Diabetes mellitus (DM) has become a significant worldwide public health problem and economic burden because a great proportion of healthcare costs has been spent on the treatment of DM and its related complications. The aim of this study was to examine the costs and length of stay (LoS) of hospitalizations due to diabetes-related complications in Taiwan.  This study is a retrospective claim database analysis using the Longitudinal Cohort of Diabetes Patients, with 2012 used as the base year. The hospitalization costs and LoS per admission were estimated for each complication of interest using data from the LHDB 2004 to 2012 cohorts. The presence of eight DM-related complications were identified using the ICD-9-CM codes and procedure codes. ANOVA was used to examine the relationships of diabetes duration with the LoS and costs of the complications.  A total of 27,473 DM patients who were hospitalized in 2012 due to one of the examined DM-related complications were identified. The most common complications that caused the hospitalizations were nonfatal stroke (34.7%) and nonfatal ischemic heart disease (IHD) (28.7%). Amputation was the complication with the longest hospital stay, with a mean ± SD of 21.6 ± 14.1 days, followed by nonfatal stroke (13.6 ± 11.3), ulcer (12.7 ± 11.8), and fatal IHD (12.2 ± 13.6). The complications with the greatest hospitalization cost were fatal IHD  and fatal myocardial infarction .  This study indicates that DM-related complications are associated with significant hospital LoS and costs. The study results could be useful for economic evaluations of diabetes treatments and the estimation of the overall economic impact of diabetes. Hospital care is usually the the U.S. . In Canathe U.S. . Furtherthe U.S. .highly correlated with hospitalization, it is important to examine the hospitalization costs of DM-related complications. However, previous estimates of the costs associated with the management of DM-related complications in Taiwan have been based on aDCSI scores rather than individual complications. As such, the objective of our study was to examine hospitalization costs and length of stay (LoS) of hospitalizations in Taiwan for each DM-related complication.As inpatient costs are likely to account for the majority of overall DM-related medical costs, and DM-related complications are Ethical approval was obtained from the Shin-Kong Memorial Hospital Institutional Review Board .In this retrospective database analysis, we used the data from the Longitudinal Cohort of Diabetes Patients (LHDB) 2004 to 2012 cohorts. The LHDB is a database of patients with diabetes who met the following criteria: (1) history of hospitalization for DM or an in-hospital prescription for an antihyperglycemic medication; (2) two or more diagnoses of DM in outpatient settings within one year; or (3) both an outpatient diagnosis of DM and another outpatient visit with a prescription for an antihyperglycemic medication. A cohort of LHDB consisted of 120,000 newly diagnosed DM patients randomly selected each year from the National Health Insurance Research Database (NHIRD) , which cThe International Classification of Diseases, Ninth Revision, Clinical Modification ICD-9-CM) codes and procedure codes were used to identify DM complication-related hospitalizations. The codes associated with the eight common DM-related complications examined in this study are listed in -CM codesFor hospitalizations due to each DM-related complication, the following outcome measures were assessed: (1) length of stay, (2) total costs of hospitalization, and (3) inpatient drug costs. As only one patient was hospitalized in 2012 as a result of blindness, this complication was excluded from this part of analysis. Baseline characteristics of the selected patients were reported by descriptive statistics. The relationship of DM duration with the LoS and costs of complications was examined by ANOVA.All monetary values were reported in Taiwan dollars (TWD) (1TWD = 0.034USD). The significance level of this study was set at 0.05. All statistical analyses were performed using SAS software, version 9.4 .A total of 27,473 DM patients who were hospitalized in 2012 due to one of the study's DM-related complications were identified, and these patients had a total of 29,582 DM complication-related hospitalizations in 2012. The characteristics of the patients are summarized in n = 25,509). As shown in For the estimation of costs and LoS per hospitalization, only the patients who were hospitalized for a single complication in 2012 were included in the analysis (p < 0.01).The mean LoS, total hospital costs, and inpatient drug costs over different lengths of DM duration for each complication are shown in A few studies have examined the economic impact of DM-related complications. Chen et al.  reportedIn our study, the mean LoS in nonfatal IHD patients was 4.0 days, the shortest hospital stay among the DM-related complications. A significant difference in LoS was reported in a study in Japan where a mean LoS of IHD patients ranged from 10.9 days to 15.1 days depending on the patients' age . The difIn terms of inpatient drug costs, 17.9% of total hospitalization costs of ulcer resulted from drug costs, which proportion was the highest among all DM-related complications, followed by amputation (17.7%). These two complications had similarly high drug cost proportions, and foot ulcers can lead to amputation. In DM patients, the lifetime incidence of foot ulcers could be as high as 25% . DiabetiBao et al. reported that laboratory tests and prescribed drugs were the two major contributors to hospitalization costs, accounting for 22.4% and 36.2% of the costs, respectively . HoweverThere are a few limitations to this study. First, the identification of DM-related complications was based on ICD-9 codes listed in the study claim database, where errors and incompleteness may have existed and resulted in the misclassification of patients in each complication group. However, a previous study evaluating the accuracy of the NHIRD for a DM-related complication has shown high levels of diagnostic accuracy . Second,In conclusion, this study indicates that DM-related complications are associated with significant hospital LoS and costs. The study results could be useful for economic evaluations of diabetes treatments and the estimation of the overall economic impact of diabetes.


Tolerance is defined as an interaction in which viruses accumulate to some degree without causing significant loss of vigor or fitness to their hosts. Tolerance can be described as a stable equilibrium between the virus and its host, an interaction in which each partner not only accommodate trade-offs for survival but also receive some benefits . This equilibrium, which would be associated with little selective pressure for the emergence of severe viral strains, is common in wild ecosystems and has important implications for the management of viral diseases in the field. Plant viruses are obligatory intracellular parasites that divert the host cellular machinery to complete their infection cycle. Highjacking/modification of plant factors can affect plant vigor and fitness. In addition, the toxic effects of viral proteins and the deployment of plant defense responses contribute to the induction of symptoms ranging in severity from tissue discoloration to malformation or tissue necrosis. The impact of viral infection is also influenced by the virulence of the specific virus strain (or strains for mixed infections), the host genotype and environmental conditions. Although plant resistance mechanisms that restrict virus accumulation or movement have received much attention, molecular mechanisms associated with tolerance are less well-understood. We review the experimental evidence that supports the concept that tolerance can be achieved by reaching the proper balance between plant defense responses and virus counter-defenses. We also discuss plant translation repression mechanisms, plant protein degradation or modification pathways and viral self-attenuation strategies that regulate the accumulation or activity of viral proteins to mitigate their impact on the host. Finally, we discuss current progress and future opportunities toward the application of various tolerance mechanisms in the field. Tolerance to biotic stresses caused by pathogens, including viruses, is well-documented in plants . ToleranPlant viruses should not only be viewed as pathogens. In fact, experimental evidence documenting the beneficial impact of accommodating long-term virus infection is accumulating, especially in natural environments . Indeed,While many plant antiviral resistance genes (R genes) have been characterized , the genA recent review focused on plant–pathogen co-evolution in tolerant interactions . In thisThe majority of plant viruses are considered generalists as they can infect a large variety of plant hosts . HoweverRNA silencing is often considered the most important basal adaptive plant antiviral defense response . RNA silMost viruses encode a viral suppressor of silencing (VSR) to counteract the plant antiviral RNA silencing. Characterized VSRs show tremendous diversity in their protein sequence and mode of actions . VSRs caSalicylic acid (SA) is a key signal molecule in plants that mediates defense responses associated with basal innate immunity and with inducible immunity directed by antiviral dominant R genes . Basal iThe second line of SA-mediated defense responses is often referred to as the effector-triggered defense (ETI). ETI requires the recognition of pathogen avirulent proteins, also termed effectors, by plant intracellular receptors, which are encoded by dominant R genes . Most knSeveral plant viruses have been shown to suppress the oxidative burst and the expression of defense genes associated with PTI or ETI . A repliTm-1 gene binds to the tomato mosaic virus replication proteins and inhibits viral RNA replication  gene down-regulates the expression of the R-like gene SNC1 , but also from chloroplasts, peroxisomes or vacuoles, depending on the specific plant–virus interaction . Cell-toIn addition to the gross alterations in sub-cellular structures described above, a large network of interactions between plant and virus proteins has been characterized . In factAlthough many viral proteins contribute to virulence, VSRs are often virulence factors and symptom determinants. VSRs were first discovered in the context of synergistic interactions between two plant viruses. The potyvirus HC-Pro protein was shown to assist a potexvirus with counter-defense responses to the plant antiviral RNA silencing, resulting in increased symptom severity . The virIn silico analysis, and in some cases further functional validation, revealed many plant mRNA targets of vsiRNA in several plant–virus interactions (Nicotiana tabacum. This was correlated with the down-regulation of a gene involved in chlorophyll biosynthesis (ChlI) which is targeted by small RNAs derived from the satellite RNA (Nicotiana benthamiana eukaryotic translation initiation factor (NbeIF4A) was shown to be associated with the stunting phenotype of N. benthamiana plants infected with rice stripe virus  (Plants exhibit more tolerance to disease as they age. The maintenance of TGS and PTGS can differ in plants that are in vegetative or reproductive stages and some VSRs are not active in older plants at the reproductive stage . For exaN gene) . FurtherN gene) . In natu2 affect viral symptom expression  are temperature-sensitive directly affecting the defense response   , 2011. C element .2 is another factor that influences plant defenses to pathogen infection  or genes that are associated with tolerance. For example, several genes and QTLs have been linked to tolerance to barley yellow dwarf virus in barley, oat, and wheat . While iyviruses . HoweverA. thaliana-cucumber mosaic virus interaction, plants with longer vegetative growth cycles  are more tolerant to infection . In the nfection . Tolerannfection . Howeverspecific . In addispecific . AdditioVirus infection induces global changes in the plant transcriptome and proteome in both susceptible and resistant interactions . To dateNahG gene that down-regulates SA signaling, showed more severe symptoms upon virus infection and a diminished induction of photosynthesis genes at early stages of infection , creating a virus front that invades naïve cells  are associated with several viruses and have been shown to attenuate symptoms induced by the parent virus. The diRNAs contain non-contiguous segments from the parent viral RNA and are produced by template-switching of the viral RNA-dependent RNA polymerase (RdRp) during viral RNA replication . They coilencing . In tombilencing . As descilencing . Howeverilencing . Interesilencing . Indeed,ilencing .N. benthamiana , which is related to leucine-rich repeat receptor-like kinases implicated in the innate immune PTI response . NIK1 wahe plant . As a cohe plant . Intereshe plant .In addition to the plant responses described above, viruses minimize the accumulation of viral virulence factors  using sub-optimal translation initiation codons or inefficient frameshift or read-through translation mechanisms  and the autophagy pathway are key regulators of plant–virus interactions . By contA. thaliana). Depending on the nature of the ubiquitination (mono- or poly-ubiquitination), proteins are selectively targeted to the 26S proteasome for degradation. Cellular E3 ubiquitin ligases are common interactors of plant virus proteins, including, MPs and RdRps, many of which are destabilized by the UPS  (Direct evidence for a role for the UPS in facilitating tolerance is exemplified in the interaction between (TYLCCV) . The TYL(TYLCCV) . However(TYLCCV) . Importa(TYLCCV) . HoweverAutophagy is another highly conserved protein degradation pathway implicated in many aspects of plant–pathogens interactions including the regulation of programmed cell death . ProteinA. thaliana  to degradation . Thus, pA. thaliana  reduces its silencing suppression activity and diminishes symptom severity . Similarthaliana . In the thaliana . On the thaliana . While tN. benthamiana allowed increased systemic accumulation of potato virus X and potato virus Y and exacerbated systemic necrosis symptoms indicating that the UPR is induced to release ER stress, control virus accumulation, and prevent cell death . Although natural sources of tolerance are available for some economically important crops, they are generally poorly characterized and have been of limited use. The next question becomes: is it feasible to engineer tolerance for practical field applications?Only a few examples of engineered tolerance to virus diseases can be found in the literature. Most relate to the identification and manipulation of plant genes involved in signal transduction pathways associated with basal innate immune defense responses. Some are broad-spectrum and also provide tolerance to abiotic stress, in part because some of the signaling pathways are overlapping. We have already discussed how the ectopic expression of a phosphomimic mutant of AtNIK1, an immune receptor kinase, conferred broad-spectrum tolerance to begomovirus infection in tomato . Other kOverexpression of a transcription factor, the soybean GmERF3 gene, in tobacco also conferred increased tolerance to tobacco mosaic virus . This trN. benthamiana plants infected with a begomovirus  could be affected differently. Further research aimed at elucidating the molecular mechanisms associated with tolerance, in particular in wild plant–virus interactions, may identify novel targets for engineering tolerance or assist in the development of improved agriculture practices.DP and HS jointly wrote the manuscript, and 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.


