (H5N1) virus is endemic in poultry in Egypt. The winter of 2014/2015 was particularly worrying as new clusters of HPAI A (H5N1) virus emerged, leading to an important number of AI A (H5N1) outbreaks in poultry farms and sporadic human cases. To date, few studies have investigated the distribution of HPAI A (H5N1) outbreaks in Egypt in relation to protective / risk factors at the farm level, a gap we intend to fill. The aim of the study was to analyse passive surveillance data that were based on observation of sudden and high mortality of poultry or drop in duck or chicken egg production, as a basis to better understand and discuss the risk of HPAI A (H5N1) presence at the farm level in large parts of the Nile Delta.The probability of HPAI A (H5N1) presence was associated with several characteristics of the farms. Vaccination status, absence of windows/openings in the farm and the number of birds per cycle of production were found to be protective factors, whereas the presence of a duck farm with significant mortality or drop in egg production in the village was found to be a risk factor.Results demonstrate the key role of several prevention and biosecurity measures to reduce HPAI A (H5N1) virus circulation, which could promote better poultry farm biosecurity in Egypt.The online version of this article (10.1186/s12917-018-1519-8) contains supplementary material, which is available to authorized users. The highly pathogenic avian influenza (HPAI) A H5N1) virus has been enzootic in Egyptian poultry for about ten years  virus ha. DespiteAfter nearly 10 years of continued evolution of the HPAI A H5N1) virus, several distinct antigenic clusters have emerged in Egypt within the clade 2.2, notably the clades 2.2.1, 2.2.1.1 and 2.2.1.2 N1 virus,, 9 may bA comprehensive review of risk factors of HPAI A H5N1) at different levels  in countries other than Egypt  found du at diffeThe aims of this study were to analyse the risk of HPAI A (H5N1) presence at the farm level in a large part of the Nile Delta, with a particular emphasis on simple factors such as the type of poultry production system, vaccination status, closed vs. open status of the farm, farm size and the presence or absence of duck farms reporting abnormal mortality or drop in egg production in the village in which the farms were located.The study was based on passive surveillance data recorded from 2014 to 2015 in commercial poultry farms in the Western-side of Nile delta in Egypt. Suspected premises were visited following reporting of sudden and high mortality in broiler or duck flocks, or of a drop of egg production in layers or breeders. During investigation of the suspected cases, 3–5 pooled samples from sick or freshly dead birds were tested for HPAI A (H5N1) virus by laboratory tests and confirmed by reverse transcription polymerase chain reaction (RT-PCR). The samples which consisted of a set tracheal and cloacal swabs and organs from trachea, lung and kidneys were processed for virus isolation according to the OIE manual . Each fan = 746) and duck (n = 364) farms were visited and the following information were collected at the time of the visit: species (chicken or duck), farm type , location (name of the village), date of report, number of animals in the farm, average age and weight of the birds, number of dead birds at the time of the visit, preventive measures implemented in the farm . In an open farm system, windows for the purpose of ventilation are present in the farm structure. Such open farm systems are generally lacking in rodent and peri-domestic bird control. In the closed farm system, ventilation is provided by electric fans or pad cooling systems. The advantage of such a system is improved biosecurity in farms.The study area is located on the Western-side of Nile delta, on the Mediterranean coast of Egypt, ranging from 29°25′ to 31°20′ East, 30°12′ to 31°36’ North. The samples were collected from 2014 to 2015 in five governorates (regions), namely Alexandria, Behera, Kafr El-Shikh, Gharbia, Menoufia : i) broiler chickens: day 5–10 (first round); ii) layer chickens: day 10, 40 and 110; iii) meat ducks: day 5, 10; and iv) layer ducks: day 10, 50 and 100. The number of booster doses and the vaccine strain were not available for our dataset. For the geographical coordinates of the farms included in this study, we used the village centroids for protection of privacy.n = 1110 farms), a binomial generalized linear model (GLM) was formulated. Initially investigated variables were the type of poultry production (egg or meat) and species (chicken or duck) information, which were combined into a single variable describing the production type . The other independent variables included the presence or absence of vaccination for influenza A (H5Nx) virus (“Vaccination”), the closed vs. open farm status (“Closure”), the number of birds per production cycle in the farm (“Nb_Birds”), the presence (1) or absence (0) of at least one visited duck farm in the village  in which the farm was located (“HasDuck”). A multivariate GLM binomial model was developed using all individual risk factors. In addition, the bivariate interactions between production type and vaccination, production type and closure status, and production type and the number of birds were tested. The average age and weight of birds of the flocks were not tested because the largest part of the variability in age and weight was confounded by the poultry type variable. We quantified the effects of all simple and interaction terms from a full model  using an analysis of deviance upon removal and a chi-squared test with type II sum of squares procedure. Insignificant variables and interactions were then taken out according to the Akaike’s information criteria (AIC) to derive the final most parsimonious model with a stepwise selection procedure. The final model was also used to visualise the role of each explanatory variable on the prediction, having adjusted for other variables presented in model [In order to test the effect of a list of independent variables on the probability of presence of HPAI A (H5N1) virus at farm level  in the years 2014–2015 in 5 Egyptian provinces in the delta of Nile River  virus was about 42.43% (471/1110): 43.91% (220/501) for the broiler chicken farms, 48.57% (119/245) for the layer chicken farms, 40.63% (78/192) for the meat duck farms and 31.40% (54/172) for the laying duck farms. The proportion of closed farms was 44.05%: 50.10% (251/501) for broiler chicken farms, 38.37% (94/245) for layer chicken farms, 43.75% (84/192) for meat duck farms and 34.88% (60/172) for laying duck farms.5 in broiler chicken farms, from 2349 to 8.7 × 104 in layer chicken, from 2401 to 9.9 × 104 in meat duck farms and from 2588 to 7.7 × 104 in laying duck farms. Figure The mean number of birds per farm ranged from 101 to 1.3 × 10The goodness of fit of GLM model was high with a mean AUC value of 0.93 and the model residuals showed no evidence of spatial autocorrelation  virus at poultry farm level. In this study, the biosecurity level of farms was estimated through a list of biosecurity practices and biosecurity risks: the absence of windows/openings in farm, the number of birds per cycle of production (practices) and the presence of duck farms in the village (risk). Every variable tested in the GLM model were found significant to explain the probability of HPAI A (H5N1) presence and the discriminatory capacity of model was good with AUC about 0.93. It should be recalled that the model is based only on five independent variables. Figure  virus atVaccination for HPAI was associated with a decrease in the probability of presence of HPAI A (H5N1) virus and can explain the high goodness of fit of the GLM model. Indeed, the virus was detected in 22.08% (104/471) of vaccinated farm while 91.71% (586/639) of unvaccinated farms were infected showing the good discriminatory capacity of this factor. However, in a few cases (104/471) vaccination failed to prevent the virus from being introduced into the farm. The absence of information about the number of booster doses and the vaccine strain used creates difficulties in interpreting this result. Vaccination in poultry using a non-matching virus vaccine strain to the circulating strain and lack of booster doses have been shown to possibly reduce clinical disease signs, without reducing the effect on virus transmission , 