A 73-year-old man was confirmed to have an influenza A (H7N9) virus infection, and the causative agent A/Beijing/02/2014(H7N9) virus was isolated. Genetic and phylogenetic analyses revealed that the virus belonged to a novel genotype, which probably emerged and further reassorted with other H9 or H7 viruses in poultry before transmitting to humans. This virus caused a severe infection with high levels of cytokines and neutralizing antibodies. Eventually, the patient was cured after serially combined treatments. Taken together, our findings indicated that this novel genotype of the human H7N9 virus did not evolve directly from the first Beijing isolate A/Beijing/01/2013(H7N9), suggesting that the H7N9 virus has not obtained the ability for human-to-human transmissibility and the virus only evolves in poultry and then infects human by direct contact. Hence, the major measures to prevent human H7N9 virus infection are still to control and standardize the live poultry trade. Early antiviral treatment with combination therapies, including mechanical ventilation, nutrition support and symptomatic treatment, are effective for H7N9 infection. Recent studies showed that the internal genes of the H7N9 virus have continued to undergo dynamic reassortments with the poultry H9N2 viruses345338Since March 2013, novel influenza A H7N9) viruses have emerged in China and spread quickly, causing severe respiratory disease in humans1N9 viruseA 73-year-old man who worked as a live poultry seller and butcher in Beijing, China was diagnosed with an influenza A (H7N9) virus infection confirmed by detection of the H7N9 virus in the laboratory. The patient was an alcoholic with a past medical history of chronic bronchitis and coronary heart disease. The illness began with flu-like symptoms, including a high fever (38.3 °C), cough with yellow-white phlegm and feeling fatigued on 30 January 2014. Because the detection of the influenza A virus universal antigen was negative on the throat-swab by means of the immune colloidal gold technique and the radiologic findings revealed bronchitis, the patient was treated with anti-infective therapy by an intravenous injection of moxifloxacin. However, that treatment did not take effect, and the symptoms gradually worsened.On 5 February 2014, the patient appeared with hyperpyrexia (maximum temperature 40.0 °C), coughing with bloody sputum and dyspnoea with a low oxygen saturation (88.8%). The H7N9 viral RNA was positive in the oropharynx swab confirmed by the real-time RT-PCR method according to the protocol of the Chinese CDCA H7N9 virus, A/Beijing/02/2014(H7N9) (abbreviated as BJ02 thereafter), was isolated and identified after one passage propagation in eggs. To further study the gene evolution of the H7N9 virus, the whole genome was amplified and sequenced. Genetic alignments showed that the HA, NA, PB1, PA, NP and NS genes possessed the highest genetic similarities (99.33–99.74%) with other H7N9 virus genes. However, the PB2 and M genes possessed the highest nucleotide similarities  with H9N2 isolates . FurtherThe phylogenetic analyses showed that the HA and NA gene sequences of BJ02 clustered with previously identified human-infecting H7N9 viruses . The phyThe level of serum neutralizing antibody (NAb) and cytokines in flu patients play pivotal roles in disease progression and recovery101112131415161718The evaluation of serum cytokines in the H7N9 patient revealed that the levels of IL-6, IL-12p40, MCP-1 and IP-10 were significantly increased on 7, 15 and 18 d.a.o. compared with the normal ranges, whereas the IFN-γ, IFN-α and IL-17A levels did not notably increase . The levThe present data documented that the novel genotype G2.9 of the H7N9 virus (BJ02) could infect and cause severe disease in humans. In addition to the G1.5 virus (BJ01), there are at least two genotypes of H7N9 virus existing in Beijing. To date, only one G1.5 and one G2.9 virus from humans have been isolated; and the other G2.9 strain (SC537) was first isolated from chickens. Although high homology is shared between BJ02 and SC537 , the PB2To date, a total of 29 genotypes of H7N9 viruses, including BJ01 (G1.5) and BJ02 (G2.9), have been identified according to our classification and nomenclature systemIn addition, the oseltamivir resistance mutation (R292K) in the NA protein of BJ02 could not be found after 3 days of oseltamivir usage; however, the mutation emerged in the A/Shanghai/1/2013(H7N9) strain just after one day treatment with oseltamivir10In general, viral and host factors contribute to disease severity and outcomes. A previous multivariate analysis revealed that the presence of coexisting medical conditions (such as chronic heart disease and chronic obstructive pulmonary disease) were the only independent risk factors for severe illness with H7N9 infection212324252728113110112333435Although this was a severe H7N9-infected case with cytokine storm-like appearances and multiple organ failure, the patient was eventually cured after combination therapy with antivirals, mechanical ventilation, supportive nutrition and symptomatic treatment. In our study, the 73-year-old patient was not prescribed oseltamivir until day 6 d.a.o. because the H7N9 virus was not identified at the onset of his illness. According to the clinical finding reports of the H7N9-infected patients in 2013, the median time of the initiated antiviral therapy was 7 days after the onset of illnessA previous study showed that the early and rapid induction of NAb was correlated significantly with better clinical outcomes14The H7N9 influenza virus is spreading, evolving and becoming widespread among chickens in ChinaA suspected case of H7N9 influenza virus infection was confirmed by a real-time RT-PCR assay in the Beijing Centers for Disease Control and Prevention (CDC). The epidemiologic and clinical data were collected. A confirmed case was defined as evidence of pneumonia with H7N9 viral RNA or isolation of H7N9 virus from respiratory specimens. Informed consent was obtained from all participating individuals. This experimental protocol was approved by the local Ethics Committee of Beijing Ditan Hospital, Capital Medical University. The methods were carried out in accordance with the approved guidelines.http://www.megasoftware.net). The genotype analysis of the H7N9 virus was classified by the criteria previously describedAn oropharynx swab, collected on day 8 after the onset of flu-like symptoms, was used for virus isolation. The virus was propagated in 10-day-old specific pathogen free (SPF) embryonated chicken eggs and MDCK cells for 48 to 72 hours at 35 °C, respectively. For genetic analysis, the complete gene segments were amplified using improved primers  based on38Sera from the H7N9 patient were consecutively collected on 7, 15 and 18 d.a.o. for kinetic evaluations of the neutralizing antibodies and cytokine levels. A pandemic H1N1 severe case, onset on 2 February 2014, was used as a control, and the serum was collected on 15 d.a.o. The neutralizing antibodies (NAbs) were detected by Microneutralization Assays with A/Anhui/1/2013 (H7N9) and A/California/04/2009 (H1N1) viruses in MDCK cells, according to the previously described methodHow to cite this article: Bi, Y. et al. A new reassortment of influenza A (H7N9) virus causing human infection in Beijing, 2014. Sci. Rep. 6, 26624; doi: 10.1038/srep26624 (2016).