Background: There is paucity in the literature regarding the role of the interoceptive pathway through the insular cortex (IC), as well as heart rate variability (HRV) in relation to Osteopathic Manipulative Therapy (OMT) and deep-touch.Aims: The present study investigated whether both OMT treatment and deep-touch (a newly hypothesized treatment option) was effective at altering the interoceptive pathway and HRV, whilst OMT was only expected to be effective for increasing Range of Motion (ROM).Methods: Thirty-five healthy volunteers were randomly allocated into three conditions in a repeated measures crossover design; a control (laying supine on a plinth); deep-touch (head cradling); and an osteopathic mobilization therapeutic technique on the temporomandibular joint (TMJ). Interoceptive accuracy (IAc), HRV, as well as range of motion (ROM) for the TMJ area as well as the cervical spine (Csp) right and left measures were taken pre and post each condition setting.Results: Significant condition effects emerged from the deep-touch and mobilization interventions for IAc where increases were identified through planned comparisons. For the HRV measure (RMSSD), a significant effect emerged in the deep-touch condition (increase) but not in the mobilization or control conditions. ROM did not increase for any condition. IAc correlated with post-ROM outcomes in many cases and HRV moderated some of these relations.Conclusion: These results are discussed in the context of clinical practice, where cranial deep-touch maybe an effective treatment and modulator of the parasympathetic nervous systems, as well as the interoceptive system. Interoception can be defined as the moment-to-moment representation of all bodily sensations , and invAltered interoceptive awareness is associated with a wide range of problematic conditions such as pain and mental health disorders. For example, on the negative side, it is associated with chronic pain  as well From a psychophysiological perspective, These signals are then decoded and travel through the spine via synaptic relays of lamina I and II, and then to the brain stem homeostatic regions, thalamus, and finally to the anterior insular of the insular cortex (IC) , 2003. FHowever, despite these interesting findings in relation to the activation of the IC pathway during OMT touch, very little is known about how manual therapy affects the interoceptive system . One typThe physiological aspects of how this therapy interacts with the body is understood well but is has only been until quite recently that sufficient knowledge has been discovered about the interoceptive system. The exteroceptive system has been studied extensively by manual therapists but the newly understood interoceptive system has been ignored . This isThis interoceptive state is referred to as interoceptive accuracy (IAc). It is a measure of interoception and is usually assessed by measuring the individual’s accuracy to perceive their own heart rate . IAc is Interestingly, higher heart rate variability (HRV) which is a measure of the parasympathetic nervous system (PNS) is also related to attentional, emotional, and behavioral self-regulation. It is suggested that the vagal tone index measures the efficacy of central-peripheral neural feedback mechanisms . SpecifiThe impact of OMT on HRV has previously been investigated and has shown that OMT and other manual therapies typically lead to an increase in parasympathetic dominance . OMT hasWhat has not been explored in great detail is how many newtons of pressure each OMT technique or touch provides and a detailed record of the vlocity of movement. It is perhaps important to understand the different types of touch and how they may impact on the ANS and IAc systems so to make specific and accurate hypotheses. This is because studies have shown differences in ANS responses based on the type of pressure used, i.e., whether it is light or deep touch. In example of this, in one study, only 0.8 N (newtons of force) and a velocity of 0.2 cm/s was applied to the forearm and thigh areas and led to a significant increase in the sympathetic responding as recorded by electrodermal activity . HoweverThe touch procedure in this current study involves cradling a person’s head which involves placing pressure on the suboccipital muscles (rear head muscles), as the weight of the head forms its own downward pressure through gravity. As the average head weights approximately 10 lbs  this conSo, in summary, the primary hypothesis is that it is predicted that OMT and deep-touch will affect IAc as they will activate the CT afferent fibers, and thus lead to an increase in IAc. It is predicted that both will increase, but there will be little differences between these conditions as they will active the CT fibers in a similar way. For the second outcome measure, it is hypothesized that the parasympathetic dominated vagal tone (increase in HRV), will be observed after both the deep-touch and OMT conditions, as this has been observed in previous studies . Again, In addition to these, baseline HRV and IAc will be correlated with post-ROM outcomes, where it was predicted that both baseline measures will associate with the post-ROM outcomes as they are both involved in behavioral self-regulation. HRV will also be explored as a moderator between pre–post ROM, pre–post IAc and Pre-IAc with post ROM where it is predicted that HRV will moderate these relations.M = 21; F = 14) healthy, asymptomatic 1st and 2nd year undergraduate students participated in this study. Participants were included in the trial if they were aged between 18–45 and were English speaking to acquire valid informed consent. Participants with any previous direct trauma to the TMJ, whiplash to the cervical spine (Csp), symptoms of jaw, facial or neck pain, history of Csp disk prolapse, surgical procedures to the Csp, dental surgery or orthodontics within the last year, any form of chronic pain and currently taking any analgesic or anti-inflammatory medication were excluded from the trial. Prior to commencement of the study, participants were randomly assigned to a treatment intervention  through use of a computer research randomiser (see randomization section) into a sequence of the three condition arms .A purposive sample of 35  and two covariates  by 3 intervention measures. This is appropriate for a 2 × 3 repeated measure, within factors ANCOVA (two covariates), according to Dattalo’s text book on determining sample size using G∗Power . The main advantage of the crossover design when compared to a between design is that participants can act as their own controls, and thus variation in participant individual differences between conditions can be removed. In addition to this, a repeated crossover design has greater power when compared with the same sample size but with a between groups design and so can reduce the chances of obtaining a type 2 error .Ethics were approved through the University Research Ethics Council (REC), which included participant consent, right to withdraw and a full debriefing at the end of the study.Researcher 1 (R1) conducted the measurements only, whilst Researcher 2 (R2) conduced the intervention only. R1 was blinded to the intervention by leaving the laboratory during the intervention phase. R2 who conducted the interventions was blind to the measurements by leaving the laboratory during the measurement phase.The area for the OMT mobilization in this present study is called the temporomandibular joint (TMJ) (the jaw joint area), which involves multi-vector guided movement of the TMJ. It is typical to use a measure called ROM when measuring the effectiveness of OMT treatments, which is the ROM of a bodily limb or other appendage at a fixed point (such as the head pointing forward) to a tolerable starching point in a fixed direction  .Figure 2) which has been found to have high intra-assessor reliability (ICC = 0.9–0.98)  at the cervical spine (Csp). Digital inclinometry (DI) has been previously used in several trials assessing cervical, thoracic and lumbar spine ROM where all authors concluded moderate to good inter-rater reliability (ICC = 0.6–0.9)  . It was 0.6–0.9) , 2015.Interbeat interval data were recorded to measure HRV and interoception using an FDA certified ECG measurement system (BIOPAC MP160) with the patient in a supine position and with conventional Ag/AgCl electrodes. The reference ECG signals were acquired at the sampling rate of 1000 Hz. After being recorded with the BIOPAC’s AcqKnowledge software, R–R interval data were analyzed using Kubios HRV Analysis Software 2.0 , 2014.In terms of the best possible way to determine IAc, heartbeat detection has emerged as the dominant method . This isThe recordings were measured at baseline and immediately following each intervention for a period of 5 min. HRV primarily measure PNS, and is an indirect measures of cardiac autonomic balance, where higher values generally indicate higher vagal tone and parasympathetic dominance. One measure of HRV is to use time domain; root mean squared of the successive  differences (RMSSD) or the R–R interval to measure vagal nerve parasympathetic response, as recommended by the Task Force of the European Society of Cardiology and The North American Society of Pacing and Electrophysiology . Some reFollowing inspection of the inclusion and exclusion criteria, and a completed consent form, participants were randomized into one of the six condition sequences (see randomization). The following conditions were then conducted with a 1-week washout period between them, based on those determined randomized sequences, as follows:1.’ In a supine position with a pillow supporting the head and neck, R2 then instructed each participant to open and relax their mouth with broad contact over the body and ramus of the participants’ mandible. R2 followed by mobilizing the TMJ into combined vectors of elevation, depression, protraction, retraction and medial and lateral gapping . The mobilization was carried out for a period of 90s. Following this, R2 left the room and R1 returned to measure post-intervention outcome measures .All baseline outcome readings  and demographic data (in the case that this was the first condition they participated in) were initially assessed by R1. Following this, R1 left the room and R2 entered to conduct the mobilization intervention. Participants in this condition were told the following; ‘Today you will be receiving an osteopathic technique directed toward your jaw joint. Breath normally and relaxFigure 3). Following this, R2 left the room and R1 re-entered to reassess the outcome measures .All baseline outcome readings  and demographic data (in the case that this was the first condition they participated in) were initially assessed by R1. Following this, R1 left the room and R2 entered to conduct the deep-touch intervention. Participants in the deep-touch treatment group were told the following; ‘Today you will be receiving an osteopathic technique. Breath normally and relax.’ R2 instructed the patient to lie supine, with a pillow supporting the head. The deep-touch technique consisted of R2 gently placing both hands over the base of the head, lightly palpating the sub-occipital musculature for a period of 90s  and demographic data (in the case that this was the first condition they participated in) were initially assessed by R1. Participants in the control group were told the following, ‘Please lay on the plinth until instructed otherwise, breath normally and relax.’ The participants lay supine on the plinth with their head on a pillow for 90s and then were instructed to sit up. R1 then re-assessed the outcome measures .p > 0.05) and thus justifies the use of parametric tests.All data analysis was conducted through IBM’s SPSS software version 23. Firstly, to correct for positive skewed distributions (when identified through a significant Shapiro–Wilk test), a logarithmic transform (log 10) was used before proceeding with the main analysis to ensure a normal distribution. Correcting non-normality can be made through transforming the data, where it is suggested  that corFor the pre-processing of the HRV data, this was first visually inspected for artifacts caused by ectopic beats, and poor conductivity. A very low correction threshold was chosen for artifact correction  so not to distort natural variability. Less than 1% of beats were identified as artifacts and no cases were removed from the analysis. RMSSD was than calculated through the Kubios software which utilized the MATLAB R2011 software.For the pre-processing of the IAc data, the following transformation was used on the perceived and actual heart beats as used in other studies  whereby post hoc tests were conducted to determine the nature of the interaction. In addition to this, a series of bivariate correlations were conducted exploring the relation between baseline IAc and RMSSD with post-ROM outcomes for each of the three areas . Finally, moderation analysis was conducted using the SPSS PROCESS macro by Andrew Hayes  by three (OMT/deep-touch/control) general linear models, consisting of a repeated measures analysis of covariance (ANCOVA), for each of the three separate areas  for ROM and for the two measures of IAc and HRV . BMI, age and gender were covariates in the model. When interactions were significant appropriate ew Hayes  to exploTable 1 for the descriptive statistics of the mean and standard deviations of the ROM measure, and Table 2 for the mean and standard deviations of the HRV and IAc measures.The demographics (mean and standard deviation) of the population were recorded as; Age (Years) = 21.52 (5.12); Weight (KG) = 73.47 (18.01); Height (M) = 1.72 (0.16); and BMI = 24 (5.42). See F = 0.187, p = 0.669, F = 0.197, p = 0.660, F = 1.654, p = 0.209, F = 2.287, p = 0.120, Figure 4), post hoc Bonferroni comparisons (pre–post interventions) identified that IAc significantly increased in those receiving the mobilization  and deep-touch  interventions. This increase did not occur in the control condition .IAc did not differ according to the covariates BMI  but not in the mobilization  or control conditions . To check the reliability of the time domain index RMSSD, then pre and post measures of this for all conditions were correlated with the frequency domain index HF (another index of parasympathetic dominance) and were found to be highly correlated .None of the covariates were significant: BMI ; an effect not observed in the deep-touch  or mobilization  conditions. The interaction Time by Intervention was not significant [F = 0.457, p < 0.638, Older participants had reduced ROM [F = 1.237, p < 0.305, F = 7.175, p < 0.012, F = 0.002, p < 0.969, F = 1.379, p < 0.250, The interaction Time by Intervention was not significant [r = 0.259, p = 0.084); Baseline RMSSD sham with post-ROM TMJ sham was not significant but in a positive direction ; Baseline RMSSD control with post-ROM TMJ control was significant but in a negative direction . Baseline RMSSD mobilization with post-ROM Csp right mobilization was not significant but in a positive direction ; and so was Baseline RMSSD sham with post-ROM Csp right sham ; Baseline RMSSD control with post-ROM Csp right control was not significant but was in a negative direction . Baseline RMSSD mobilization with post-ROM Csp left mobilization was not significant but in a positive direction ; this was the same for Baseline RMSSD sham with post-ROM Csp left sham ; Baseline RMSSD control with post-ROM Csp left control was significant and in a negative direction ; Baseline RMSSD mobilization with post-IAc mobilization was not significant ; however, baseline RMSSD sham with post-IAc sham was significant and in a positive direction ; Baseline RMSSD control with post-IAc control was not significant but was in a negative direction .There were very few significant relations between baseline RMSSD and post-ROM outcomes. Baseline RMSSD mobilization with post-ROM TMJ mobilization was just outside of significance and in a positive direction .A series of bivariate correlations were also used to also explore the relation between baseline IAc and post-condition ROM, which showed these were several significant relations. Baseline IAc mobilization with post-ROM TMJ mobilization was significant and negatively related . Baseline IAc control with post-ROM TMJ control was significant and in a negative direction . Baseline IAc mobilization with post-Csp right mobilization was significant and in a negative direction . Baseline IAc sham with post-Csp right sham was also significant and in negative direction . Baseline IAc control with post-Csp right control was not significant . Baseline IAc mobilization with post-Csp left mobilization was significant and in a negative direction ; and so was baseline IAc sham with post-Csp left sham ; as well as baseline IAc control with post-Csp left control .Baseline IAc sham post-ROM TMJ sham was not significant  would be effective at altering the IAc, HRV with little difference between them, whilst OMT would be effective for the increased ROM (as this was a specific measure of OMT effectiveness).We did not find what we were expecting in some cases. Hypothesis one was found to be true, where a significant increase in IAc was observed for the deep-touch and mobilization conditions (but this was only identified through the pre–post Bonferroni tests). Hypothesis two was not found to be true, and the null was accepted as there was a significant increase in RMSSD for the deep-touch condition only and not the OMT treatment as predicted. Hypothesis three was also found to be false, and the null was accepted as there was no increase in ROM for the OMT condition (or deep-touch).In addition to these, there were very few associations which were significant between RMSSD and post-ROM outcomes. One of these was a negative correlation between baseline RMSSD control with post-ROM TMJ control; another was a negative correlation between baseline RMSSD control with post-ROM Csp left control. For the relations between RMSSD and IAc, there was only a positive correlation between baseline RMSSD sham with post IAc sham. Many of the IAc scores correlated with post-condition ROM scores. These included negative correlations for the following; baseline IAc scores with post-ROM mobilization TMJ; with post-ROM TMJ control; post-ROM Csp right mobilization; post-ROM Csp-right sham; post-ROM Csp left mobilization; post-ROM Csp left sham; post-ROM Csp left control.As baseline IAc demonstrated many significant associations with ROM outcomes, RMSSD was also explored as a moderator between the associations of pre–post ROM, pre–post IAc, and pre-IAc with post ROM. RMSSD did not moderate pre–post ROM for any of the conditions or areas, but it did moderate pre–post IAc mobilization and sham conditions, as well as pre-IAc and post-ROM TMJ mobilization and control conditions, pre-IAc with post-ROM CspR control, pre-IAc and post-ROM CspL sham and control conditions.The significant increase of HRV in the deep-touch condition only and not in the OMT condition was unlike other studies which found a significant increase after the OMT intervention. The reason why the OMT condition did not lead to an increase in RMSSD is perhaps because this was a mobilizing of the jaw joint intervention (rotating the jaw joint) rather than a stroking of the skin intervention such as soft tissue massage as conducted in, for example, the Toro-Velasco study . As for As light and deep touch seem to affect the ANS system differently, where light touch of only 0.8 N of pressure and a velocity of 0.2 cm/s on the forearm and thigh leads to a sympathetic response . Whilst,The correlations also produced some interesting findings which showed that in nearly all cases IAc was significantly negatively related with post-ROM outcomes, which means that potentially IAc is a reliable predictor for post-intervention ROM outcomes. Perhaps equally interesting is that RMSSD moderated some of these relations, which shows that HRV scores may strengthen the IAc post-ROM relation outcomes and therefore increase the ability to predict ROM outcomes.There were also some inconsistencies in the literature (mentioned in the introduction) regarding behavioral self-regulation where both increases in IAc and HRV are related to increased behavioral self-regulation. The inconsistency is that high IAc involves a sympathetic response whilst higher HRV is a parasympathetic response, which of course counterintuitive. The findings of this present study does not claim to explain this inconsistency but it has identified HRV as a possible moderator of the relation between IAc and post-ROM outcomes as well as pre–post IAc mobilization and sham outcomes. Further studies would be needed to specifically explore these relations in the context of behavioral self-regulation, perhaps where HRV is explored as a moderator to perhaps explain these inconsistencies.The increase in IAc after the deep-touch and OMT demonstrates that touch has an effect not just with deep-touch but also with this particular OMT technique and in this location. Therefore, maybe osteopathic practitioners should be more aware of this general effect in practice, and more research should focus in this area. Awareness is increasing as osteopathic practitioners are beginning to realize that they are missing knowledge in relation to the emotional body through the interoceptive system . HoweverAccordingly, these findings could translate into a clinical setting of pain management delivered in the form of OMT and other manual therapy. For instance, in a recent systematic review  it was sThere are also important questions around chronic pain and the co-morbidity of psychological health problems. Recently, a study was conducted which showed that OMT reduced chronic pain related psychological issues such as anxiety and mental health dysfunction, but was less effective for fear avoidance and depression . UnderstThe vagal nerve (not just as a moderator for IAc) seems to be closely linked to psychological function and positive mental health outcomes. For example, several trials have demonstrated that individuals with anxiety, obsessive compulsive disorders (OCD) and components of chronic pain exhibit lower baseline HRV , which dOn the whole, this study offers some interesting findings in relation to OMT, touch and its impact on ROM, IAc and HRV. From the literature, it is clear that IAc and vagal tone have a very complex relation with touch, pain perception as well as affective mental health states. However, further integration studies are needed. Integrating biomechanical, spinal, peripheral and neurophysiological components into a single model maybe needed for a complete understanding of how touch and OMT impact psychophysiological outcomes of pain perception, vagal tone and interoception. This has also been supported by previous researchers . Before In perhaps the only OMT-neuroscience study of this kind , a high Ethical approval: All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki Declaration and its later amendments or comparable ethical standards. Informed consent was obtained from all individual participants included in the study.DE, HY, RJ, and AC designed the study. DE wrote the majority of the paper with contributions from HY and AC. HY and DE conducted the majority of the statistical analysis. AC contributed to the research design and some aspects of the writing of this manuscript including some aspects of the statistical analysis.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.