24. AltThen, the risk of HPAI A (H5N1) infection was higher in farms with windows/openings than farms without such types of ventilation in GLM models. This result may suggest that a strict isolation of poultry from wildlife is needed to prevent the farms from infection by the HPAI A (H5N1) virus. Indeed, bridging host species may exist in the peri-domestic populations  frequently found around the poultry farms and some are susceptible to HPAI A (H5N1) . In EgypIn the study area 97.57% of studied farms had less than 20,000 birds per cycle with the exception of layer chicken farms,  virus in poultry is dependant of the type of poultry production. The GLM predicted that the probability of virus presence was higher for the layer chicken farms while the observed prevalence for this category was lowest (57.14%) compared to other poultry type. The observed virus prevalence is the result of a sum of multiple factors and cannot be directly interpreted. A multiple regression model was needed to separate the effects affecting the probability of farm infection. For example, layer chicken farms have a higher number of poultry per production cycle in average, and the effects associated with this factor can be predominant compared to some other effects linked to the type of poultry production. The higher probability of HPAI A (H5N1) virus in layer chicken farms may be linked to the higher mean age of birds Fig.  in this The presence of a duck farm reporting abnormal mortality or a drop in egg production in the village where the farm is located was associated with a higher probability of presence of the HPAI A (H5N1) virus in the GLM. In Egypt, duck production is mainly in small commercial or backyard farms with relatively low biosecurity levels. Ducks have long been considered an important risk factor for HPAI A (H5N1) virus presence, and show various levels of clinical signs at the individual level, ranging from high mortality to the absence of clinical signs accompanied with virus shedding . Recent The study analysed data from passive surveillance and all surveyed farms had therefore reported abnormal mortality or drop in egg production. So, the results this study should be considered in that specific context, which entails a number of limitations. First, the virus was detected in 62.16% of the tested farms and this figure can therefore not be compared to prevalence figures obtained through cross-sectional surveys that consider all farms. Two types of farms may not have reported abnormal mortality or drops in egg production, for entirely different reasons. On the one hand, some farms with very high biosecurity and thorough implementation of vaccination may never report clinical signs. On the other hand, farms with domestic ducks or specific chicken breeds that have lower susceptibility may also not present clinical signs in birds, and would not be included. So, depending on the relative abundance of those two situations, our apparent farm-level prevalence of 62.16% may be an overestimation or an underestimation of the true prevalence. For the same reason, our measure of the benefits of vaccination at the farm level could be underestimated if many vaccinated farms never report any clinical signs to be included in the survey, or/and were not infected at all, or, conversely, overestimated, if inefficiently vaccinated farms had silent circulation of the virus. Other factors may be at play in influencing the vaccination coverage. For example, in parts of the Nile Delta other than the survey area that experienced fewer recent HPAI A (H5N1) outbreaks, farmers may have a lower perception of the usefulness of vaccination, which could result in lower vaccination coverage.The analysis focuses on only a relevant but limited subset of protective or risk factors at the farm level for different type of poultry production systems. However, many other potentially important factors could be considered, such as, for example, the presence of other pathogens, the number of visits to the farms, the turnover of poultry production, the extent of inward (inputs) and outward (outputs) movements, or the position of the farm in the poultry value-chain network. Future investigations could consider such information to provide a more comprehensive assessment of the local risk of AI transmission in poultry.Additional file 1:Receiver operating characteristic from the logistic regression computed with the training data (black line) and computed with a cross-validation (CV) method (blurred lines). A stratified random sampling of the dataset into training and test sets was used for the CV and the AUC was bootstrapped with 50 different data splitting. (PNG 84 kb)Additional file 2:The results used in assessing spatial autocorrelation. A) Map of residual deviances of GLM (top): the dot sizes are proportional to the deviance value of each farm. The red dots represent negative residual deviances while the blue dots are the positive residual deviances. B) Correlogram of model residuals (bottom-left). C) Moran’s test for spatial autocorrelation using a spatial weights matrix based on the neighbourhood relations showed in the figure (bottom-right). Two farms were linked together if the distance between these two farms was less than 0.08°. (PNG 202 kb)


CRISPR-Cas9 has been harnessed to confer virus interference against DNA viruses in eukaryotes, including plants. In addition, CRISPR-Cas13 systems have been used to target RNA viruses and the transcriptome in mammalian and plant cells. Recently, CRISPR-Cas13a has been shown to confer modest interference against RNA viruses. Here, we characterized a set of different Cas13 variants to identify those with the most efficient, robust, and specific interference activities against RNA viruses in planta.Our data show that LwaCas13a, PspCas13b, and CasRx variants mediate high interference activities against RNA viruses in transient assays. Moreover, CasRx mediated robust interference in both transient and stable overexpression assays when compared to the other variants tested. CasRx targets either one virus alone or two RNA viruses simultaneously, with robust interference efficiencies. In addition, CasRx exhibits strong specificity against the target virus and does not exhibit collateral activity in planta and demonstrate its suitability for studying key questions relating to virus biology.Our data establish CasRx as the most robust Cas13 variant for RNA virus interference applications  Plant viruses are obligate parasites that rely mostly on host cells to complete their life (infection) cycle. They infect many economically important crops, leading to a significant decrease in crop yields worldwide. It is estimated that plant diseases cause 10 to 15% reduction in global crop yields each year, and 47% of this loss is caused by viruses . TherefoBacterial and archaeal species employ CRISPR-Cas systems to fend off foreign genetic elements from invading phages and nucleic acids . CRISPR-Because class II CRISPR-Cas systems are composed of a single effector to mediate the interference and immunity against the invader’s genomes, their engineering is simple; therefore, they are widely adopted in genome and transcriptome engineering. In recent years, class II CRISPR/Cas systems have been extensively developed to provide powerful and versatile tools for various genome engineering purposes –14. SeveClass II type VI CRISPR-Cas systems are RNA-guided and RNA-targeting machineries that provide prokaryotes with immunity against RNA  and DNA Leptotrichia shahii was the first Cas13 orthologue to be harnessed for programmable RNA-targeting activities [LshCas13a to engineer immunity against the tobacco turnip mosaic RNA virus (TuMV) in different plant species, including Nicotiana benthamiana and Arabidopsis thaliana [Ruminococcus flavefaciens (CasRx) orthologue has shown consistently robust activity and specificity in mammalian cells compared to the previously characterized Cas13 proteins, namely LwaCas13a and PspCas13b [CRISPR/LshCas13a from tivities . We prevthaliana , 33. We thaliana , 35. Sinthaliana –38. Thesthaliana , 38–40. thaliana . Recentlthaliana , 40 and thaliana . Notablyin planta. Here, we investigated nine different Cas13 variants for robust virus interference to establish the use of this adaptive immunity mechanism for engineering plant immunity against single and multiple RNA viruses. We identified