LTP-like activity induced by iTBS in AH was more frequent in females. Gender influences the functional excitability changes that take place after human stroke and the level of LTP that can be induced by repetitive stimulation. This knowledge is of high value in the attempt of individualizing to different genders any non-invasive stimulation strategy designed to foster stroke recovery.The acute phase of stroke is accompanied by functional changes in the activity and interplay of both hemispheres. In healthy subjects, gender is known to impact the functional brain organization. We investigated whether gender influences also acute stroke functional changes. In thirty-five ischemic stroke patients, we evaluated the excitability of the affected (AH) and unaffected hemisphere (UH) by measuring resting and active motor threshold (AMT) and motor-evoked potential amplitude under baseline conditions and after intermittent theta burst stimulation (iTBS) of AH. We also computed an index of the excitability balance between the hemispheres, laterality indexes (LI), to evidence hemispheric asymmetry. AMT differed significantly between AH and UH only in the male group ( Gender related functional asymmetries between the two cerebral hemispheres have been documented in healthy human brain  and the propensity of the cortex to undergo LTP- and LTD-like plasticity by means of a repetitive TMS (rTMS) paradigm, known as intermittent theta burst stimulation (iTBS). iTBS produces LTP-like changes in the stimulated hemisphere and LTD-like changes in the contralateral hemisphere . Inclusion criteria were: (1) single ischemic stroke  involving the middle cerebral artery territory; (2) less than 10 days post-stroke; (3) hand weakness; (4) recordable muscle evoked potential (MEP) after TMS of the AH. Exclusion criteria were: (1) history of seizure; (2) hemorrhagic stroke; (3) concomitant neurological or other severe medical problems; (4) complete paralysis of the hand; (5) inability to give informed consent; (6) concomitant treatment with drugs acting on the central nervous system; (7) contraindications for TMS studies. In order to identify at risk patients for post-stroke epilepsy, all patients underwent an EEG before entering the study and none of them showed any epileptic abnormality , and lesion size was estimated by using the Alberta Stroke Program Early CT Score (ASPECTS) . A figure-of-eight coil with external loop diameters of 9 cm was held over the motor cortex at the optimum scalp position to elicit MEPs in the contralateral first dorsal interosseous muscle (FDI). The induced current flowed in a postero-anterior direction. We evaluated the threshold and amplitude of MEPs. The resting motor threshold (RMT) was defined as the minimum stimulus intensity, expressed as the percentage of the maximal output intensity deliverable by the stimulator, which produced a liminal MEP  at rest Rossini, . The actiTBS was delivered over the affected motor cortex “hot spot” for MEPs in the contralateral FDI muscle using a MagPro stimulator (Medtronic A/S Denmark) connected to a figure-of-eight coil (MCF B65). The magnetic stimulus had a biphasic waveform with a pulse width of about 280 μs and a maximum magnetic field strength of 1.5 T. The initial direction of the current induced in the brain was anterior to posterior. The stimulation intensity was defined in relation to the AMT evaluated using the MagPro stimulator. An intensity of 80% AMT was used. We applied the iTBS protocol in which 10 bursts of high frequency stimulation (3 pulses at 50 Hz) are delivered at 5 Hz every 10 s, for a total of 600 pulses /(MEPUH + MEPAH). On the contrary, in the case of AMT and RMT the correlation with excitability is opposite (the lower are the thresholds the higher is the excitability). Thus, LI is calculated as follow: LIRMT= (MEPAH - MEPUH)/(MEPUH + MEPAH) and LIAMT= (MEPAH - MEPUH)/(MEPUH + MEPAH). LI ranges between −1 and +1; positive values always indicate higher excitability of the UH. The bigger the difference from 0, the higher is the inter-hemispheric imbalance. Gender effect on baseline RMT, AMT, and MEP is evaluated applying a mixed model ANOVA with Hemisphere (two levels: Affected -AH and Unaffected -UH) as within-subjects factor and Gender  as between-subjects factor. A two-tailed independent sample t-test is used to assess the LI between groups difference. iTBS effect is tested on RMT, AMT, and MEP amplitude by using a mixed model ANOVA with Hemisphere (Affected -AH and Unaffected -UH) and iTBS (Pre and Post) as within-subjects factor and Gender  as between-subjects factor. The same model without the factor hemisphere is applied to study iTBS-related LI changes. Differences between females and males for non-normal distributed data were checked applying Mann-Whitney tests. In order to better address the variability of iTBS effects on MEP amplitude, the proportion of iTBS-induced AH excitability increase and UH excitability decrease are compared between groups, by means of Chi-Square test. The correlation between the clinical status and the neurophysiological data was performed using Pearson's correlation coefficients and partial correlations. The statistical distribution of all the variables is tested by means of Kolmogorov and Smirnov test. The significance level is set to 0.05. Descriptive statistic is reported as Mean ± Standard Error of the Mean (SEM).Main aim of the statistical analysis is to assess the effect of gender on excitability and plasticity measures. Baseline and iTBS-dependent excitability changes were tested on RMT, AMT, MEP amplitude and on the Laterality Index (LI)  and clinical status (NIHSS) at stroke onset . Groups were also matched regarding the percentage of patients with different lesion site , a pure cortical lesion was present in 3 out of 15 female patients (20%) and in 4 out of 20 male patients (20%). This is relevant because functional changes in cortical excitability may be influenced by stroke location and distribution  and resulted 7.47 ± 0.47 for females and 7.40 ± 0.36 for males. In a subgroup of 7 females and 8 males we measured the stroke volume using the procedure described in Di Lazzaro et al. .The average NIHSS at onset was 5.21 ± 0.413. Gender groups were matched regarding age  = 4.449, p = 0.043], with a trend toward significance for RMT , not significant for MEP amplitude . The post-hoc analysis reveals that AMT is significantly lower over the UH only in the Male group (p = 0.004), not in the Female group (p > 0.200) . This effect does not differ depending on the Hemisphere . The significant iTBS by Gender interaction is motivated by a cumulative (both hemispheres together) mild increase of brain excitability for Females and decrease of brain excitability for Males  regardless of the Gender . More in details:RMT:Pre-iTBS 0.04 ± 0.04; Post-iTBS 0.03 ± 0.04, p = 0.022;LIAMT:Pre-iTBS 0.05 ± 0.04; Post-iTBS 0.03 ± 0.04, p = 0.001; after iTBS LIAMT remains negative for Female and positive for Male ;LIMEP: Pre-iTBS 0.41 ± 0.06; Post-iTBS 0.30 ± 0.08; p = 0.014.LIFemales  than in Males . Even if the comparison does not reach a significant level (Chi-square p = 0.266), the rate of iTBS-induced UH excitability decrease is higher in Males  than in Females .To better characterize iTBS effects, we also looked at the rate of subjects reporting iTBS-related effects in the two groups. The rate of iTBS-induced AH excitability increase is higher in The individual level of brain excitability and iTBS effects are reported in Supplementary Figure R = 0.328, p = 0.029) and after iTBS . This correlation pattern did not survive the correction by sex, suggesting that sex might in fact play also a role in the relationship between measure of cortical excitability and clinical status. The subsequent analysis performed independently on the two groups showed: (i) absence of correlation in the male group (ii) strong correlation in the female group. More in details, in this subgroup we confirmed the relationship between LI and NIHSS . In other words, a worst clinical condition is associated to stronger interhemispheric unbalance toward higher excitability of the UH. Additionally, both before and after iTBS higher NIHSS scores were associated to higher AH AMT .Pooling together all patients, NIHSS showed a significant correlation with the AMT LI both before iTBS  increase of brain excitability, while males a decrease of it. In the female group, there is a higher rate of increase of AH excitability than in the male group and a tendency for a lower rate of decrease of UH excitability. Again men showed a pronounced effect in UH with a partial correction of the hyper-excitability associated with a comparable increase in the excitability of AH. In women, instead, we observed a more pronounced increase in the excitability of the AH that was associated with a slight increase in excitability, and not a suppression, of UH.This is a further demonstration that the establishment of inter-hemispheric imbalance after stroke