Foamy viruses (FVs), also known as spumaretroviruses, are complex retroviruses that are seemingly nonpathogenic in natural hosts. In natural hosts, which include felines, bovines, and nonhuman primates (NHPs), a large percentage of adults are infected with FVs. For this reason, the effect of FVs on infections with other viruses (co-infections) cannot be easily studied in natural populations. Most of what is known about interactions between FVs and other viruses is based on studies of NHPs in artificial settings such as research facilities. In these settings, there is some indication that FVs can exacerbate infections with lentiviruses such as simian immunodeficiency virus (SIV). Nonhuman primate (NHP) simian FVs (SFVs) have been shown to infect people without any apparent pathogenicity. Humans zoonotically infected with simian foamy virus (SFV) are often co-infected with other viruses. Thus, it is important to know whether SFV co-infections affect human disease. Spumaretrovirinae subfamily of the family Retroviridae and are also designated spumaretroviruses [Spumaretrovirinae the oldest known extant vertebrate virus subfamily. Foamy viruses (FVs) comprise the oviruses . FVs areoviruses . Interesoviruses . The coeoviruses , making FVs are apparently nonpathogenic in their natural hosts, which include NHPs (reviewed in ), felineSimian FVs (SFVs) replicate primarily in tissues of the oral mucosa ,16, and While humans have contacts with felines and bovines, there is no evidence for actual infection of humans with FVs from these species (reviewed in ). ZoonotInterestingly, SFV replication takes place in the most terminally differentiated superficial epithelial cells of the oral mucosa—those about to slough off into saliva . The lacEach organism is host to a multitude of microbes, known as the microbiome. Organisms are also host to a collection of viruses, known as the virome (reviewed in ). Since,There is evidence for zoonotic transmission of SFVs to humans (reviewed in ). Many oSFVs are endemic in all NHP species examined to date in Africa, Asia, and the Americas ,35. In ASTLV-1 has not been as extensively studied in its natural NHP hosts as human T-cell lymphotropic virus-1 (HTLV-1) has been in humans. HTLV-1 is pathogenic in only a small fraction of infected humans (reviewed in ), and STMacaca mulatta, as a model for HIV infections. In the wild, SIV is not known to exist in Asia [Researchers have used the rhesus macaque (RM), also known as  in Asia ,48, the  in Asia  and has  in Asia ,25. A ke in Asia . HoweverIt is also possible that SFV can affect the pathogenicity of other viruses, such as SIV. In natural settings, most adult NHPs are naturally infected with SFV. Therefore, this issue was addressed using RM individually housed in primate center facilities. The researchers examined SFV− and SFV+ RM infected with a pathogenic SIV strain (SIVmac239) . The SFVThe mechanism by which SFV increases SIV pathogenesis is unknown. One tissue target of SIV pathogenesis in RM is the jejunum . WhetherFree-living chimpanzees (cpz) in multiple countries of Africa, such as Cameroon and Gabon, have been analyzed for infection with SFVcpz and SIVcpz . The resA cohort of naturally SFV-infected baboons were studied. Some of the baboons were also naturally infected with STLV-1, while others were not, with 18 baboons studied per group. It was found that SFV DNA levels in the blood, but not in the saliva, were increased in the STLV-1-infected baboons . It is nSRV D infections in Asian primates such as macaques sometimes causes an immunodeficiency-like syndrome ,45. SRV One study identified HTLV-1 and SFV co-infected hunters in Central Africa . Fifty-sThere is a publication presenting evidence that SFV-infected bushmeat hunters from Cameron seem to have different levels of hematological markers, including hemoglobin, creatine phosphokinase, and bilirubin, than SFV-uninfected bushmeat hunters . The SFVMost work on FV transmission has been done in NHPs. This has served as the general model for FV transmission in other natural species. While the transmission route for non-primate FVs has been speculated to include saliva, other transmission modes seem to occur. For example, bovine foamy virus (BFV) has been detected in bovine breast milk . In factA serious illness of unknown origin in dairy cows is non-responsive post-partum metritis (NPPM). It is thought to be caused by a virus, as it is unresponsive to antibiotics. There is some evidence that bovine gammaherpesviruses (BoHVs) are associated with this disease . Since many cows are infected by BFV , it is pFeline leukemia virus (FeLV), a retrovirus of cats, often leads to fatal diseases, including lymphomas . One stuThere are cats co-infected with FFV and feline immunodeficiency virus (FIV), a lentivirus causing decreases in CD4+ T cells and feline acquired immune deficiency syndrome (FAIDS) . In one In natural hosts, FVs are highly prevalent and therefore finding uninfected adult animals is difficult. Thus, all information about the effect of FVs on other viral infections in these species involves artificial situations in which some animals are kept FV-free. Most of the studies on FV effects on other viral infections use lab-adapted pathogenic viruses. In research laboratory settings, there is clear indication that FV infections exacerbate lentiviral outcomes. There is also evidence that SFV has an expanded tissue tropism in lentiviral co-infected NHPs. The prevalence of FV co-infected individuals has been described in natural hosts. However, these studies did not report whether FV co-infection affects pathogenesis induced by other viruses. Therefore, it is not known whether the results from lab settings are relevant to what occurs in natural settings. The mechanism of how FV infections may affect other viral infections is not clear. Humans can be zoonotically infected with SFVs from NHPs. While it has been reported that there are people co-infected with SFV and HIV or HTLV-1, there are almost no data about whether SFV infection of humans can exacerbate other viral infections. Overall, more studies in natural settings and of human zoonotic FV co-infections would be important to understand whether FVs contribute to the pathogenicity of other microorganisms. One example of a FV pathogenic effect on hosts is in RM, using lab-adapted strains of a lentivirus, SIV. In the future, it will be important to see whether HIV pathogenesis is worse in humans co-infected with SFV. It is also possible that in humans, FV infections could exacerbate other viral infections such as HTLV-1, HBV, or even CMV or other herpesviruses. The understanding of how SFVs affect other human viruses is important, given the potential use of FV vectors for gene therapy to treat human diseases.


Microcephaly and other birth defects have been identified among infants born to women with Zika virus infection during pregnancy  and infant number 8 (31 cm) were below the third percentile for gestational age and sex. Zika virus test results (serum IgM and RNA and urine RNA) were all negative for infant number 6 at birth; the infant received a diagnosis of microcephaly at age 1 week, but head circumference was normal at ages 2, 6, and 12 months, and a cranial ultrasound at age 3 months was unremarkable. A pediatrician classified the infant as normal at age 12 months. Infant number 8 was born at 38 weeks gestation, weighing 2.2 kg. Zika virus test results (serum IgM and RNA and urine RNA) at birth were negative. The infant received a diagnosis of symmetric growth retardation and was admitted to the neonatal intensive care unit for respiratory distress but was discharged home in good health at age 4 days. A pediatrician found this infant to be healthy and with normal head circumference at age 12 months.Among 11 infants born to women in LAC with evidence of confirmed or probable Zika virus infection during pregnancy, the nine who participated in follow-up through age 12 months had no apparent adverse health effects at that time. Subtler health effects, or health effects occurring later in life, would not be captured with this surveillance activity. In addition, mothers with Zika virus infection who did not seek medical care, as well as those who chose not to participate in, or did not complete, the surveillance, limited the generalizability of these findings. Ongoing assessment of the health of infants born to women with evidence of Zika virus infection during pregnancy is important to assess the public health impact of Zika virus and to guide interventions.