CasRx as the most effective Cas13 orthologue for RNA virus targeting in N. benthamiana. CasRx confers robust RNA virus interference compared to other variants in transient assays targeting a highly replicating RNA virus. In addition, transgenic plants overexpressing CasRx exhibited efficient interference against TuMV RNA compared to plants overexpressing other Cas13 variants, including LwaCas13a and PspCas13b, using cognate, position-matched crRNAs. Importantly, the catalytically deactivated (dCasRx) version could not mediate any virus targeting and, thus, inhibition, indicating that the HEPN-dependent catalytic activity of CasRx is required for virus RNA degradation. Finally, using synchronous coinfection of two non-competing RNA viruses, we show that CasRx RNA virus targeting is highly specific to the targeted virus and lacks collateral activities in planta. In addition, CasRx mediated efficient multiplexed virus interference by targeting two different RNA viruses simultaneously. This work establishes the use of CasRx for efficient RNA virus interference applications in plants.Because most viruses infecting plants possess RNA genomes, and reasoning that other uncharacterized Cas13 proteins might exhibit more robust activity in plants against RNA viruses, we sought to identify a more robust RNA-targeting CRISPR/Cas13 system by characterizing a set of Cas13 family members to assess their virus RNA-targeting activity in planta expression of multiple Cas13 proteins of three different Cas13 subtypes, including subtypes A, B, and D , two orthologues of Cas13b from Bergeyella zoohelcum (BzCas13b) and from Prevotella sp. P5-125 (PspCas13b), and one orthologue of Cas13d from Ruminococcus flavefaciens XPD3002 (CasRx). The selection of these different Cas13 orthologues was based on several reports showing improved RNA-targeting activity of these variants in comparison to LshCas13a protein in vitro and in mammalian cells [pK2GW7 binary vector, and their expression was driven by the 35S cauliflower mosaic virus promoter. Because the PspCas13b orthologue has been previously shown to be most active for RNA interference with the NES signal [Different CRISPR-Cas13 variants have been identified through computational pipelines searching for effector proteins possessing two HEPN domains. Cas13a, Cas13b, and Cas13d have been identified and tested for RNA manipulation in vitro and in vivo , 40–42. an cells , 40, 42.S signal , we procS signal , where tin planta, generation of transgenic plants overexpressing the CRISPR/Cas13 machinery is normally needed. However, such an approach is time-consuming and challenging when multiple and different CRISPR/Cas13 variants need to be evaluated, especially when considering the evaluation of multiple and different crRNAs for each variant. Therefore, the development of a transient assay that allows the assessment of different Cas13 variants and crRNAs simultaneously and systemically prior to the generation of transgenic plants would be valuable. We adopted a tobacco mosaic virus (TMV)-RNA-based overexpression (TRBO-G) system expressing the GFP gene to serve as a reporter construct to measure Cas13 interference activity in transient assays in plants. TRBO-G is a positive-sense, single-stranded RNA plant-infecting virus that has been engineered to replace the CP-encoding sequence with a GFP-encoding sequence, rendering the virus unable to move systemically in the infected plants, but retaining its ability to highly and efficiently replicate and express GFP protein in the infected (infiltrated) leaves  with each of the Cas13 variants and measured the virus-expressed GFP signal 3 days post-infiltration. Notably, we observed a broad range in the interference levels between the different Cas13 variants. Our data show that both CasRx variants (CasRx-NLS and CasRx-NES) exhibited the highest level of interference against TRBO-GFP using GFP-T1 and GFP-T2 crRNAs compared to the other Cas13 orthologues. In addition, other variants including LwaCas13a-NLS, LwaCas13a-NES, and PspCas13p-NES exhibited an efficient interference against TRBO-GFP using also the two GFP-targeting crRNAs  and showed that LwaCas13a-NLS, LwaCas13a-NES, and PspCas13b-NES mediated strong interference against the RNA virus. Intriguingly, CasRx mediated the highest and most efficient interference when compared to these or other Cas13 variants, as indicated with the low GFP protein level of the targeted virus Fig. c, d.Because the CRISPR-Cas13 machinery targets the RNA transcripts of the virus for degradation, we wanted to assess the interference at the RNA level. Therefore, we conducted reverse transcription-quantitative PCR (RT-qPCR) to assess the interference activities of different Cas13 variants. Consistent with the level of GFP signal, the RT-qPCR data showed that CasRx mediated robust and highly efficient RNA interference when compared to other Cas13 variants Fig. e. These In our previous interference experiments using crRNA against the GFP sequence of the virus genome, we identified three Cas13 orthologues with the highest level of interference activity against the TRBO-GFP RNA genome, namely LwaCas13a, PspCas13b, and CasRx. However, CasRx mediated the highest interference efficiency among these variants. Next, we wanted to test whether this interference activity and efficiency are applicable to other virus sequences and whether targeting other (conserved) virus sequences would result in similar interference efficiencies. Therefore, we designed three crRNAs targeting the replicase gene of the virus  and tested the activity of LwaCas13a, PspCas13b, and CasRx variants for RNA virus interference. Our data showed that CasRx mediated the highest level of interference activity, followed by LwaCas13a proteins  as evident with the Rep-T2 crRNA  identified from our previous experiments to confer strong RNA virus interference activities, with their cognate crRNAs targeting different genomic sequences of virus genome, and TuMV-GFP as a target. Cas13-mediated interference against the TuMV-GFP genome would result in attenuated replication and, thus, spread of the virus, which can be measured by monitoring the spread of the virus-expressed GFP to systemic leaves [Next, we tested whether the interference activities of the Cas13 variants conducted in transient assays in inoculated leaves were similar when using a different virus capable of systemic spread throughout the plant. To this end, we conducted a set of transient assays in wild-type c leaves . Our datN. benthamiana plants expressing Cas13 variants, including LwaCas13a-NLS, LwaCas13a-NES, PspCas13b-NES, and CasRx-NLS, using Agrobacterium tumefaciens-mediated transformation to assess the interference efficiencies of these Cas13 variants. We confirmed the expression of Cas13 proteins in the generated permanent lines, and our western blotting analysis show that the different Cas13 proteins were expressed efficiently in N. benthamiana plants, and the correct proteins sizes were detected  variant. Consistent with the phenotypic data, we observed a significant reduction in the abundance of the virus GFP protein with the catalytically active CasRx variants compared to the dCasRx Fig. c. In addin planta. Specificity of CasRx is an important issue in its interference applications against viruses. Therefore, we sought to test the specificity of CasRx against different RNA viruses. We developed a virus-based reporter system that relies on synchronous coinfection and co-replication of two different viral vectors. The two vectors need to be derived from different RNA viruses so they can non-competitively replicate and exist in the same cell [potato virus X expressing GFP (PVX-GFP), which has been found to be non-competing with the TMV-based vectors [N. benthamiana with crRNAs providing specificity to either virus. Theoretically, by delivering virus-specific crRNAs, only the titer of the targeted virus will be mitigated, indicating specific interference against the targeted virus. However, if the interference activity is not specific, interference of both viruses will occur . Our data show that both CasRx variants were able to exhibit efficient targeting of both viruses simultaneously using the two (GFP-T2 and Rep-T3) crRNAs, while no virus interference was observed when NS crRNA was used  requirements for these different Cas13 variants [In this study, we constructed