should not be given for grant, rather it is strictly dependent on patient's individuality. We recently showed that also the haplotype of Brain-Derived Neurotrophic Factor (BDNF) gene has a profound influence on the inter-hemispheric imbalance in cortical excitability (Di Lazzaro et al., vice versa in males (Gibson, Is the imbalance in cortical excitability deleterious for recovery? As we suggested for BDNF polymorphism (Di Pino et al.,  Gibson, .Moreover, the differential functional changes that take place in the AH and UH in males and females might be adaptive or maladaptive depending on the degree of corticospinal tract damage. Gender influences many aspects of stroke including risk/incidence, diagnosis, symptoms, treatment and outcomes (Reeves et al., Those considerations warrant further studies aimed at characterizing the interactions that gender and inter-hemispheric imbalance have on recovery.In conclusion, our study suggests the existence of gender-dependent differences in the functional brain changes that take place after human stroke, in that it seems that male brain has greater asymmetry than the female's. This perfectly fits the recently advanced hypothesis of a higher strength of inter-hemispheric connection owned by the female's healthy brain (Ingalhalikar et al., Male and female individuality could conceivably arise from a complex interaction of some sort of gender-specific base with a mosaic of environmental factors. Stroke and its strong plasticity-inducing potential are, in our opinion, optimal examples of events that might unveil and amplify those gender-specific differences, that otherwise might remain unrevealed. Our findings should suggest to be cautious in designing stroke studies, especially since sex differences in stroke that might affect recovery and brain plasticity probably result from a combination of factors, including elements intrinsic to the sex chromosomes, as well as the effects of sex hormone exposure, and not less important cultural and social factors (Cox et al., VD designed the study and wrote the manuscript. GP was in charge of statistic and revised the manuscript, GDP, FR, FL, LF, and FC participated to patients' recruitment, neuromodulation and revised the manuscript. All Authors approved the final version of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The United States’ multiple-payer health care system requires substantial effort and costs for administration, with billing and insurance-related (BIR) activities comprising a large but incompletely characterized proportion. A number of studies have quantified BIR costs for specific health care sectors, using micro-costing techniques. However, variation in the types of payers, providers, and BIR activities across studies complicates estimation of system-wide costs. Using a consistent and comprehensive definition of BIR , we synthesized and updated available micro-costing evidence in order to estimate total and added BIR costs for the U.S. health care system in 2012.We reviewed BIR micro-costing studies across healthcare sectors. For physician practices, hospitals, and insurers, we estimated the % BIR using existing research and publicly reported data, re-calculated to a standard and comprehensive definition of BIR where necessary. We found no data on % BIR in other health services or supplies settings, so extrapolated from known sectors. We calculated total BIR costs in each sector as the product of 2012 U.S. national health expenditures and the percentage of revenue used for BIR. We estimated “added” BIR costs by comparing total BIR costs in each sector to those observed in existing, simplified financing systems . Due to uncertainty in inputs, we performed sensitivity analyses.BIR costs in the U.S. health care system totaled approximately $471 ($330 – $597) billion in 2012. This includes $70 ($54 – $76) billion in physician practices, $74 ($58 – $94) billion in hospitals, an estimated $94 ($47 – $141) billion in settings providing other health services and supplies, $198 ($154 – $233) billion in private insurers, and $35 ($17 – $52) billion in public insurers. Compared to simplified financing, $375 ($254 – $507) billion, or 80%, represents the added BIR costs of the current multi-payer system.A simplified financing system in the U.S. could result in cost savings exceeding $350 billion annually, nearly 15% of health care spending.The online version of this article (doi:10.1186/s12913-014-0556-7) contains supplementary material, which is available to authorized users. In a well-functioning health care system, sound administration is required to ensure efficient operations and quality outcomes. In the United States however, the complex structure of health care financing has led to a large and growing administrative burden . In 1993In the U.S. multi-payer system, insurers’ coverage, billing and eligibility requirements often vary greatly, requiring providers to incur added administrative effort and cost . These pSeveral studies have used micro-costing methods — cost estimates constructed from detailed classification of resource use or expenditures — to quantify the portion of administrative costs attributable to BIR activities in physician and hospital sectors. Though the specific set of methods used to estimate this cost varies by study, the general approach has been to identify the administrative functions related to BIR activities and use clinician interviews and/or surveys to determine the proportion of work time spent on these activities. In some studies, this process has been supplemented with additional interviews with non-clinical staff ,9 and obThough studies have documented BIR costs in physician and hospital sectors, the specific analytical methods and components included in the analyses vary, rendering estimates mostly non-comparable. Thus, results cannot be easily combined into a system-wide estimate. To address this problem, we synthesized available micro-costing data on BIR costs. We use an explicit, consistent, and comprehensive definition of BIR to calculate BIR costs in well-studied sectors; estimate the portion of BIR spending in other provider sectors; and present a system-wide estimate of total BIR costs in the U.S. health care system in 2012. We also calculate potential savings from a system with simplified financing, by comparing measured BIR in US health care sectors to lower levels observed with different financing mechanisms. This paper updates preliminary information developed for an Institute of Medicine roundtable on Value and Science-Driven Health Care . It is iDrawing on U.S. National Health Expenditures (NHE), existing research and publicly reported data, we estimated total and added BIR costs in the U.S. health care system in 2012. Our estimates included the following sectors: physician practices, hospitals, private insurers, public insurers and “other health services and supplies.” We assembled micro-costing estimates of total and added BIR costs from various studies ,8, as weWe defined the sectors using categories designated as “personal health care” in the Centers for Medicare and Medicaid Services’ (CMS) accounting of NHEs, and in the case of payers, from the categories designated as “health insurance”   , we explDue to uncertainty in some sector-specific inputs, we varied the percentage of revenue for BIR for each sector to obtain a plausible range of total BIR costs. Where available, we used lower and upper bound estimates from the literature; where unavailable, we varied the estimates by up to ten percentage points, using wider variations when data was least certain, e.g., for the “other health services and supplies” sector. Varying the estimates in tandem, we obtained upper and lower bound estimates of total BIR costs across the U.S. health care system in 2012  by 5 percentage points in either direction. For the hospital sector, we calculated a new ratio of 8.1% (Canadian: U.S. BIR costs) based on published data on total (not just BIR) hospital administrative spending in the U.S. and Canada   of annual BIR costs constitutes additional spending compared to a simplified financing system. This 80% reflects 73% savings among provider sectors [Figure If clinician coding of services is excluded as a BIR activity, total and added BIR costs in physicians’ practices are reduced minimally to $65 billion and $48 billion, respectively.Using Canada’s Medicare overhead 1.8%)  instead .8%  instVarying in tandem each of the sector-specific estimates of the percentage of revenue spent for BIR yields a plausible range for total 2012 BIR costs of $330 billion - $597 billion . Non-BIR administrative activities represent an additional 9.4% , leavingOur findings update and expand on previous estimates. Woolhandler et al. estimated total administrative spending in the U.S. health care system in 1999 at $294.3 billion, with added spending of $209 billion when compared to Canada . AdjustiOur estimates of BIR costs in physicians’ practices are higher than previous studies, due