Filoviridae) cause outbreaks of viral hemorrhagic fever in sub-Saharan Africa. The Egyptian rousette bat (ERB) is a natural reservoir host for the marburgviruses and evidence suggests that bats are also natural reservoirs for the ebolaviruses. Although the search for the natural reservoirs of the ebolaviruses has largely involved serosurveillance of the bat population, there are no validated serological assays to screen bat sera for ebolavirus-specific IgG antibodies. Here, we generate filovirus-specific antisera by prime-boost immunization of groups of captive ERBs with all seven known culturable filoviruses. After validating a system of filovirus-specific indirect ELISAs utilizing infectious-based virus antigens for detection of virus-specific IgG antibodies from bat sera, we assess the level of serological cross-reactivity between the virus-specific antisera and heterologous filovirus antigens. This data is then used to generate a filovirus antibody fingerprint that can predict which of the filovirus species in the system is most antigenically similar to the species responsible for past infection. Our filovirus IgG indirect ELISA system will be a critical tool for identifying bat species with high ebolavirus seroprevalence rates to target for longitudinal studies aimed at establishing natural reservoir host-ebolavirus relationships.With the exception of Reston and Bombali viruses, the marburgviruses and ebolaviruses (family  The family Filoviridae includes two genera of viruses that are known to cause disease in humans and/or non-human primates; the genus Marburgvirus includes one virus species comprised of two viruses (Marburg virus and Ravn virus) and the genus Ebolavirus includes six virus species comprised of one virus each 2. With the exception of Reston3 and Bombali viruses4, the marburgviruses and ebolaviruses cause outbreaks of viral hemorrhagic fever in sub-Saharan Africa10. Reston virus causes disease in non-human primates only, and its circulation appears to be limited to the Philippines and China1. Far less is known about Bombali virus, as its genomic viral RNA was only recently detected in Angolan free-tailed bats (Mops condylurus) and little free-tailed bats (Chaerephon pumilus) in Sierra Leone4. To date, Bombali virus has not reportedly been isolated.Following spillover from a zoonotic source, the filoviruses can be transmitted person-to-person through direct contact with infectious bodily fluids, resulting in large outbreaks of viral hemorrhagic fever with high case fatality5, many of the index cases of marburgvirus disease reported entering bat-inhabited caves or underground mines prior to becoming ill13. Retrospective investigation of three outbreaks of marburgvirus disease among miners working in Goroumbwa Mine in northeastern Democratic Republic of the Congo (DRC) from 1998–200014, miners working in Kitaka Mine in southwest Uganda in 200715, and two tourists that had separately visited the nearby Python Cave, Uganda in 200817 revealed that all of these locations were inhabited by large populations of the Egyptian rousette bat . A longitudinal ecological investigation later identified this bat species as a natural reservoir host for the marburgviruses and a source of virus emergence in humans18.Since the discovery of Marburg virus in 1967Mops trevori) directly over the working area of the index case19. The putative index case of the 2007 Ebola virus disease outbreak in the Kasaï-Occidental province of the DRC was said to have purchased bats for consumption following a reported annual migration of hammer-headed bats (Hypsignathus monstrosus) and Franquet’s epauletted fruit bats (Epomops franquetti)20. Lastly, the presumed index case of the 2014 Ebola virus disease outbreak that started in Guinea was reported to have played in a tree hollow, where DNA traces of Mops condylurus were later identified21.Unlike the spillover events leading to marburgvirus disease, those for ebolavirus disease have been difficult to associate with a specific environment, such as caves. Furthermore, the evidence linking ebolavirus spillover to a particular species of bat is largely circumstantial. For example, the 1976 and 1979 index cases of Sudan virus disease both worked in a cotton factory in Sudan, where a retrospective investigation eight to nine months after the initial outbreak identified a large roof colony of Trevor’s free-tailed bats 22 and Reston virus RNA has been reportedly detected in four bat species captured in the Philippines 23. Yet, infectious virus was not isolated from any of these species23, indicating that they are either dead-end virus hosts, had cleared virus infection prior to sampling, or that current filovirus isolation techniques lack the sensitivity to recover infectious virus from specimens with low viral loads. Serological reactivity of bat sera with ebolavirus antigens using indirect ELISAs, indirect fluorescent tests, bead-based multiplex assays and/or western blots has been reported in 375 bats representing at least 21 species throughout sub-Saharan Africa and Asia32. However, interpretation of this data has been exceedingly difficult due to multiple reasons that include: (1) the absence of a panel of positive and negative bat sera for initial serological assay validation and continuing quality control, (2) the use of various uncharacterized recombinant filovirus antigens, and (3) the application of different statistical methods for establishing cutoff values for seropositivity. Nonetheless, ebolavirus serosurveillance offers several distinct advantages compared to surveillance for active virus infection including a longer diagnostic window , no knowledge of virus-tissue tropism and no requirement for destructive sampling.The majority of research directed towards the identification of the natural reservoir hosts of the ebolaviruses has consisted of cross-sectional surveillance of the sub-Saharan African and Asian bat population for evidence of active and past ebolavirus infection. The predominance of cross-sectional studies can be attributed to the large number of bat species residing within the geographic range of ebolavirus circulation that require investigation, as well as the challenges associated with obtaining diagnostic specimens from a mammal that is difficult to capture due to its elusive nocturnal nature and ability to fly. Collectively, Ebola virus RNA has been detected in three bat species captured in the Republic of the Congo and Gabon  at euthanasia was allowed to clot and then centrifuged at 1,500 g for 10 min. The sera were transferred into polypropylene CryoElite Wheaton vials , and then temporarily stored under liquid nitrogen vapors in the biosafety level four laboratory (BSL-4) until they were double bagged, passed through a dunk tank containing 5% Micro-Chem Plus  and transported to the gamma-cell irradiator. The doublebagged serum aliquots were sandwiched between layers of dry ice and then exposed to 5 × 106 rads of gamma-cell irradiation using a cobalt-60 source.The bats used to generate the filovirus-specific antisera in this study originated from an ERB breeding colony37 were used as filovirus-naïve sera. These sera were collected and processed in the same manner as outlined above.Sera obtained from filovirus-naïve bats from the breeding colony and negative control bats from previous experimental studiesAll animal procedures were approved by the Institutional Animal Care and Use Committee (IACUC) and performed in compliance with the Guide for Laboratory Animal Care and Use . The animal research facilities at the CDC have received full accreditation by Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC).Procedures conducted with filoviruses or filovirus-infected bats were performed in accordance with Select Agent Regulations  in a BSL-4 laboratory at the CDC. All bat cages were further contained within bio-flow isolator units with HEPA-filtered inlet and exhaust air supplies .ad libitum.All bats were group-housed according to filovirus inoculum in a climate controlled BSL-4 animal area, with a 12 h day/12 h night cycle. Bats received a quantity of fresh fruit, supplemented with protein/vitamin powder, equal to their body mass daily and water 40. Briefly, roller bottles of Vero E6 cells were inoculated with virus and the cultures were harvested when ≥90% of cells exhibited evidence of infection by immunofluorescence assay. Filovirus antigen lysates were prepared from the cultures by detergent basic buffer extraction of infected cells. Uninfected control antigen lysate was generated in the absence of virus, but otherwise prepared in the exact same manner.Filovirus antigen lysates Table  and uninEach filovirus-specific antiserum (n = 124) and filovirus-naïve serum (n = 19) was tested for reactivity against each filovirus antigen lysate (n = 7) and uninfected control antigen lysate  by sex, age category, or the interaction between sex and age category .The breeding colony was founded from Ugandan wild-caught, filovirus-naïve adult (≥1 yr) ERBs18. We assume that the seroprevalence of the ebolaviruses in their respective natural reservoir bat hosts approximates this value. However, the prior probability has relatively little influence on determining the magnitude of the cutoff value for seropositivity. The cutoff value for each filovirus-specific indirect ELISA represents the value where the posterior probabilities for the reactivity of the filovirus-specific antisera with homologous antigen and the reactivity of filovirus-naïve sera with this same antigen are equal.For each filovirus-specific IgG indirect ELISA, quadratic discriminant analysis was used to determine a cutoff value for seropositivity by considering: (1) the reactivity of the filovirus-specific antisera group with homologous antigen, (2) the reactivity of the filovirus-naïve sera group with that same antigen, and (3) the prior probability of the filovirus-specific antisera group . The prior probability of each filovirus-specific antisera group was set at 0.15. This value represents the overall population seroprevalence of marburgvirus in its natural reservoir host, the ERBFor each indirect ELISA at its specified seropositivity cutoff value, the filovirus-specific antiserum and filovirus-naïve serum samples were classified as true positive, false positive, false negative or true negative. The sensitivity of each assay was then calculated by dividing the number of individuals with a positive test result by the number of individuals with a history of past infection , and the specificity of each assay was calculated by dividing the number of individuals without a history of past infection (filovirus-naïve group) by the number of individuals with a negative test result.The level of serological cross-reactivity between each group of filovirus-specific antisera and each heterologous virus antigen was calculated by dividing the number of antisera positive for reactivity with a virus antigen by the total number of antisera tested against that antigen , and was expressed as a percentage.After the nucleotide sequences of the filovirus isolates used to inoculate the bats in this study were retrieved from GenBank, the coding regions were translated into amino acids and aligned using the MUSCLE algorithm . Percent identity values were then calculated from pairwise amino acid comparisons generated from the virus protein alignment. A Pearson’s Product-Moment Correlation was performed to assess the relationship between percent serological cross-reactivity and percent amino acid identity .Quadratic discriminant analysis was used to predict which one of the seven filoviruses in the system was most antigenically similar to the filovirus responsible for past infection by considering the relative reactivity of each filovirus-specific antiserum and filovirus-naïve serum with each filovirus antigen, as well as their covariance . First, prior probabilities were calculated for the eight classes used in the analysis (seven filovirus antigen classes and one negative class). Second, classification