different Cas13 variants, including Cas13a (LshCas13a and LwaCas13a), Cas13b (PspCas13b and BzCas13b), and Cas13d (CasRx), and their corresponding crRNA scaffolds for variants . We seleN. benthamiana overexpression lines of these Cas13 variants and conducted interference assays. Our data showed that CasRx is indeed the most robust Cas13 variant to confer virus interference followed by the LwaCas13a-NES variant. These data indicate that CasRx is an excellent candidate for RNA virus interference applications in plants.To test whether these variants limit systemic spread of the virus with similar efficiencies, we used TuMV-GFP as a target and co-delivered the Cas13 variants with their corresponding crRNAs. CasRx provided robust interference against the TuMV virus, thus significantly limited the spread of the TuMV-GFP virus into systemic leaves. In addition, the cytoplasmic-localized LwaCas13a provided strong interference compared to PspCas13b. To corroborate these data, we generated Francisella novicia, to target RNA viruses have shown that the binding, but not the cleavage, ability of these proteins is sufficient to inhibit virus replication in mammalian and plant cells [Previous studies harnessing CRISPR-Cas proteins, such as FnCas9 from nt cells , 49. Ournt cells . Similarnt cells , 51.in planta, and our data show that the CasRx targeting activity is highly specific to the targeted virus, where the transcript abundance of the co-delivered, non-targeted, virus was not affected. Cas13 variants possess robust collateral activities in vitro, enabling the development of pathogen detection systems that have revolutionized molecular diagnostics [in planta. This interference system can further be improved by determining the molecular basis of how the type VI system mediates antiviral defenses within their own hosts (spacer acquisition and adaptation) and how these effectors might function in eukaryotic hosts. These questions warrant detailed studies to understand the molecular functions, and the basis of the catalytic activities, of these proteins for broader applications in endowing immunity to eukaryotes.The specificity of CasRx for in vivo application is an important point to consider. Therefore, we evaluated the specificity of CasRx in the context of virus targeting gnostics , 53. Altgnostics , such acgnostics , 38. HerIn addition, we have shown that CasRx can be used to target either one virus alone or two RNA viruses simultaneously, thereby extending the applicability of this interference system. Such system might have the potential use in crop species for virus interference of mixed infections of RNA viruses that occur in the field and under natural conditions. It is worth noting that recent studies have shown the ability of DNA viruses to evolve as a result of Cas9 targeting , 54. TheIn conclusion, CRISPR-CasRx enables programmable RNA virus interference in plants and has the potential to be optimized and developed in the future for transcriptome engineering applications. CRISPR-CasRx expands the power of CRISPR systems for virus interference applications and may help to address basic plant virology questions.in planta expression. The DNA sequences of the LshCas13a variants were plant codon optimized and subsequently cloned into binary vectors, as described previously [pK2GW7 binary vectors using LR Gateway recombination cloning to generate pK2GW7-BzCas13b, in which expression is controlled by the constitutive 35S cauliflower mosaic virus promoter  using primers that remove NLS sequences from the N and C termini of CasRx-NLS ORF, leading to the removal of the HA tag and GFP-encoding sequences, and BsaI sites were added at both ends of the isolated ORF  with primers that add AseI and NotI restriction sites to the 5′ and 3′ ends of the amplified BFP sequence, respectively  sequences was purchased from Sigma as ssDNA oligonucleotides  were used for all transient experiments. To generate transgenic Nicotiana benthamiana plants overexpressing different Cas13 orthologues, Agrobacterium tumefaciens GV310 strains harboring the selected constructs were transformed into N. benthamiana leaf discs following previous published protocols [Two- to 3-week-old wild-type rotocols . GeneratpK2GW7-Cas13 constructs, the TRV RNA1 genome, the engineered TRV RNA2 genomes harboring crRNA sequences under the pPEBV promoter, and the different infectious clones of the RNA viruses, including TRBO-GFP, TRBO-BFP, PVX-GFP, or TuMV-GFP) were individually electroporated into A. tumefaciens strain GV3101. Single colonies grown overnight in selective medium were centrifuged and suspended in infiltration medium  and incubated at ambient temperature for 2 h. For infiltration into leaves of N. benthamiana plants, cultures were mixed at a final OD600 of 0.5 for pK2GW7-Cas13, 0.1 for RNA1 and RNA2-crRNAs, 0.05 for TuMV-GFP and PVX-GFP, and 0.005 for TRBO-GFP and TRBO-BFP, relative to the suspended culture grown overnight. Healthy and fully developed leaves were selected for experiments, and agroinfiltration was done with a 1-ml needleless syringe.The different constructs . For the quantitative measurements of the GFP signal intensity in images, the mean pixel values of the TRBO-GFP gene expression images were analyzed with ImageJ software to estimate GFP expression across samples. For each sample (each target of Cas13 variants), three biological replicates, represented as three different infiltrated leaves from three different plants, were analyzed. For each leaf, the GFP signal intensities were obtained as the corrected total cell fluorescence (CTCF) following this formula: CTCF = integrated density − (area of selected cell × mean fluorescence of background readings). The CTCF values of all three biological replicates for each sample were then averaged, and the normalized average of each target was compared to the normalized average CTCF value of the NS target. All values were limited to max = 100.2VO3, NaF, MG132, and MG115). Proteins were separated on a 10% polyacrylamide gel. Immunoblot analysis was conducted using mouse α-GFP antibody  for virus-expressed GFP and rat α-HA (1:1000) antibody for detection of Cas13 proteins. The antigens were detected by chemiluminescence using an ECL-detecting reagent (Thermo Scientific). The quantitative graphs summarizing the average of three independent biological replicates were produced by calculating the densiometric data generated by the relative quantification of protein bands from immunoblot membranes using ImageJ software. The average data of three independent immunoblot replicates were calculated as previously described [Total proteins were extracted from 100 mg of sample using extraction buffer  with protease inhibitors  values from target Ct values to normalize for total input, resulting in ΔCt levels. Relative transcript abundance was computed as 2Ct−ΔΔ [Total RNA was extracted from infiltrated leaves (in transient assays) or systemic leaves (in Cas13 overexpression lines) using Direct-zol RNA Miniprep Kits (Zymo Research) following the manufacturer’s instructions. Viral RNA was quantified with one-step RT-qPCR using the iTaq Universal SYBR Green One-Step Kit (Bio-Rad). All RT-qPCR reactions were performed in 10 μl reactions with three technical replicates in either 384-well or 96-well format and read out using a StepOnePlus™ Real-Time PCR System (Applied Biosystem) for the 96-well plates or the CFX384 Touch™ Real-Time PCR detection system (Bio Rad) for the 384-well plates. Expression levels were calculated by subtracting the housekeeping reference gene (tobacco PP2A ) cycle ts 2−ΔΔCt . All samAdditional file 1: Figure S1. TRBO-GFP virus genome. Figure S2. Cas13 proteins are required for RNA virus interference. Figure S3. CasRx mediates efficient interference against TuMV-GFP virus by preventing its systemic spread in wild type N. benthamiana plants. Figure S4. Confirmation of Cas13 protein expression in permanent N. benthamiana lines. Figure S5. Multiplexed targeting of TRBO-BFP and PVX-GFP viruses through delivery of two different crRNAs with CasRx. Sequence S1. BzCas13b amino acid sequence (3xHA-BzCas13b-NLS). Sequence S2. BzCas13b full-length plant codon optimized DNA sequence (3x-HA-BzCas13b-NLS). Map S1. BzCas13b in pK2GW7 (HA-BzCas13b-NLS). Table S1. crRNA sequences used in this study. Table S2. primers used in this study.Additional file 2. Review history.