to the more complete set of BIR activities, payers, and costs quantified in our analysis. Morra et al. estimated total BIR costs per U.S. physician of $82,975, translating to total and added BIR costs of $38 billion and $28 billion, respectively  analysisSeveral caveats apply to our estimates. First, BIR estimates in the published literature are most robust for physicians’ practices, with limited information available for hospitals and almost no data for categories within the sector defined as “other health service and supplies.” Hence, we explored the effect of uncertainty in our estimates in sensitivity analyses.Second, our analyses assume that BIR can be distinguished from other administrative functions. This seems a fair assumption, given that consistent findings were obtained for similar activities using varied methods. Qualitative claims by physicians of the burden of BIR lend further support to this assumption .Finally, our estimate of total and added BIR is likely conservative on three accounts. First, we assume no BIR spending outside of the direct health sector, e.g., by employers or patients. Since employment-based coverage is pervasive in the U.S., documentation of BIR costs of employers might well augment the estimates presented here. Second, for providers, we assume that costs such as public relations and marketing are incurred for non-BIR reasons. This assumption might underestimate added BIR costs, since such non-BIR administrative costs might also be lower in a simplified financing system. Finally, new evidence comparing total hospital administrative costs in the U.S. to Canada and other OECD countries suggests that added BIR for hospitals may be substantially higher than our base case estimate .Since these added costs are a function of the structure of the U.S. multi-payer system, some might characterize these costs as excess, in that that they provide little to no added value to the health care system. If BIR functions produce secondary benefits, such as enhanced quality or utilization management, the high BIR costs in the U.S. might be justified. Some research suggests, for example, that prior authorization can reduce over-utilization of brand-name medications without reducing patient satisfaction . It is aEliminating added BIR costs of $375 billion per year  would provide resources to extend and improve insurance coverage, within current expenditure levels. Since uninsured individuals have utilization of about 50% of insured individuals , the curUnfortunately, recent reforms incorporated in the Affordable Care Act (ACA) and the American Recovery and Reinvestment Act (ARRA) are unlikely to substantially reduce BIR costs and administrative burden. Data on the BIR portion of administrative costs is not yet available in the published literature. Using the BIR cost percentages identified in this analysis as a starting point, we projected BIR costs under the ACA in 2014 and 2018. Our projections were based on estimated increases in the insured population in each health sector  . AssuminMoreover, greater use of deductibles under the ACA will likely further increase administrative costs, since each claim will require processing and value adjustment before determining whether the deductible has been met. Thus, the new system will incur some new BIR costs for both the insured and uninsured portion of care. Empirical evidence from similar reform in Massachusetts is not encouraging: exchanges added 4% to health plan costs , and theWhile it was hoped that the ARRA’s incentives for adoption of health information technology (HIT) would reduce costs, partly by streamlining billing and administration , savingsA recent estimate suggests that simplifying administrative activities within the existing multi-payer system by implementing a range of standardization, automation and enrollment stabilization reforms could save $40 billion annually . While tWhile the estimates presented here should continue to be refined through additional sector-specific research on BIR costs, the cost burden of BIR activities in the existing U.S. multi-payer health care system is clear. Implementation of a simplified financing system offers the potential for substantial administrative savings, on the order of $375 billion annually, which could cover all of the uninsured  and upgr


This study was undertaken to quantitate a single virus suspension in culture medium without any pre-processing. The electrical capacitance per virus particle was used to identify the kind of virus present by measuring the suspension (virus plus medium) capacitance, de-embedding the medium contribution, and dividing by the virus count. The proposed technique is based on finding the single virus effective dielectric constant which is directly related to the virus composition. This value was used to identify the virus type accordingly. Two types of viruses thus tested were further quantified by a biochemical technique to validate the results. Furthermore, non-organic nanoparticles with known concentration and capacitance per particle were identified using the proposed method. The selectivity of the method was demonstrated by performing electrical measurements on a third virus, revealing that the proposed technique is specific and sensitive enough to permit detection of a few hundred virus particles per milliliter within a few minutes. For efficient control and treatment of viral infections, a rapid method of virus identification (within less than a minute) equipped with continuous monitoring capability is highly desirable. Most of the existing techniques require pre-processing steps which increase the cost and are highly time consuming to perform13Recently, we have identified and categorized different types of viruses by constructing a concentration-mobility relationship which should be unique for each kind of virusSC) (virus plus medium) is calculated from the addition of the two capacitances: bulk  viral capacitance (vC) and mock medium capacitance (mC):Viruses are basically complex particles composed of proteins, nucleic acids, and lipids in addition to other minor constituentsviC) can thus be computed as follows:De-embedding the relative capacitance of the mock medium from that of the suspension (virus plus medium) starts with virus volume determination which can be computed from the concentration measurements. Next, the proposed technique computes the relative medium capacitance by quantifying the virus counts inside the suspension structure, and determining its volume as well as that of the medium (since the dielectric constant of the medium is known). The corresponding average virion capacitance  or the virus suspension (virus plus medium). To probe single virus particles is quite expensive and requires dedicated tools and well-trained professional researchers. Additionally, such a process requires heavy sample preparation and single virus particle isolation. Our present technique overcomes this handicap by utilizing a conductive substance such as the liquid medium to pass the current through the virus particles and get them charged. Employing this methodology, we could by-pass the need for going through these cumbersome procedures. Thus, the methodology presented here is a lot more superior when compared to the other state-of-the-art techniques, in addition to being quick, simple and easy to handle, not to mention, very economical.SC) (virus plus medium) relative to the frequency are shown in To demonstrate the concept and operational principle of the proposed technique, two different viruses, feline immunodeficiency virus (FIV) and human immunodeficiency virus (HIV)10111210 and 3.6–6.2 × 1010 for HIV and FIV, respectivelyNanocomposix, USA) with a diameter of 120 nm and adjusted to a concentration of 2.4–5.4 × 1011, were suspended in water and their suspension concentrations were measured using the more classical approach of the Agilent 8453 ultraviolet/visible spectrophotometry (UVIS).The concentration measurements were performed and the results are shown in The effective  capacitance shown in To further analyze the electrical properties of the organic virus particles, the HIV virus stock was diluted by a factor of 2 and 4 using the mock medium. The capacitance versus frequency of the concentrated and diluted virus stocks were then measured compared to the mock control. As can be observed from The effective capacitance measured was composed of two types of capacitances added together - the main virus single particle capacitance (self-capacitance) and the virus-virus capacitance through the conductive medium . The virus-virus capacitance depends on the virus particle size and the distance between them. However, the motion of the virus particles inside the suspension exhibits a random Brownian motion, which is unpredictable and therefore could not be used to measure the distance between the individual particles. Nevertheless, if the distance between two adjacent virus particles are larger than the virus diameter, then this