was performed by calculating posterior probabilities for the inclusion of an antisera/sera sample in each class and then assigning the sample to the class with the highest probability.35 or whole blood36 samples collected from experimentally inoculated ERBs. Five of these samples were collected four weeks post primary Marburg virus inoculation36, while two of the samples were collected at 23 and 27 weeks post primary inoculation following a “natural” boost 36.To evaluate the ability of our system comprising seven filovirus-specific indirect ELISAs to predict the filovirus species most antigenically similar to the species responsible for past infection, we tested seven Marburg virus convalescent serumA total of 124 ERBs were used in this study that was performed over a two-year time span. The total number of bats dedicated to this study, as well as the size and composition of each group was dependent on the reproductive capacity of the ERB breeding colony, as well as the characteristics and number of bats needed for other experimental studies. The 124 bats were divided into seven groups, with group sizes ranging from 15–20 individuals  generated from bats intentionally infected with each of the seven known culturable filoviruses to develop a classification system (fingerprint) to predict which filovirus elicited the antibody response. Supplementary Table To further assess the performance and predictive ability of our filovirus IgG indirect ELISA system, we tested five Marburg virus convalescent sera or whole blood samples collected four weeks post primary Marburg virus experimental inoculation and two whole blood samples collected at 23 and 27 weeks post primary experimental inoculation following a “natural” boost . As expected, marburgvirus-specific IgG antibody levels were markedly lower in these seven convalescent serum and whole blood samples compared to marburgvirus-specific IgG antibody levels in the serum samples from the prime-boosted bats in this study Fig. . However43, varying levels of serological IgG cross-reactivity between the ebolavirus species (20–100%)44 and strong serological IgG cross-reactivity within the species Marburg marburgvirus (95%). The low level of serological IgG cross-reactivity between some of the ebolavirus species underscores the necessity of performing all assays within the system to avoid false negative results. Although significant levels of serological IgG cross-reactivity were observed between the prime-boost filovirus-specific antisera and some of the filovirus antigens, when the overall covariance of the seven-individual indirect ELISAs in the system were considered, we were able to predict the filovirus species responsible for past infection 100% of the time using as little as 25 μL of sera (each serum was tested against each antigen in duplicate). Further evaluation of the performance and predictive ability of our system using seven Marburg virus convalescent serum or whole blood samples collected after primary experimental infection with Marburg virus  confirmed that the system was able to predict the filovirus species most likely responsible for past infection. As expected, the filovirus IgG indirect ELISA system was not able to correctly predict whether past infection was due to Marburg virus or Ravn virus 100% of the time. This finding supports the current classification of Marburg and Ravn viruses into a single virus species, Marburg marburgvirus2.In this study, we generated filovirus-specific antisera from prime-boosted ERBs and collected filovirus-naïve sera to validate and characterize an indirect ELISA system that utilizes non-recombinant, infectious-based filovirus antigens for the detection of virus-specific IgG antibodies from bats. Initial validation of the system revealed that the individual filovirus-specific IgG indirect ELISAs exhibited high sensitivity (90–100%) and specificity (100%). Similar to previous studies using human sera, we observed limited to no serological IgG cross-reactivity between the filovirus genera (0–15%)31. Alternatively, false positive serological results can occur when recombinant virus antigens unknowingly share similar epitopes with other virus antigens for which the study population possesses antibodies against46. False positive results can also arise from failing to account for non-specific serological reactivity resulting from the presence of non-virus contaminants in an antigen preparation. Here, we negated non-specific serological reactivity to Vero E6 cell components by subtracting the reactivity of wells coated with uninfected antigen lysate from the reactivity of corresponding wells coated with filovirus antigen lysate. While the majority of previously published ebolavirus serosurveys of bats used recombinant filovirus antigens generated in bacterial expression systems, not all of the studies implemented procedures to negate non-specific reactivity between residual bacterial epitopes in the antigen preparation and antibacterial antibodies in bat sera29. Like the majority of published ebolavirus serosurveys of bats, we performed serial serum dilutions to assess for linearity and specificity30. Most importantly, we included pooled filovirus-specific bat antisera as a positive control and pooled filovirus-naïve bat sera as a negative control in every run to ensure that our filovirus IgG indirect ELISA system continually performed as expected.The robustness and discriminatory power of our system results from its underlying components and various control points. We employed infectious-based filovirus antigens, rather than recombinant virus protein antigens, as a strategy to increase both the sensitivity  and specificity  of the system. Although recombinant filovirus antigens can be generated in large quantities and do not require a BSL-4 laboratory for production, the use of single recombinant virus proteins for antibody detection can lead to false negative results if an individual’s antibody repertoire is not directed against the particular recombinant virus protein that was employed32. Four out of the 11 previously published ebolavirus serosurveys of bats used Ebola virus antigen only to detect ebolavirus IgG antibodies26 and another four of these serosurveys used Ebola and Reston antigens only to detect ebolavirus IgG antibodies29. Based on the low serological cross-reactivity between Ebola virus antigen and Sudan virus antisera (68.4%) and Reston virus antigen and Sudan virus antisera (57.7%) observed in our study, we predict that the serosurveys using Ebola virus antigen or Ebola and Reston virus antigens only would have missed the opportunity to detect bats previously infected with Sudan virus. Likewise, due to the high level of serological cross reactivity between ebolavirus-specific antisera and heterologous ebolavirus antigen that we observed in our study, it is possible that the serological reactivity of bat sera with Ebola or Reston virus antigens in the previous serosurveys was due to past infection with one of the other known ebolaviruses or an undescribed ebolavirus. Although we successfully used quadratic discriminant analysis to predict which of the filovirus species in the system was most antigenically similar to the species responsible for past infection, only nucleotide sequence data can be used to ascertain infection with a particular filovirus. Furthermore, incrimination of an ebolavirus-natural reservoir host relationship requires consistent detection of both active (RNA and virus isolation from tissues/bodily fluids suggestive of a transmission mechanism) and past infection with a particular ebolavirus through space and time.Using filovirus-specific antisera collected from bats that were prime-boosted at the same time not only allowed us to thoroughly examine the level of serological cross-reactivity between the virus-specific antisera and heterologous filovirus antigen, but also provided the opportunity to use this knowledge to evaluate previously published ebolavirus serosurveys of bats36. Others have reported periodic boosting of rabies virus neutralizing antibody titers in a wild colony of big brown bats (Eptesicus fuscus)47. Second, we used a goat anti-bat IgG conjugated to horseradish peroxidase as the secondary antibody in our system. This antibody was generated using sera from ERBs, as well as sera from nine other bat species comprising four chiropteran families (manufacturer product datasheet). While this antibody has been confirmed to react with sera from these 10 chiropteran species and has been used with success to detect IgG antibodies in sera from >27 bat species comprising seven chiropteran families57, it is possible that its reactivity with IgG antibodies from divergent bat species might diminish. If this is the case, the concentration of secondary antibody may need to be optimized prior to testing a new species of bat or an alternative secondary antibody may need to be used. Third, our filovirus IgG indirect ELISA system includes seven virus antigens that represent all of the reported culturable filoviruses. Given the recent discoveries of filovirus RNA from divergent bat families in geographically distant locations , we expect that there are a number of undiscovered ebolaviruses that continue to circulate in nature. However, we believe that the genetic diversity of ebolavirus antigens (up to 61.6%) included in our system will allow for the detection of past infection with a novel ebolavirus, and prediction of the ebolavirus species that is most antigenically similar to the species responsible for past infection.While our filovirus IgG indirect ELISA system enables the robust detection of virus-specific IgG antibodies from bats and was able to predict the filovirus species most likely responsible for past infection, it does have some limitations. First, this system was validated using virus-specific antisera from prime-boosted bats and sera from filovirus-naïve bats. Although all seven of the Marburg virus convalescent whole blood and serum samples collected from ERBs after primary experimental Marburg virus inoculation or following a “natural” boost were marburgvirus IgG antibody positive, these samples had markedly lower marburgvirus-specific IgG antibody levels compared to samples collected from the bats in this study that were prime-boosted with one of seven filoviruses. This suggests that the sensitivity of the individual filovirus IgG indirect ELISAs may be lower than what was reported here (90–100%) using filovirus-specific antisera from prime-boosted bats and filovirus naïve bat sera. However, statistical determination of seropositivity cutoff values for the indirect ELISAs were largely driven by the variance surrounding the mean reactivity of the filovirus-naïve sera with each of the virus antigens. It is likely that circulation and long-term maintenance of the ebolaviruses in their natural reservoir hosts leads to multiple virus exposures throughout the lifetime of an individual, resulting in boosting of virus-specific IgG antibody levels. “Natural” boosting of virus-specific IgG antibody levels in ERBs that had been previously infected with Marburg virus was observed during a recent experimental transmission study shortly after documenting virus infection and seroconversion in naïve contact batsWe intend to use our filovirus IgG indirect ELISA system to screen archived and incoming specimens from cross-sectional serosurveys of the bat population of sub-Saharan Africa and Asia for evidence of past filovirus infection. If other specimen types exist for bat species identified as filovirus seropositive, they will be tested for evidence of active infection using virus-specific qRT-PCR, pan-filovirus RT-PCR and/or virus isolation techniques. Bat species positive for evidence of active filovirus infection or species exhibiting a seroprevalence equivalent to or larger than that of marburgvirus in ERBs  will be targeted for longitudinal studies aimed at collecting a wide range of specimens for evidence of active and past ebolavirus infection.While most of the virus-specific antisera generated in this study will be pooled to use as positive controls in our indirect filovirus ELISA system, some will be reserved for investigations aimed at determining the mechanisms by which bats clear and control filovirus infections through virus neutralization and cross-neutralization assays, as well as epitope mapping studies. Any remaining filovirus-specific bat antisera may be used to develop a pan-ebolavirus indirect ELISA that utilizes a mixture of non-recombinant, infectious based antigens from culturable ebolaviruses, as well as validate the performance of indirect ELISAs that use recombinant filovirus proteins to detect filovirus antibodies.Supplementary information