Flaviviruses are the most medically relevant group of arboviruses causing a wide range of diseases in humans and are associated with high mortality and morbidity, as such posing a major health concern. Viruses belonging to this family can be endemic , but can also cause fulminant outbreaks . Intense research efforts in the past decades uncovered shared fundamental strategies used by flaviviruses to successfully replicate in their respective hosts. However, the distinct features contributing to the specific host and tissue tropism as well as the pathological outcomes unique to each individual flavivirus are still largely elusive. The profound footprint of individual viruses on their respective hosts can be investigated using novel technologies in the field of proteomics that have rapidly developed over the last decade. An unprecedented sensitivity and throughput of mass spectrometers, combined with the development of new sample preparation and bioinformatics analysis methods, have made the systematic investigation of virus–host interactions possible. Furthermore, the ability to assess dynamic alterations in protein abundances, protein turnover rates and post-translational modifications occurring in infected cells now offer the unique possibility to unravel complex viral perturbations induced in the infected host. In this review, we discuss the most recent contributions of mass spectrometry–based proteomic approaches in flavivirus biology with a special focus on Zika virus, and their basic and translational potential and implications in understanding and characterizing host responses to arboviral infections. Viruses are highly adapted intracellular pathogens that co-evolved with their hosts over millions of years. This resulted in a sophisticated adaptation that allowed viruses to engross cellular machineries for their own replication, with the benefit to encode their genetic information with high efficiency. However, this strict dependency on cellular factors comes with the obligation to replicate intracellularly and the requirement of intimate interactions between viral and cellular proteins. Moreover, most pathogens are preferentially replicating in specific organs and cause virus-associated pathologies. Technological developments in the fields of genomics and proteomics became available in recent years, allowing to systematically map interactions between viruses and their hosts in an unbiased manner. Such interactions are of particular interest especially when uncharacterized pathogens strike in a naïve population.Flaviviridae family, causing the first large unexpected outbreak in Asia and French Polynesia in 2013 [Flaviviridae family like Dengue (DENV), Yellow fever (YFV), Japanese encephalitis virus (JEV) and West Nile virus (WNV), ZIKV is also transmitted by mosquitoes. Furthermore, analogously to JEV, WNV and tick-borne encephalitis virus (TBEV), Zika virus infections are associated with neurological diseases, although ZIKV is the only Flaviviridae member with documented ability to cross the placental barrier and be sexually transmitted [One such pathogen is the Zika virus, a positive stranded RNA virus belonging to the  in 2013 ,5. Follo in 2013 ,7. Howevnsmitted .Technical advancement in mass-spectrometry analysis and the availability of replication competent epitope-tagged viruses and individual viral proteins have prompted several studies to investigate persistent modulation of protein abundances, post-translational modifications and protein–protein interactions on a global scale and with unprecedented sensitivity and quantitation ,11,12,13The recent technological revolution allows acquiring knowledge on biological processes at incredible speed. Several systematic and unbiased genetic approaches to identify host factors involved in flavivirus replication have been conducted to date ,19,20,21Development of sample preparation methodologies, highly sensitive mass analyzers as well as tailored bioinformatics processing pipelines (comprehensively summarized in ) have caFlaviviridae family are considered highly pathogenic infectious agents and knowledge on their interactions is pivotal to understand these pathogens in order to identify new forms of therapy. This is particularly true for epidemic viruses such as Zika virus, which since 2016 has left a devastating impact on large populations in southern America. In an effort to understand the pathogenicity of this virus, a number of laboratories employed systems analysis to elucidate the functions of individual proteins and the virus as a whole.Some members of the Systematic studies are of paramount importance to identify virus–host protein–protein interactions (PPIs), since they provide an unbiased view of molecular complexes and therefore insights into viral mechanisms of replication, host resources exploitation and immune evasion strategies ,12,31,32Among the other novel capsid interactors, additional work by the Ott and the Ramage groups identified the nonsense-mediated decay (NMD) pathway and the exon-junction complex (EJC) as targets of the WNV, DENV and ZIKA virus capsids ,46. WhilIn addition to proteins recruited by several flavivirus capsids, the ZIKV capsid is the only one reported to specifically associate with cellular factors that are involved in neuronal development or neurological disorders . These pAltogether, flavivirus capsids evidently evolved evasive mechanisms through binding/sequestration of ZC3HAV1 and other RNA-binding proteins . Capsid proteins are apparently exploiting their versatile subcellular localization (both nuclear and cytoplasmic) and their intrinsic hydrophobic character to simultaneously highjack and diverge regulation or expression of specific transcripts.Another interesting ZIKV protein that appears to have both conserved and unique cellular interactors across flaviviruses and plays a central role in virus–host adaptation, is the non-structural protein NS4B. Indeed, ZIKV-NS4B associates with subunits of the ATP synthase , voltage-dependent anion selective channel proteins  and calcium-binding mitochondrial proteins , as well as several components of the cytochrome C oxidase complex  c. These NS4B also appeared to associate with cellular proteins that could be associated with the whole spectrum of ZIKV-associated pathogenesis including neurodegenerative disorders and retinal degeneration , neuronal differentiation defects  and axonal dysfunction  . While tIn addition to capsid and NS4B, several novel ZIKV NS5 binders were recently identified in these proteomics surveys. Among these, multiple members of the PAF1C complex , confirming similar observations previously made on DENV-NS5 by the Gamarnik group  and receFurthermore, among the novel cellular targets identified in the ZIKV NS4A interactome is ANKLE2, a protein previously shown to cause autosomal recessive microcephaly in humans . Interesbona fide interactors in diverse cellular backgrounds as a selection criterion to pinpoint targets relevant for in vivo infections. This study identified a strong enrichment of the CCT complex (cytosolic chaperonin-containing T complex), and subunits of the OST (oligosaccharyl transferase) complex such as DDOST, STT3A, STT3B and RPN2, in the NS1 interactome. Previous reports identified some of these proteins as host-dependency or druggable targets in genetic [An important aspect is that AP-LC-MS/MS often identifies protein complexes rather than binary protein–protein interactions. This is also illustrated in an interactome study of DENV-NS1  in the c genetic ,56 and d genetic , and the genetic ,40,41,42 genetic ). Likewi genetic ) and NS4 genetic ). ProteoIn addition to PPI, flaviviruses were also shown to exploit their own genomic RNA (gRNA) or subgenomic derivatives, such as the subgenomic flavivirus RNA (sfRNA), to “sponge” critical RNA-binding host proteins out of the cellular pool, or to specifically inhibit or redirect their homeostatic