mutual capacitance lends itself to an open circuit capacitance; i.e., has no considerable influence on the effective capacitance, it therefore could be ignored.Next, another type of virus was examined to demonstrate the selectivity of the proposed technique. Finally, the dielectric constant and the capacitance density of the viruses under study along with silica nanoparticles were extracted from the isolated sphere capacitance condition∈0, ε and r are the free space dielectric constant, the relative dielectric constant and the radius of the spherical particle, respectively. The capacitance densities per cm2 were computed by dividing the capacitance per particle over its surface area.To exclude the geometry effect of the capacitance structure, it was prudent to identify the virus particle based on the dielectric constant rather than the capacitance itself. The dielectric constant is directly related to the particle composition. By default, each virus is unique with its own protein/nucleic acid composition. That is what makes its individual capacitance unique. Therefore, the effective dielectric constant rather than the capacitance structure of the virus was used for identification of the virus type. Moreover, with the de-embedding process, the geometry of the assay would not affect the capacitance per particle measurements, although the geometry will affect the limit of detection.The dielectric constant profiles up to 100 Hz versus frequency of all the materials tested showed a smooth behavior . The effThe presented model accounts for possible virus-virus particle interactions which are by default represented by the cumulative effective capacitance. In case of the co-existence of different types of virus particles in the same suspension (which is not the case in our study), the interactions should be re-modeled and considered specifically.In our earlier work, we have shown that different virus particles can be ascribed a “signature” value based on their electrical characterization parameters; however, since these parameters were based on “bulk” and not “per particle” values, it was possible that two different viruses could end up with the same value due to changes in their concentrations. Therefore, this study was undertaken to test whether signature sequences particular to a specific virus particle could be obtained using electrical methods on a per particle rather than bulk bases. Towards this end, two related viruses, FIV and HIV, were chosen to demonstrate the sensitivity of the current method. These viruses belong to the lentivirus group of retroviruses1212−5 and 10−6 F/cm2) as indicated in The data presented in this study have been compared with what has been reported so far in literature. For example, Gomila and colleagues recently attempted to extract the dielectric constants of different types of nanoparticles of similar shapes and sizes in the same order of magnitudes, including polystyrene, silicon oxide, and aluminum oxide using electron force microscopy−21). Furthermore, the sensitivity of the method was evaluated by diluting a known concentration of the virus with the corresponding mock control medium. The detection limit was observed to be as low as hundreds of particles per milliliter, as previously reportedThe accuracy and reproducibility of the current approach has been validated by repeating both the capacitance and concentration measurements for multiple virus stocks prepared at different times. The virus count originally was determined using a biochemical method that measures the viral nucleic acid in a suspension. The capacitance measurement was of high-precision, up to zepto-Farad  which is used to conduct electrical measurements, is a high performance, high current USB potentiostat which has 11 current ranges from 3 amps to 300 picoamps and a compliance voltage reaching to 32 volts. The measurements from the Reference 3000 can perform in a frequency range from 10 μHz to 1 MHz since it is equipped with on-board electronics for electrochemical impedance spectroscopy. For the current experiment, applied oscillation voltage of 15 mVpp was used for impedance measurement in the range of 100 mHz to 100 KHz. Earlier measurements at a high frequency from 1 MHz up to 13.5 GHz for different types of viruses (FIV and HIV), helped us to choose this frequency range. Significant identification record for different virus frequency response was obtained at a range of 100 mHz to 100 KHz, which simplified the procedure further to achieve specific 'signatures' that facilitated identifying and characterizing the different viruses and also investigating the feasibility of titer quantitation using the current method. The radiofrequency generators of the Reference 3000 are capable of producing power signals at different frequencies and also help to measure current voltage, capacitance voltage, polarization in addition to charging/discharging profiles with the ability to change different parameters. The calibration of the system was performed with the aid of manufacture-provided calibration kit2–1011 virus particle/ml in its relevant mock free medium. Viral capacitance over this range was measured and then the medium were de-embedded. When the diluted capacitance values were plotted as a function of the quantity of the virus, it exhibited a linear relationship with the intercept not significantly deviating from zero, as previously reportedTo determine the limit of detection and sensitivity, a known quantity of the virus was diluted to different concentrations ranging from 10To further establish the “specificity” of the current method, in addition to the two viruses belonging to the lentivirus group namely FIV and HIV, another more distantly related retrovirus was also tested by this methodology: MPMV. Viral particles counts were measured by the previous electrical proposed method inThe speed, accuracy and reproducibility of the current technique has been checked by repeating the electrical measurements against multiple virus stocks prepared at different times; i.e., all the measurements have been conducted within the mentioned specific frequency range and applied bias voltages. The accuracy of the presented method is comparable with other conventional techniques and may in fact be better in the lower range of detection.An algorithm was developed in our previous study to extract electrical parameters of bulk virus preparations when chargedCapacitance measurements were conducted in a coaxial adaptor connected to coaxial cables. The structure of the coaxial adaptor comprised of inner and outer conductors with dimensions of 2 and 5 mm, respectively, and a length of 7mm. The control or viral suspension media were loaded into the adaptor serving as the dielectric material. The advantage of the coaxial topology is that the radio frequency signal and the electrostatic field propagations are confined and protected from outside interferences and the signals do not escape space between inner and outer conductorstrans complementation strategy that is routinely used in the laboratory2021The virus particles used in this study were produced using a genetic The virus particles thus produced were then used for further electrical signature parameters. The number of FIV and HIV lentiviral particles released in the tissue culture media were estimated by using QuickTiter™ Lentivirus Quantitation Kit  using manufacturer's directions. This assay is valuable over other antibody-based methods since both FIV and HIV virus particles can be measured using the same method  thereby nullifying any difference in specificity and sensitivity that may arise when different assays are used. Briefly, the mock or viral supernatants obtained from transfected cultures were centrifuged (300 × g for 5 minutes at 4°C) and filtered through a 0.2 micron filter to remove any cellular debris. The clarified supernatants thus obtained were digested with nucleases to completely get rid of any residual nucleic acids from ruptured cells. Finally, the virions were lysed and the viral RNA thus released from virions was detected using a CyQuant® GR RNA binding dye using the Perkin Elmer VICTOR™ X3 Multilabel Plate Reader using a 485/538 nm filter set. The RNA standard provided in the kit for quantification provided a linear curve in the range of 1–1000 ng of lentiviral RNA which was used for the quantitation of FIV and HIV RNAs. The values thus obtained were used to determine viral titers (virus particles/ml) as described in the kit.MA conceived the concept and performed the electrical experimental work. MA, FM, and TAR supervised the project and contributed towards writing and editing the manuscript. JVK and LMA made the virus preparations. FM performed the viral quantitation assays. All authors discussed the results and commented on the manuscript.How to cite this article: Ahmad, M.A., Mustafa, F., Ali, L.M., Karakkat, J.V. & Rizvi, T.A. Label-Free Capacitance-Based Identification of Viruses. Sci. Rep.5, 9809; DOI:10.1038/srep09809 (2015).