Phenuiviridae have been detected in bats, including the highly pathogenic Rift Valley fever virus and Malsoor virus, a novel Banyangvirus with close genetic relation to Huaiyangshan banyangvirus (BHAV) and Heartland virus (HRTV), both of which have caused severe disease with fatal casualties in humans. In this study we present the whole genome of a novel Banyangvirus, named Zwiesel bat banyangvirus, revealed through deep sequencing of the Eptesicus nilssonii bat virome. The detection of the novel bat banyangvirus, which is in close phylogenetic relationship with the pathogenic HRTV and BHAV, underlines the possible impact of emerging phenuiviruses on public health.Bats are reservoir hosts for several emerging and re-emerging viral pathogens causing morbidity and mortality in wildlife, animal stocks and humans. Various viruses within the family  Phenuviridae family in the order Bunyavirales, is characterized by a tri-segmented, negative- or ambisense ssRNA genome of 11–19 kb length in total. Four structural proteins are encoded on the genome: the L protein (RNA-dependent RNA polymerase) on segment L, two glycoproteins Gn and Gc on segment M, the nucleocapsid protein on segment N and the nonstructural protein NSs on segment S. Banyangviruses can infect vertebrates and invertebrates and have caused febrile infections, encephalitis and severe fevers with fatal outcome in humans, therefore being increasingly reported as emerging pathogens of public health importance3. Recently, two novel tick-borne phenuiviruses (Huaiyangshan banyangvirus [BHAV] and Heartland virus [HRTV]) were detected and characterized; they form, together with Guertu banyangvirus a new genus within the Phenuiviridae genus: the Banyangviruses clade2. BHAV was initially reported in 2011 in the Henan and Hubei Provinces, China. Patients showed fever or hemorrhagic fever, thrombocytopenia, leukocytopenia and multi-organ dysfunction with an initial case fatality rate of 30%4. By then, the etiological virus was isolated from patients’ blood and Haemaphysalis longicornis and Rhipicephalus microplus ticks throughout China, South Korea and Japan6. Similar symptoms were recognized in two men from Missouri, USA. The respective virus, named Heartland virus (HRTV), was isolated in 2012 from patients’ blood and Amblyomma americanum ticks collected in the field7. A third novel banyangvirus with close relation to BHAV and HRTV was detected in Tasmania State, Australia. The virus, named Hunter Island Group virus (HIGV), was isolated from Ixodes eudyptidis ticks which were blood-feeding on a shy albatross  colony with signs of unknown disease outbreak8. Despite the identification of ticks as vectors for BHAV, HRTV and HIGV, the amplifying or reservoir hosts of the viral pathogens remain unknown.The Banyangvirus genus, currently classified as part of the 9. So far, two members of the family Phenuiviridae are associated with bats. The highly pathogenic Rift Valley fever virus was detected in Micropteropus pusillus and Hipposideros caffer bats in Guinea10. Malsoor virus, a fourth novel banyangvirus within the genus Banyangvirus, was recently isolated from a Rousettus leschenaultii bat in India11.Bats are the primary reservoir host for several zoonotic viruses with a high impact on public health, including numerous filoviruses, paramyxoviruses, coronaviruses and bunyaviruses12. Novel sequencing technologies allow massive parallel sequencing of all organisms in one sample, also allowing the detection of viruses with low sequence similarity to already known viruses14.Bats may be the source of many more yet unknown viruses which can cause morbidity and mortality in wildlife, animal stocks and humans(Eptesicus nilssonii). Phylogenetic analysis of the ZbbV polymerase protein showed that the closest relationship was to Malsoor virus, HRTV and BHAV.In this study, organs of microchiropteran bats, which were moribund or found dead, with potentially virus-related histopathological changes were used for deep sequencing of the bat virome. A novel banyangvirus, named Zwiesel bat banyangvirus (ZbbV) after the town of bat origin in Germany, was detected in the virome of several Northern bats The combination of sequences obtained from virome data and gap-filling PCR revealed the partial genome sequence of the novel ZbbV with 6,277, 3,434 and 1,793 nt for the L, M and S segment, respectively. The L segment open reading frame encodes for the RNA polymerase, the M segment for the two glycoproteins. Two open reading frames in ambisense orientation were determined on the S segment, which code the N protein (sense) and the nonstructural NSs protein (antisense).Phylogenetic tree reconstruction of the ZbbV L, M and S segment revealed a monophyletic relationship with Malsoor virus, HRTV and BHAV within the genus banyangvirus. No signs of genetic reassortment were observed. Comparison of the L segment showed 84% aa (74% nt), 71% aa (67% nt) and 69% aa (66% nt) identity to Malsoor virus, HRTV and BHAV, respectively. Comparison of the M segment showed 70% aa (68% nt), 57% aa (58% nt) and 56% aa (59% nt) identity to Malsoor virus, HRTV and BHAV, respectively. Comparison of the S segment showed 38% aa (69% nt), 10% aa (48% nt) and 16% aa (49% nt) identity to Malsoor virus, HRTV and BHAV, respectively.Eptesicus nilssonii bats used in this study , after the town of Zwiesel in Bavaria, where one of the Eptesicus nilssonii bats. After 3 passages, no cytopathic effect was observed and cell culture supernatants were tested negative for ZbbV.To obtain the virus isolate of the novel banyangvirus, Vero and Paki cell lines were inoculated with pooled organs of all 12 Eptesicus nilssonii bats. Genetic characterization showed that the virus is related to the tick-borne zoonotic phenuiviruses BHAV and HRTV, responsible for severe disease and death in humans, as well as to Malsoor virus which was recently isolated from a Rousettus bat in India.In this study we identified a novel banyangvirus, named Zwiesel bat banyangvirus, in the virome of Aedes aegypti mosquitoes15. However, although Haemaphysalis longicornis ticks are supposed to be the vector of BHAV, most infected patients did not report tick bites prior to disease onset1. Moreover, investigation of a cluster of BHAV cases suggests evidence of person-to-person transmission15, making the further study of BHAV- and BHAV-like viruses imperative regarding human public health.Since similar viruses have been detected in far apart countries such as North America, India, China, Japan, South Korea and Australia and in ticks of different genera, numerous zoonotic phenuiviruses may be widely distributed in different parts of the world. So far, ZbbV is the first banyangvirus detected in bats of northern Europe. Grouping of ZbbV within the tick-transmitted genus Banyangvirus suggests ticks to be the vector of the novel virus. Additionally, we have not found any evidence that ZbbV does encode for a NSm protein on segment M, this is corresponding with all other members of the genus Banyangvirus. As shown before, NSm is important for infectivity in It is tempting to suggest that viruses discovered in insectivorous bats are only remnants of the bats’ diet. Although we found sequences of ZbbV in the intestines of the bats, we also detected the virus in bat liver, lung, brain and spleen using specific PCR assays.Eptesicus nilssonii bats, which are the most abundant bats in northern Eurasia, the potential zoonotic impact of emerging banyangviruses on public health should be further investigated.Considering the close relationship of ZbbV to BHAV and HRTV as well as the broad distribution of zoonotic phenuiviruses and https://ec.europa.eu/environment/nature/legislation/habitatsdirective/) and the Agreement on the Conservation of Populations of European Bats (www.eurobats.org), investigative research on bats was permitted by local government bodies. We herewith confirm, that all permits to investigate carcasses of deceased bats and all experimental protocols on the dead bats were approved by the respective local governmental authorities . We herewith further confirm that all experiments were performed in accordance with relevant guidelines and regulations. All bats in a broad study on diseases of native bats in Germany (n = 375) were found dead or moribund with subsequent euthanization and were kindly provided by bat researchers and bat rehabilitation centers16. 12 Eptesicus nilssonii bats collected throughout urban and suburban areas of Bavaria, showed potentially virus-related histopathological changes and have been further examined in this study.In compliance with the species protection through the European Commission (17. Deep sequencing was performed using the Illumina HiSeq. 1500 (Illumina Inc.) technology according to the Illumina sequencing protocol. Raw reads were trimmed for primers, adapters, low-quality bases and length (>30 bp)18. Sequences that mapped to RefSeq sequences of eukaryotes, fungi and bacteria using Bowtie 2 version 2.2.6 with default parameters were discarded. Remaining reads were de novo assembled using Velvet assembler 1.2.10 in default mode20. Contigs and remaining sequences were aligned to the NCBI RefSeq viral protein database using DIAMOND v0.7.121. All contigs and sequences with similarity to members of the genus Banyangvirus were assembled into larger contigs. The complete genome sequences of the L, M and S segments were obtained by using newly designed gap-filling PCR primers (sequence information available on request). Reagent concentrations were as follows: 5 µl of cDNA, 0.4 µM of each forward and reverse primer, 0.4 mM of desoxynucleoside triphosphates (Invitrogen), 2.5 ml of 10 × Platinum® Taq buffer, 4 mM of MgCl2 and 1.25 U Platinum® Taq polymerase (Invitrogen) with water added to a final volume of 25 µl. Cycling conditions were 95 °C for 10 min, 45 amplification cycles at 94 °C for 45 s, 55 °C for 45 s, 72 °C for 45 s and a final extension for 10 min at 72 °C. Open reading frames of the viral genome were predicted with Geneious 7.1.4. , 2.5 ml of 10 × Platinum® Taq buffer, 5 mM of MgCl2 and 1.25 U Platinum® Taq polymerase (Invitrogen) with water added to a final volume of 25 µl.All work was performed at Biosafety level-2 conditions with appropriate precautions. After obtaining the result that Zwiesel virus is related to SFTS virus (rated BSL-4 in Germany), further work on the original material was restricted to BSL-3. Homogenized organs n = 31] with potentially virus-related histopathological changes (data available on request) were pooled 28. Posterior probabilities are depicted at the branches.The nucleic acid sequence  of the L segment, M segment  and S segment  where used for phylogenetic analysis. Alignments were built with ClustalW before model prediction with JModel test