regulatory functions ,62,63. IOnly one study to date reported the systematic analysis of ZIKV and DENV gRNA-associated proteins, using the ChIRP-MS (comprehensive identification of RNA-binding proteins by mass spectrometry) method. In this method, target RNA and associated proteins are retrieved after formaldehyde-based cross-linking using hybridization with antisense biotinylated oligos and streptavidin-conjugated beads, and protein complexes eluted via incubation with biotin . This apSeveral studies systematically investigated the impact of flavivirus infections on protein abundance on a global scale ,69. GarcA similar unbiased study investigating the proteome of ZIKV-infected iPSC-derived human neuronal progenitors, confirmed specific downregulation of neuronal factors upon ZIKV infection, as well as activation of general antiviral response pathways, such as upregulation of type-I interferon stimulated genes  . InteresRecently, additional reports investigated further the impact of ZIKV infection on human mesenchymal stem cells using the MudPIT method . This identified a profound downregulation of PI3K/AKT and mTOR, as well as dysregulation of several components of the phosphatidylinositol pathway . AlternaA unifying theme crystallizing from proteomic and transcriptomic analysis of ZIKA virus infected neuronal cells is that ZIKV infection has a profound effect on expression of proteins involved in neurogenesis or on proteins that are markers of neuronal differentiation. At this stage it is not clear whether this effect is limited to ZIKA virus or whether other viruses elicit similar effects in a neuronal background. Along these lines would be important to test whether neuropathic flaviviruses share certain binding patterns that may be etiologically linked to their pathology. Similarly, it would be of great interest to investigate the differential proteomes of diverse flaviviruses from primary biopsies of infected patients.Initial studies by the Cao group focused on the global protein phosphorylation status in WNV- and JEV-infected cells ,75. In tIn the case of ZIKV, only one study so far reported a time-resolved phosphoproteomic analysis of ZIKV-infected cells . SimilarFurthermore, a profound upregulation of the ATM (ataxia-telangiectasia mutated)/ATR (ATM- and Rad3-related) DNA-damage pathway, was observed at multiple levels  including a number of proteins involved in cell-cycle regulation and DNA damage checkpoint . Altogether, these effects provide possible explanations for the observed ZIKV-induced cell-cycle arrest and apoptosis  and p53 Another important PTM is ubiquitination, which becomes more and more accessible to proteomics analysis and has recently been studied in the context of flavivirus infections . For insCollectively, the analysis of PTMs in flavivirus-infected cells starts to unravel unappreciated cellular activities. Development of techniques to study PTMs as well as development in bioinformatic tools allowing better interpretation of PTM activities, hold promise to dramatically accelerate our understanding of virus–host interactions and to appreciate additional important pathways engaged during virus infections.Large-scale approaches allowing the accurate assessment of virus–host interactions have revolutionized the way we investigate and deconvolute the complex cellular changes triggered by virus infections. While central linear pathways were the main research focus in the past, it becomes more evident that innate immune signaling, and cellular responses in general, engage many interconnected pathways in parallel. The knowledge of these pathways is paramount to holistically understand how viral pathogens interact with their hosts. Certainly more work on viral–host interactions, protein turnover as well as post-translational modifications is required to further expand our understanding of the intracellular landscape of flavivirus-infected cells. This requires well-controlled comparative studies in relevant cell culture systems of multiple members of this highly diverse arbovirus family. Similarly, rigorous validation methods by alternative approaches , robust and transparent cut-off criteria, and functional assays on selected candidates are increasingly needed to pinpoint physiologically relevant host factors. In this respect, further efforts are required to integrate and standardize data generated by mass spectrometry based surveys in easy-to-mine databases, ideally providing critical annotations on virus isolates and cell culture systems as well as integration with other -omics data . Integra


To verify whether serum levels of reactive oxygen metabolites (ROM) are predictive of future clinical remission in patients with rheumatoid arthritis (RA) receiving tocilizumab (TCZ) therapy.A total of 46 patients with RA receiving TCZ therapy were enrolled in this study. Patients were divided into remission and non-remission groups based on disease activity score 28 (DAS28)-erythrocyte sedimentation rate (ESR) or clinical disease activity index (CDAI) at 52 weeks. Associations between serum levels of ROM, C-reactive protein (CRP), and matrix metalloproteinase-3 (MMP-3) at 4 and 12 weeks and the remission by DAS28-ESR and CDAI at 52 weeks were investigated.There were no significant differences in CRP and MMP-3 between DAS- or CDAI-remission and non-remission groups at 12 weeks. However, ROM in DAS-remission group were significantly lower than those in the non-remission group. For ROM, the area under the curve of the receiver operating characteristic curve was 0.735 and the cut-off value that distinguished DAS-remission group from non-remission group was 305.5 U. Carr . A multivariate logistic regression analysis revealed that ROM at 12 weeks was associated with DAS-remission at 52 weeks .Serum levels of ROM at 12 weeks during TCZ therapy may be predictive of DAS-remission at 52 weeks in patients with RA. Tocilizumab (TCZ) is a humanized monoclonal antibody against the IL-6 receptor, and inhibits the binding of IL-6 to its soluble and transmembrane receptors , 2. TCZ TCZ ameliorates inflammatory manifestations and normalizes acute phase protein levels, including C-reactive protein (CRP), thus confirming the observation that IL-6 is essential for the production of CRP . SufficiOxidative stress occurs in many autoimmune diseases such as RA, along with the excess production of reactive oxygen species (ROS) and reactive nitrogen species (RNS). Both ROS and RNS play a prominent role in signaling pathways in inflammatory cells and contribute to the destructive, proliferative synovitis of RA –13. ReceRegarding the efficacy of TCZ on the production of reactive oxygen species, it is reported that patients using TCZ had dramatically lower hydroperoxide levels than those using non-biological DMARDs, and that TCZ showed significantly lower ROS levels when compared to anti-TNF-α . HoweverA total of 46 patients with RA  receiving treatment with TCZ, including 32 naïve and 14 switched patients from other biologics, were enrolled in this study. In switched patients, a washout period of the previous biological DMARDs (bDMARDs) was not provided. Associations between the serum levels of ROM, CRP, and matrix metalloproteinase-3 (MMP-3) at 4 and 12 weeks and the remission as measured by DAS28-ESR and clinical disease activity index (CDAI) at 52 weeks were investigated.Blood samples were collected for the measurement of serum CRP and MMP-3. In our hospital, the normal reference value for CRP is 0.3 mg/dL. ROM was also measured as described below.To assess RA disease activity, measurements of DAS28-ESR and CDAI were obtained during the same visit at which the blood samples were collected. Remission based on the DAS28 and CDAI were defined as scores ≤2.6 and ≤ 2.8, respectively. Furthermore, a health assessment questionnaire (HAQ) was also assessed temporally.This study was approved by the Ethics Committee of Toho University Sakura