Acute maculopathy is a rare condition of unknown aetiology and Coxsackie virus is known to be associated with this macular chorioretinitis.We report a case of acute unilateral maculopathy in a 35-year-old woman with concurrent hand foot and mouth disease. Furthermore, we display multimodal imaging  charting the course of the disease. The source of the virus was thought to be the patient's child. Empirical treatment with oral corticosteroids was commenced and the inflammation resolved, leaving a residual macular scar.We present this case combined with the review of literature of adult onset Coxsackie-virus-associated retinitis. This case reiterates the fact that Coxsackie virus is an uncommon but important consideration in the differential diagnosis of chorioretinitis and posterior uveitis with atypical retinopathy. A taxonomically diverse group of viral agents are known to affect the posterior segment of the eye, either as a primary infection or secondary to vascular sequelae . VirusesCoxsackie virus can lead to hand, foot and mouth disease (HFMD) which is a highly contagious condition predominantly affecting children . It is cWe report a case of acute maculopathy in a 35-year-old female with concurrent hand, foot and mouth disease. Multimodal imaging, including previously undescribed autofluorescence findings, displays the outer retinitis and ensuing resolution.A 35-year-old woman presented with a 1-day history of a right central scotoma and metamorphopsia which was preceded by viral exanthem and a macular rash over the hand and in the mouth. On examination, the dermatological findings were characteristic of HFMD, and there was an associated history of contact with the virus from the patient's daughter. Visual acuity was 20/20 in both eyes with right central scotoma. Fundus examination revealed right macular intraretinal haemorrhage, intraretinal thickening and retinal pigmentary changes temporal to the fovea (Figure Coxsackie virus infection has been implicated in vitritis and posterior uveitis. Concurrent systemic infection, decreasing titers of B3 and B4 Coxsackie virus with resolving systemic and ocular disease has been reported ,6. ThereAn overview of the existing literature on the adult cases with possible Coxsackie virus related to outer retinitis or uveoretinitis is presented in Table Corticosteroids were administered in our patient given the rapid deterioration in vision, and there have been conflicting reports in the literature on the use of corticosteroids in unilateral acute idiopathic maculopathy with possible viral etiology. Our experience highlights that there was no progression of the viral retinopathy after administration of corticosteroids and also there was rapid recovery of vision.This case reiterates the fact that Coxsackie virus is an uncommon but important consideration in the differential diagnosis of chorioretinitis and posterior uveitis with atypical retinopathy. A careful history of dermatological manifestations and appropriate examination should be undertaken as well as ascertainment of contact history of those with characteristic HFMD signs. In this case, autofluorescence was a useful non-invasive monitoring tool in monitoring of this outer retinal disease, and vision improved dramatically following systemic corticosteroid treatment.The patient has given consent for the report to be published.


Varroa destructor mite, and determined that a large proportion of LSV2 is found in the honey bee gut, suggesting that vector-mediated, food-associated, and/or fecal-oral routes may be important for LSV dissemination. Pathogen-specific quantitative PCR data, obtained from samples collected during a small-scale monitoring project, revealed that LSV2, LSV1, Black queen cell virus (BQCV), and Nosema ceranae were more abundant in weak colonies than strong colonies within this sample cohort. Together, these results enhance our current understanding of LSVs and illustrate the importance of future studies aimed at investigating the role of LSVs and other pathogens on honey bee health at both the individual and colony levels.Honey bees are critical pollinators of important agricultural crops. Recently, high annual losses of honey bee colonies have prompted further investigation of honey bee infecting viruses. To better characterize the recently discovered and very prevalent Lake Sinai virus (LSV) group, we sequenced currently circulating LSVs, performed phylogenetic analysis, and obtained images of LSV2. Sequence analysis resulted in extension of the LSV1 and LSV2 genomes, the first detection of LSV4 in the US, and the discovery of LSV6 and LSV7. We detected LSV1 and LSV2 in the  Apis mellifera) are the primary pollinators of US crops, valued over $17 billion per year [Nosema) prevalence and abundance [Throughout the globe, insects pollinate plant species that enhance landscape biodiversity and agricultural crops valued at $153 billion annually [per year . Therefoper year ,5. In thper year ,7,8,9,10per year ,12,13. Abundance ,15,16. Lbundance ,19,20,21bundance ,23,24.Picornavirales order [The majority of honey bee infecting viruses are small (+) sense RNA viruses of the es order ,26,27,28es order  and the es order ,23,24,30es order . These ves order . In thates order . While tes order . Since tes order , LSV-Naves order , as welles order .i.e., DxSRFD and SG amino acid motifs), and all eight conserved viral RdRp domains are present in the RdRp encoded by all sequenced LSV genomes [Nodaviridae family, which includes plant, fish, and insect-infecting viruses [Lake Sinai virus genomes were first identified via high throughput sequencing of libraries generated from RNA isolated from virus-enhanced honey bee lysates . LSVs ar genomes ,30,33. P viruses ,22,25,30 viruses ,25.Varroa destructor mites. In honey bees LSV2 levels were highest in gut tissue, readily detected in the thorax and abdomen, and present at low levels in the head. Lastly, to begin to investigate a potential role of LSVs and other pathogens on honey bee colony health, we monitored colony health  and the prevalence and abundance of common honey bee pathogens. LSV2 and LSV1 were the most abundant pathogens evaluated in this sample cohort and the abundance of these and other pathogens  was greater in weak or collapsed colonies as compared to healthy or recovered colonies. This result underscores the importance of including LSVs in future longitudinal monitoring studies aimed at determining the role of pathogens on honey bee colony health.To further characterize this group of viruses we sequenced LSVs currently circulating in Western US honey bee populations, performed phylogenetic analysis, and purified LSV2. Sequence analysis resulted in extension of the LSV1 and LSV2 genomes, extension of the LSV4 RNA dependent RNA polymerase (RdRp) sequence, and identification of LSV6 (GenBank KR021357) and LSV7 (GenBank KR021355). The presence of the replicative intermediate forms of LSV1 and LSV2 in honey bee samples were confirmed by negative-strand specific PCR. To better understand the distribution and transmission of LSVs, we determined the prevalence and abundance of LSVs in honey bee colonies located in Montana and California, examined the distribution of LSV2 in individual bees, and screened mites for LSVs. We determined that LSVs were very common and abundant in bee samples, and detected LSV1 and LSV2 in n = 203) were obtained from commercially managed colonies from October 2013 to June 2014 (n = 54). Colony strength, using colony population size as a proxy for strength, was measured at each sample collection event. We defined weak colonies as those that had less than five frames covered with bees at the time of sampling (n = 41) and strong colonies as those that had nine or more frames covered with bees at the time of sampling (n = 81). We tested these samples for 16 honey bee pathogens , Deformed wing virus (DWV), Sacbrood virus (SBV), Chronic bee paralysis virus (CBPV), Israeli acute paralysis virus (IAPV), Kashmir bee virus (KBV), Acute bee paralysis virus (ABPV), Nosema spp., Paenibacillus larvae, Melissococcus plutonius, and trypanosomatids  usina passim . There wi.e., LSV1, HQ871931, 5508 nt, and LSV2, HQ888865, 5355 nt)  . Since t5355 nt) . Currentectively . We confectively . The newi.e., Montana and California), but did not detect LSV5. We sequenced 576 nt of the RdRp encoding region of LSV4 2014MT, isolated from honey bees sampled in Montana (MT) in 2014 (GenBank KP892556). This is the first report of LSV4 in the US, and we extended the previously characterized LSV4 (GenBank JX878492) sequence by 164 nt. The overlapping 460 nucleotides (nt) of LSV4 2014MT and LSV4 are 94.3% identical . LSV7 is only 63.2% identical to LSV1 and 65.3% identical to LSV2. LSV7 is less than 62%–65.1% identical to new LSV genomes obtained from Belgian samples [To characterize the diversity of currently circulating LSVs, we sequenced the RNA-dependent RNA polymerase (RdRp) and Orf1 encoding regions . We detedentical , and 92.dentical . We sequdentical  and 98.6dentical , and cordentical . LSV6 shdentical . We also samples  , each form phylogenetically isolated lineages on this RdRp-based phylogenic tree. The 15 LSV RdRp sequences, including both full-length and partial RdRp sequences, did not cluster based on the geographic regions from which they were identified, but did cluster together into a monophyletic clade, which likely warrants designation of a new Sinaivirus genus.We examined the relationship between LSVs and other plant and animal infecting viruses by performing phylogenetic analyses based on the virus RNA dependent RNA polymerase (RdRp) amino acid (aa) sequence . The phy8 copies/1 µg RNA) and abdomen (2.72 × 107/1 µg RNA) (n = 22). Although reduced, there were readily detectable levels of LSV2 genomes in the heads (1.56 × 104/1 µg RNA) and thoraxes (1.96 × 106/1 µg RNA) of naturally infected honey bees   B.n = 50) from an LSV2-positive colony were homogenized, filtered, and subjected to cesium chloride (CsCl) step-gradient centrifugation to enrich the number of virus particles in specific fractions based on their particle density [9 and 2.72 × 108 copies, respectively   A.i.e., human keratin), a total of eight unique peptides identified the LSV2 capsid protein in the staining band  was detected  were imaged using a transmission electron microscope (TEM) at 37,000× magnification. Fraction 4 contained the most LSV2 genome copies and 5477× more LSV2 genome copies than BQCV. The TEM images indicated that LSV2 capsids are icosahedral with an average diameter of 27.7 ± 3 nm B. To furing band . The eiging band . In addidetected . This prVarroa destructor harbors LSVs, we screened individual mites for LSV1 and LSV2. We detected LSV1 or LSV2 in 13.2% and 14.7% of mites, respectively (n = 68). In one LSV2 positive colony, we detected LSV2 in 58% of the mites tested (n = 12) (To investigate if the ectoparasitic mite (n = 12) . These dCrithidia mellificae/Lotmaria passim), and the microsporidia Nosema ceranae (n = 9) and strong colonies as those that had nine or more frames covered with bees at the time of sampling (n = 11) [Nosema ceranae using pathogen-specific PCR, and performed qPCR analysis for LSV2, LSV1, BQCV, and Nosema ceranae, since the majority of samples tested positive for those pathogens (Nosema ceranae were greater in weak colonies as compared to strong colonies (Crithidia mellificae/Lotmaria passim) in weak and strong colonies  but had recovered by the end of the study had a decrease in LSV1 and LSV2 loads while one colony that was strong at the onset of the study but had weakened and was lost at the end of the three-month sampling period  had greater LSV2, LSV1, and Nosema ceranae loads at the end of the study. The data presented herein describe a small sample cohort, therefore extrapolation beyond this population is unwarranted. Additional studies are needed to better understand the effects of pathogens, including the recently discovered LSV virus group, on honey bee health and to assess their potential roles in colony loss.We monitored six commercially managed colonies located in California from January–March 2013. Pathogen-specific PCR was performed to screen for six pathogens: viruses , trypanosomatids ( ceranae . Colony (n = 11) ,42. We dathogens . LSV2 anathogens . Overallathogens , LSV1, Bcolonies ; there wcolonies . One colNodaviridae, Luteoviridae, and Tombusviridae families [et al., recently demonstrated that individual bees can be infected with LSVs representing three different phylogenetic clades [i.e., LSV1, LSV2, LSV3, LSV4, LSV6, and LSV7) from samples obtained from commercial beekeeping operations based in either California or Montana. In addition to honey bees, LSVs infect solitary bee species including O. cornuta [Lake Sinai viruses are a recently identified, globally distributed group of honey bee infecting viruses. In this study, we sought to further characterize LSVs from the molecular level to the colony level. We sequenced currently circulating LSVs in honey bee populations from Montana and California, which resulted in the extension of the LSV1 and 2 genomes. We also detected LSV4, and discovered LSV6 and LSV7. Phylogenetic analysis based on the virus RdRp sequences indicates that LSVs group in a monophyletic clade that is distinct from Chronic bee paralysis virus and other insect and plant infecting viruses of the families ,25,38,43c clades . Likewis cornuta . FurtherVarroa destructor mites are major vectors for several honey bee viruses, including DWV and IAPV [Varroa destructor mites. We detected both viruses in our