Gossypium hirsutum varieties that are susceptible to attack by this virus. Several approaches have been employed to explore putative resistance genes in another cotton species, G. arboreum. However, the exact mechanisms conferring disease resistance in cotton are still unknown. In the current study, we used various approaches to identify possible resistance genes against CLCuV infection. We report the identification and isolation of a set of genes involved in the resistance response to viral infestation. PCR products containing genomic DNA gave multiple amplifications with a single primer in most reactions, and 38 fragments were cloned from G. arboreum and G. hirsutum. The sequences of cloned fragments belonged to various pathway genes and uncharacterized proteins. However, five amplified fragments  showed similarity with R genes. Maximum homology (94 %) was observed with G. raimondii toll/interleukin receptor-like protein. BLAST search showed the homology of all resistance gene analogues (RGAs) with more than one chromosome, and multiple hits were observed on each chromosome for each RGA. Expression analysis through RT–PCR identified variable expression levels of the different RGAs in all tested genotypes. The expression level of RGAs differed between symptomatic and asymptomatic plants, with the exception of RGA 395, whose expression level was the same in both diseased and healthy plants. Knowledge of the interaction of these genes with various cotton pathogens could be utilized to improve the resistance of susceptible G. hirsutum and other plant species.Cotton leaf curl virus (CLCuV) disease is one of the major limiting factors in cotton production, particularly in widely cultivated  Gossypium hirsutum, is sensitive to attack by cotton leaf curl virus. The devastating virus attacks the cotton plant when it gets a chance to get into the system, consequently resulting in curling of the leaves. Whitefly serves as a viral vector, and when feeding on the cotton plant, break the protective barrier and hence result in cotton infestation. Another species of cotton, Gossypium arboreum, is totally resistant to this viral disease. Whitefly also feed on G. arboreum and transmit virus, but this species has a natural defence system that does not allow the virus to cause infection and disease. The present study identified some genes of the defence system of G. arboreum that might contribute to make this species resistant to leaf curl disease in cotton.The cotton species,  Analogous to the vertebrate immune system, the resistance (R) genes in plants recognize pathogen effectors (avirulence factors) and awaken the defence system of plants to combat attack , charactArabidopsis thaliana is reported to contain 149 NBS-LRR  are an important tool for gene discovery , moleculG. arboreum using degenerate primers on genomic DNA and RT–PCR on RGAs detected from ESTs.In this study, we used RGA and EST homologues of R genes to reveal their expression pattern in cotton leaf curl virus (CLCuV)-resistant and -susceptible cotton genotypes. The RGA and EST homologues expressed in resistant genotypes can be further studied to find genes involved in CLCuV resistance. The aim of the current study was to explore the disease resistance genes in the published literature/GenBank and to screen for them in R genes of different classes were searched in cotton species at the NCBI. The nucleotide sequences of R genes were aligned using the multiple sequence alignment tool in the CLCBIO workbench. The conserved regions of alignments were used to design degenerate primers. Primer length was restricted to 24 mer. A maximum of four degeneracies was allowed per primer. Degeneracy was not allowed at the 3′ end of the primer. The primer sequences are shown in G. arboreum (Ravi) and five G. hirsutum ) were used for genomic DNA isolation from young cotton leaves following the CTAB (cetyl trimethylammonium bromide) method. Isolated DNA was quantified using an ultra-spectrophotometer. The PCR recipe was as follows: 5 µL genomic DNA (50 ng µL−1), 5 µL 10× Taq buffer, 4 µL of 25 mM MgCl2, 1 µL of 10 mM dNTPs, 1 µL of each forward and reverse primer (50 ng µL−1), 1 µL of Taq DNA polymerase (2 units per µL) and PCR water to make a final volume of 50 µL.Six cotton genotypes . The ligation was used to transform Escherichia coli competent cells (DH5∞). The transformed cells were spread on Luria-Bertani (LB) agar plates and the white colonies were cultured to isolate plasmid. The presence of clones was confirmed by restriction analysis using XbaI, SmaI, HindIII, EcoRI, BamHI, PstI, EcoRV and SacI.All the PCR fragments amplified in CLCuV-resistant gossypium genomes at CottonGen (www.cottongen.org) to determine their chromosomal location.Ninety-eight cloned fragments were sent for sequencing to Macrogen (Korea). M13 forward primer was used for sequencing. The sequences were trimmed from vector sequences and analysed for the presence of forward and reverse primer sites. The sequences were BLAST searched at NCBI to find their homology with sequences in the NCBI database. Later on the putative biotic stress resistance genes were also BLAST searched in the sequenced G. arboreum ESTs. The homologous ESTs were translated into amino acid sequences to find the open reading frame (ORF). The protein sequences of RGAs in G. arboreum and their homologues in G. hirsutum were aligned using the ClustalW program. Only those G. arboreum ESTs that differed from their homologues in G. hirsutum were selected. Primers were designed from the coding regions of ESTs using Beacon Designer software. Primer parameters were set as follows: primer length 18–24 nucleotides, GC content 40–50 %, melting temperature 55–60 °C and amplicon size 200–250 bp. The list of primers designed using the NBS class of previously reported RGAs is shown in Cotton RGAs of the NBS-LRR class reported by G. arboreum  and five from G. hirsutum, namely Coker (highly susceptible to CLCuV), NIBGE-2, NIBGE-115, N-253 and IR-3701 (tolerant to CLCuV). Young top leaf samples of CLCuV-susceptible and -non-susceptible plants (40–45 days old) of each genotype were taken from the National Institute for Biotechnology and Genetic Engineering (NIBGE) cotton field. Leaf samples were ground in liquid nitrogen and Invitrogen RNA purification reagent was used to isolate total RNA. The quality of RNA was checked by running on 1 % agarose gel. High-quality RNA was used to make first-strand cDNA using oligo dT primers. The cDNA was stored at −70 °C until further use.Six cotton genotypes were used for the study, including one from 4)2SO4Taq buffer, 4 µL of 25 mM MgCl2, 1 µL of 10 mM dNTPs, 1 µL of each 50 ng µl−1 forward and reverse primer, 1 µL of 2 units of Taq DNA polymerase and 36 µL of PCR water. PCR was performed on a BioRad thermal cycler and the PCR profile was set as: first denaturation at 94 °C for 4 min, followed by 40 cycles of denaturation at 94 °C for 1 min, annealing at 50 °C for 1 min, extension at 72 °C for 30 s, and last cycle of final extension at 72 °C for 10 min.The concentration of the cDNA was measured on a spectrophotometer and equal concentrations were prepared for the reaction mixture. The RT–PCR reaction consisted of 5 µL of cDNA template at an appropriate dilution, 5 µL of 10×  was observed with G. raimondii toll/interleukin receptor-like protein. RM12 and RM31 showed homology with the serine/threonine protein kinase class of cell surface receptors of G. raimondii.The sequences of cloned fragments belonged to a variety of different pathway genes and uncharacterized proteins. However, five fragments  showed similarity with R genes. RM1, RM6 and RM8 were cloned from cottongen.org) in the sequenced genomes of Gossypium species to determine their chromosomal location (The identified putative RGAs were BLAST searched (at location . All theG. arboreum as well as G. hirsutum genotypes (G. arboreum or any of the genotypes of G. hirsutum. Five RGAs showed high to moderate expression in Ravi. There were similar expression patterns in both symptomatic and asymptomatic plants of Coker. In NIBGE-2 most of the RGAs were expressed in symptomatic plants but not in asymptomatic plants. In the case of NIBGE-115 only one RGA (RGA 395) was expressed in both symptomatic and asymptomatic plants. Relatively more RGAs were expressed in asymptomatic plants of N-253 and IR-3701 compared with symptomatic plants. Resistance gene analogue 383 and 384 seem important as they showed almost no expression in either symptomatic or asymptomatic coker plants (CLCuV susceptible), as seen in The expression of 24 RGAs of the NBS class reported by enotypes . The expG. arboreum ESTs were searched in GenBank against the various R gene classes. No ESTs showed similarity to the NBS class of R genes. However, EST homologues were found against the serine/threonine kinase (STK) and LRR class of genes. One EST was also found showing similarity with Rar1 . The selected ESTs have conserved domains of PKc-STKc, LRR-8 and CHORD as found in the BLASTp search  are important ESTs as they are expressed in G. arboreum and tolerant G. hirsutum genotypes, whereas no expression was detected in susceptible Coker  while the LRR domain was represented by seven ESTs . PKc is a member of the AGC group in the protein kinase superfamily . This is an important class of kinases that are known to activate the p38 MAP kinase pathway of stress-induced signal transduction in mammals. The presence of a PKc-like domain in G. arboreum ESTs was interesting and it can be inferred that these ESTs were from the protein kinase class of genes in G. arboreum.erfamily . These eArabidopsis (Leucine-rich repeat receptor kinases (LRR-RKs) are the largest subfamily of transmembrane receptors, with over 200 members in bidopsis . Leucinebidopsis .Rar1 bearing the zinc binding CHORD domain that is found in all eukaryotes except yeast. One EST (26) showed significant similarity with the resistance signalling gene G. arboreum genome contribution. In the present study, RGAs were used to reveal the expression pattern in diploid and tetraploid cotton. Expression analysis of cell surface receptors was also conducted by utilizing their conserved domain regions of LRR and STK collected from G. arboreum ESTs. The selected ESTs were confirmed to contain the LRR and STK domains of resistance genes through BLASTX and DELTA-BLAST search as shown in Gossypium arboreum ESTs were not found in the BLAST search of the NBS class of genes. The expression of STK and LRR ESTs was higher than that of the NBS class of RGAs. It can be deduced that in G. arboreum R genes of classes other than NBS-LRR might be more active to counter biotic and abiotic stresses. Besides the importance of the NBS class of R genes, transmembrane receptors might also play a role in the recognition of and defence against CLCuV in G. arboreum. The kinase domain of disease resistance genes might play a role in phosphorylation and thus help transfer phosphate from the high-energy phosphate donor molecules.Resistance gene analogues have been used to study the genetics of resistance in plant species using R gene specific or degenerate primers  may be important for further studies on CLCuV resistance and the role of the kinase domain of R genes under similar conditions of inoculum and biotic stresses. As mentioned earlier these genes are known to be important players in plant defence, especially in the context of causing programmed cell death in infected tissues. So these RGAs and ESTs may be useful for isolating full-length genes, and their particular functions should be explored in response to biotic or abiotic stresses.Resistance gene analogues and ESTs expressed only in The following additional information is available in the online version of this article—Supplemetary DataClick here for additional data file.This research work was funded through Indigenous 5000 PhD fellowship program of Higher Education Commission, Government of Pakistan awarded to Rakhshanda Mushtaq at National Institute for Biotechnology and Genetic Engineering (NIBGE), Faisalabad in affiliation with Pakistan Institute of Engineering and Applied Sciences (PIEAS) Nilore Islamabad, Pakistan.R.M. solely done the experiments and K.S. helped in gene transformation experiments. Z.H.S., T.M. and Z.A. helped in bioinformatics tools and analysis and research write up. H.A., Y.Al-Z. and H.A.S.A. provided lab space for some of the experiments and helped in expression analysis.A.B. was the Principal Investigator of the research project. S.M. is the group leader and helped in project planning and execution of the experiments.