Medical Center , and all patients gave their written consent to participate in this study. All activities were performed in accordance with the ethical standards set forth in the Declaration of Helsinki.To measure serum levels of ROM, the d-ROM test was performed using the FRAS 4 analyzer in accordance with the manufacturer’s analytical procedures , 15. Thep values < 0.05 were considered to indicate statistical significance.Results are expressed as mean ± standard deviation. All laboratory data, DAS28, CDAI, and HAQ scores were analyzed by the last-observation-carried-forward method. Between-group differences were assessed by the Mann Whitney U-test. The receiver operating characteristic (ROC) curves for ROM, CRP, and MMP-3 refer to the values at 12 weeks, and the cut-off values for ROC curves were determined by the maximum of a Youden index. A multivariate logistic regression analysis was performed by the stepwise method to compute the odds ratios (ORs) and 95% confidence intervals (95% CIs) for achievement of remission at 52 weeks. The cut-off values for ROM and CRP at 12 weeks were used as variables. All statistical analyses were performed using SPSS (ver. 19) software , and Background characteristics of patients included in this study are shown in Table The baseline DAS28-ESR was 4.82 ± 1.00; it rapidly decreased from baseline to 4 weeks, and further decreased gradually to 1.91 ± 1.65 at 52 weeks   of the ROC curve (blue) was 0.735, and the cut-off value (indicated by the asterisk) that distinguished DAS-remission from non-remission was 305.5 U. Carr   Fig. .Fig. 3RTo calculate the ORs and 95% CIs between patients with scores higher than and lower than the cut-off values, a multivariate logistic regression analysis was performed. The cut-off values for ROM and CRP at 12 weeks were used as variables. As a result, ROM at 12 weeks was identified as being associated with DAS-remission at 52 weeks ; however, CRP at 12 weeks was not Table . Naïve oIn the present study, we demonstrated that ROM serum level at 12 weeks during treatment with TCZ could be a biomarker to predict DAS-remission at 52 weeks. The cut-off value for ROM was 305.5 U. Carr, which indicates the importance of decreasing the ROM serum level to almost within normal range (< 300 U.Carr) at 12 weeks during TCZ therapy to achieve the remission.Choosing the ideal surrogate markers for inflammation in patients receiving TCZ therapy has been controversial. ESR and CRP are well-known surrogate markers of inflammation that reflect activity of various disease states such as infection, malignancy, connective tissue disease, and coronary artery disease . ParticuIn terms of biomarkers to evaluate the clinical course during treatment with TCZ, it has been reported that serum IL-6 is a useful marker instead of CRP and ESR because TCZ is an antibody to the IL-6 receptor that does not directly suppress IL-6 production . The resRecently, several studies have documented predictors of response to TCZ at baseline. Discontinuation of TCZ within 1 year was predicted by low CRP, high HAQ and prior exposure to biological agents , while ySerum IL-6 level before and after TCZ therapy is a principal biomarker in patients with RA . BaselinMeasurement of ROM is easily performed using FRAS 4 analyzer with low costs, and investigators can know the results shortly. Instead of IL-6, serum ROM level was measured in this study; at 12 weeks, it was significantly lower in the DAS-remission group than in the non-remission group. At that time, CRP and MMP-3, two conventionally used biomarkers for monitoring the disease activity of RA, were also compared between the DAS-remission and non-remission groups, but there were no significant differences. A multivariate logistic regression analysis revealed that ROM at 12 weeks was associated with DAS-remission at 52 weeks, but CRP at 12 weeks was not. These observations suggest that ROM at 12 weeks is predictive of DAS-remission at 52 weeks during treatment with TCZ, and that ROM is superior to CRP or MMP-3 as a surrogate marker to predict DAS-remission.p = 0.158, Table In patients receiving TCZ therapy, CDAI is often used to evaluate disease activity. However, there were no significant differences in CRP, MMP-3, and ROM at 12 weeks between the CDAI-remission and non-remission groups. Why was the serum level of ROM not predictive of CDAI-remission? We previously showed that the serum level of ROM correlated with CRP in RA patients . Since CWe previously reported that the cut-off value of ROM at 12 weeks that distinguished DAS-remission from non-remission in patients receiving biological DMARDs (bDMARDs) was 381.5 U. Carr , althougThis study has some limitations. First, the sample size was small. An investigation using a large sample size will be necessary to establish whether ROM is predictive of remission. Second, although oxidative stress is involved in the pathophysiology of many diseases in humans –28, its We demonstrated that the ROM serum level at 12 weeks during treatment with TCZ was a useful biomarker to predict DAS-remission at 52 weeks. As a biomarker to predict DAS-remission, ROM was superior to CRP or MMP-3. The serum level of ROM could be a biomarker to monitor treatment efficacy of TCZ in routine clinical practice.


A. duranensis after nematode (Meloidogyne arenaria) infection were released. However, the number of LRR-containing genes present in A. duranensis and the response of LRR-containing genes to nematode infection are poorly understood.Leucine-rich repeat (LRR)-containing genes are involved in responses to various diseases. Recently, RNA-seq data from A. duranensis. The inferred phylogenetic relationships revealed that the nine types of LRR domains had two originations. The inferred selective pressure was mainly consistent with LRR domains undergoing purifying selection. Twenty-one LRR-containing genes were associated with possible resistance to nematode infection after 3, 6, and 9 days. Among them, Aradu.T5WNW, Aradu.JM17V, and Aradu.MKP1A were up-regulate at these three time points, while Aradu.QD5DS and Aradu.M0ENQ were up-regulated 6 and 9 days after nematode infection. The expression of the above mentioned five genes was significantly and negatively correlated with the number of LRR8 domain, indicating that fewer LRR8 domains are associated with the promotion of LRR-containing genes that resist nematode infection. Patterns of co-expression and cis-acting elements indicated that WRKY possibly regulate the responses of LRR-containing genes to nematode infection and that expansin genes may work together with LRR-containing genes in response to nematode infection.In this study, we found 509 amino acid sequences containing nine types of LRR domains in A. duranensis. The LRR-containing genes that were found appear to be involved in responses to nematode infection. The number of LRR8 domains was negatively correlated with expression after nematode infection. The WRKY transcription factor may regulate resistance to nematode infection based on LRR-containing genes. Our results could improve the understanding of resistance to nematodes and molecular breeding in peanuts.We identified the number and type of LRR-containing genes in The online version of this article (10.1186/s12870-018-1508-x) contains supplementary material, which is available to authorized users. Arachis hypogaea) is a major oil and protein crop worldwide. Cultivated peanut is an allopolyploid hybrid between the wild diploid species Arachis duranensis and A. ipaënsis [A. duranensis and A. ipaënsis have been sequenced and released [A. duranensis and A. hypogaea revealed high expression in A. duranensis but not in A. hypogaea under Aspergillus flavus treatment [Cultivated peanut  proteins typically contain repeats composed of 20–29 residues, such that they constitute a continuous parallel β-sheet, which forms helically twisted and solenoid-like structures . LRR-conipaënsis . Howeveripaënsis , 15. Thereleased . In thishttp://pfam.xfam.org/). These HMM models include LRR19-TM (PF15176), LRRC37 (PF15779), LRRC37AB_C (PF14914), LRRCT (PF01463), LRRFIP (PF09738), LRRNT (PF01462), LRRNT_2 (PF08263), LRR_1 (PF00560), LRR_2 (PF07723), LRR_3 (PF07725), LRR_4 (PF12799), LRR_5 (PF13306), LRR_6 (PF13516), LRR_8 (PF13855), LRR_9 (PF14580), LRR_adjacent (PF08191), and LRV (PF01816). The complete A. duranensis genome has been sequenced [https://peanutbase.org/files/genomes/Arachis_duranensis/). The 17 HMMs were downloaded and LRR-containing sequences were detected using the HMMER program [A. duranensis amino acid sequences. The LRR-containing sequences were extracted using an in-house Perl script. To exclude the false-positive sequences, all sequences were uploaded to the Pfam public database to verify LRR domains.A total of 17 hidden Markov models (HMMs) of LRR domains were documented in the Pfam public database (Ka/Ks). Generally, Ka/Ks values of 1, > 1, and < 1 indicate neutral, positive, and purifying selection, respectively.Multiple sequence alignment of full-length amino acid and LRR domain sequences was conducted using MAFFT 7.0 . PhylogeA. duranensis tissues have been released on the PeanutBase website (https://peanutbase.org/gene_expression/atlas) [https://peanutbase.org/gene_expression/atlas_nematode). The sequencing details were described in previous publications [2-transformation.RNA-seq datasets from 22 different n/atlas) . The genications , 23. In 2-fold change of greater than 2 or less than − 2 were used for WGCNA analyses. A soft threshold (β) value of 12 was used in the transformation of the adjacency matrix in order to meet the scale-free topology criteria. Co-expression modules were created with the blockwiseModules function using the following parameters: power = 6, TOMType = “unsigned,” maxBlockSize = 30,000, mergeCutHeight = 0.25, minModuleSize = 30, reassignThreshold = 0.Genes co-expressed under nematode infection were detected among 22 different tissues using a weighted gene co-expression network analysis (WGCNA) script in R . DiffereA. duranensis genome available on the PeanutBase website (https://peanutbase.org/files/genomes/Arachis_duranensis/) [Gene ontology (GO) annotations for genes in each module containing the expansin gene were extracted from the nensis/) .cis-acting element sequence of the full-length LRR-containing gene was retrieved from the PeanutBase website (https://peanutbase.org/genomes/jbrowse/?data=Aradu1.0). Transcription regulatory elements were predicted using NSITE [P-value threshold of 0.05, and transcription factor annotation was retrieved from the PlantTFDB 4.0 database [The 2 kb ng NSITE  with a Pdatabase .A. duranensis. These LRR domains included LRRNT_2 (316 sequences), LRR_1 (221 sequences), LRR_2 (10 sequences), LRR_3 (33 sequences), LRR_4 (22 sequences), LRR_5 (1 sequences), LRR_6 (155 sequences), LRR_8 (643 sequences), and LRR_9 , but this feature was absent from the LRRNT_2 domain  of these paralogs was significantly higher than the average nonsynonymous substitution rate  was 0.28. These results indicated that purifying selection has acted on these paralogs. Further, the average Ka/Ks value of LRR domains were less than 1 except for three pairs of domains . The average Ks value of LRR domains exceeded the average Ka value. Among them, LRR8 . These transcription factor binding sites included Dof, BP, bHLH, RTCS, BZZR1, Hox1a, E2F2, WRKY, GT-1, Hsf, and AGL15  and Oryza sativa subsp. japonica (1400) [A. duranensis. For example, the LRR3 domain was mainly found in the NBS–LRR gene family.LRR domains were distributed among many proteins . LRR doma (1400) . HoweverKa/Ks values exceeded 1 [A. duranensis and other plants.The LRR domain has two features that were identified by previous studies. First, the LRR domain contains a conserved consensus sequence LxxLxLxx (where x is any amino acid and L is leucine) . Second,ceeded 1 –32. In tceeded 1 . Second,To reveal the origin of LRR domains, we constructed a phylogenetic tree using all 1403 LRR domains; however, no adequately common sites were identified for construction of a phylogenetic tree (data not shown). However, the multiple alignment results indicated that the nine types of LRR domains form at least two groups (consistent with multiple origins). LRR1, 2, 3, 4, 5, 6, 8, and 9 domains contained the LxxLxL repeat unit, but LRRNT_2 contained fewer L amino acids. Nevertheless, we attempted to estimate whether LRR1, 2, 3, 4, 5, 6, 8, and 9 have an ancient ancestor. Previous studies have disputed the origin of LRR domains. Kajava  suggesteMeloidogyne arenaria) [Although some reports have demonstrated that receptor-like kinase, NBS–LRR, protein kinase, LRR receptor, and F-box gene families are involved in resistance to pathogen infection , 35–37, renaria) . Peanutsrenaria) . In thisXa21 gene has 23 LRR copies and confers resistance to the bacterial blight pathogen Xanthomonas oryzae pv. oryzae race 6 [FLS2 in Arabidopsis, with 28 LRRs, was involved in flagellin response [A. duranensis. There is at least one explanation for this difference. Pattern-triggered immunity (PTI), but not effector-triggered immunity (ETI), plays a crucial role in resistance to nematode infection. To date, the innate immunity system can be classified into two layers, PTI and ETI [R genes) and resistance to invasion of pathogens. The LRR domain in R genes have co-evolved with pathogen effectors, and their role was recognized directly or indirectly with pathogen molecules [The genes with more LRR domains tend to be associated with resistance to biotic stress . For exae race 6 , and FLSresponse . Unexpec and ETI . The forolecules  found thA. duranensis showed that more expansin genes were up-regulated after nematode infection [In this study, we found that WRKY transcription factors can regulate the response of LRR-containing genes to nematode infection. This finding was consistent with previous results. Sarris et al.  demonstrnfection . We founA. duranensis. We further estimated the substitution rate of each type of LRR domain between paralogs. LRR domains were inferred to have mainly been subject to purifying selection. In addition, we comprehensively identified the LRR-containing genes that were involved in responses to nematode infection. The number of LRR domains among these genes was negatively correlated with expression level after nematode infection. Thus, the WRKY transcription factor may possibly regulate LRR-containing genes associated with resistance to nematode infection. Our results may clarify physiological mechanisms of resistance to nematode infection and marker-assisted breeding in peanut.We identified the number and type of LRR-containing genes in Additional file 1:Table S1. Gene location, gene name, and domain type for LRR-containing genes from Arachis duranensis (XLSX 59 kb)Additional file 2:Figure S1. The phylogenetic tree inferred using ATP binding amino acid sequences. Paralogs are indicated in blue. (TIF 310 kb)Additional file 3:Figure S2. The phylogenetic tree inferred using F-box amino acid sequences. Paralogs are indicated in blue. (TIF 653 kb)Additional file 4:Figure S3. The phylogenetic tree inferred using LRR receptor amino acid sequences. Paralogs are indicated in blue. (TIF 656 kb)Additional file 5:Figure S4. The phylogenetic tree inferred using NBS–LRR amino acid sequences. Paralogs are indicated in blue. (TIF 617 kb)Additional file 6:Figure S5. The phylogenetic tree inferred using protein kinase amino acid sequences. Paralogs are indicated in blue. (TIF 668 kb)Additional file 7:Figure S6. The phylogenetic tree inferred using receptor-like kinase amino acid sequences. Paralogs are indicated in blue. (TIF 1634 kb)Additional file 8:Table S2. Gene expression, gene location, gene name, and domain type among differentially expressed LRR-containing genes 3, 6, and 9 days after nematode infection. (XLSX 12 kb)