samples, corroborating results obtained by Ravoet et al. [et al., did not detect negative strand LSV, suggesting LSVs may not replicate in mites; however additional studies are needed to further confirm these results and to assess the potential role of mites in LSV transmission. As expected, honey bee viruses have different tissue tropisms. To examine the distribution of LSV2, we dissected bees into head, thorax, abdomen, and gut and assessed viral quantity using qPCR. We determined that bees highly infected with LSV2 had the greatest amount of virus in the gut. Since LSVs have been detected in pollen [and IAPV ,46,47,48t et al. . Ravoet n pollen , consumpversus strong colonies. In addition, LSV1, BQCV, and Nosema ceranae abundances were also greater in weak colonies, as compared to strong colonies. Results from this pilot study illustrate the importance of examining the relationship between LSV prevalence and abundance in future longitudinal monitoring studies aimed at determining the relative effects of multiple biotic and abiotic factors on colony health.Both LSV1 and LSV2 abundance have been positively correlated with CCD . In a smDespite the high prevalence and global distribution of the Lake Sinai viruses, the pathology of these viruses is unknown. The development of infectious clones of LSVs would facilitate additional studies that aim to understand the effects of LSVs on honey bees at both the individual bee and colony levels. The data presented herein provide two important steps toward obtaining infectious clones, specifically extension of the LSV1 and LSV2 genomes and isolation of LSV2 from infected honey bees. LSV2 isolation enabled TEM imaging of this virus, which appears to have a capsid with icosahedral symmetry (~27 nm diameter). Characterization of the capsid protein will facilitate the development of antibodies that may prove useful for virus purification and rapid detection. By further describing the molecular characteristics of the LSVs and their interactions with the honey bee host, we may gain additional understanding of these viruses and their potential role in colony losses.Apis mellifera) and mites (Varroa destructor) were collected from privately owned, commercially managed honey bee colonies in Montana, California, and Indiana, US. Samples were collected into plastic bags or tubes, immediately put on dry ice, and stored at −80 °C until RNA extraction. To determine LSV tissue specificity, honey bees were dissected under sterile conditions into head, thorax, abdomen, and whole gut (from honey stomach to hindgut), and RNA was extracted as below.Honey bees (2O with two sterile borosilicate glass beads (3 mm) by bead beating for 1.5 min in 2 mL tubes, or with a TissueLyzer  twice for 2 min at 30 Hz. Bee samples were centrifuged for 15 min at 12,000×g at 4 °C to pellet debris, and supernatants were transferred to fresh 1.5 mL tubes containing an equal volume of Trizol reagent . Trizol was added directly to mite homogenate. RNA was extracted according to the manufacturer’s instructions and was suspended in sterile water.Mites (1 mite/tube) and bees (5 bees/tube) were homogenized in 200–400 uL sterile HRACE was performed for LSV1 and LSV2 using the SMARTer RACE cDNA Amplification kit  according to the manufacturer’s instructions. cDNA for RACE was prepared as directed using RNA from a natively infected bee sample (LSV1) or from RNA extracted from the LSV2 virus prep (described below). 5′ RACE ready cDNA was prepared for LSV1 and LSV2 using primers included in the kit, and PCR and nested PCR was performed using LSV-1 5′RACE R-273 and LSV-1 5′RACE nest R-428 for LSV1, and LSV-2 5′RACE R-232 and LSV-2 5′RACE nest R-155 for LSV2 . For 3′ cDNA synthesis reactions were performed by incubating 250–2000 ng total RNA, M-MLV reverse-transcriptase (Promega) and 500 ng random hexamer primers  for 1 h at 37 °C, according to the manufacturer’s instructions. cDNA was diluted 1:1 with sterile water prior to PCR or qPCR analysis.LSV1 and LSV2 positive samples were analyzed for the presence of negative-strand RNA using strand-specific RT-PCR . RNA froPCR was performed according to standard methods . In brieTo obtain additional sequence of the LSV4 RdRp, random hexamer-primed cDNA samples were PCR amplified using the Advantage 2 Polymerase mix (Clontech) using primers specific for LSV4 (LSV4-F-1896) and a primer specific for both LSV1 and LSV2 (qLSVU-R-2477), according to the manufacturer’s instructions. Cycling conditions were 94 °C for 30 s; 94 °C 30 s, 57 °C 30 s, 68 °C 30 s, 35 cycles; final elongation at 70 °C for 10 min. PCR product was directly sequenced with the same primers using standard sequencing methods.i.e., initial honey bee lysate (lysate), virus pellet from ultracentrifugation (ultra), and fractions F2*, F2, F3, and F4 from the CsCl gradient). Five hundred ng of RNA from each of these samples was reverse transcribed with M-MLV as described above. All qPCR reactions were performed in triplicate wells with 2 μL of cDNA template. Each well was a 20 μL reaction containing 1× ChoiceTaq Mastermix (Denville), 0.4 μM each forward and reverse primer, 1× SYBR Green (Life Technologies), and 3 mM MgCl2. The plate was run on a CFX Connect Real Time instrument  with a thermo-profile of a pre-incubation 95 °C for one minute, 40 cycles of 95 °C for 10 s, 58 °C for 20 s, and 72 °C for 15 s, before a melt curve for 65 °C for 5 s to 95 °C before ending. To quantify viral genome copy numbers in the samples, LSV2 or BQCV plasmid standards were used as templates, with titer ranging from 109 to 103 copies to create standard curves for each. The detection limit was 103 copies for both LSV2 (qLSV2-F-1722 and qLSV2-R-1947) and BQCV (qBQCVorf2F_6664 and qBQCVorf2R_6805) primer sets. The host gene Apis m. Rpl8 was amplified in triplicate for each sample for comparison, using primers RPL8-Fw1 and RPL8-Rev1. Wells containing no template were run as negative controls. qPCR specificity was verified through melt point analysis and via gel electrophoresis. The linear standard equations for the plasmid standards were Cp = −3.7x + 42.1, R2= 0.998 for BQCV, and Cp = −3.8x + 44.8, R2= 0.996 for LSV2.qPCR was used to analyze the relative abundance of LSV2 and BQCV in select samples representing distinct stages in virus purification . The objective for pathogen screening in our study was to identify the most prevalent pathogens associated with honey bees sampled from individual colonies at each sampling event. Based on empirical data, literature values, and practical sample handling considerations, we assayed five bees per colony per sampling event [et al., 2013, N = ln(1 − D)/ln(1 − P)  predicts that with a sample size of five bees, pathogenic infections affecting 45% or more of the individuals within a colony would be detected with 95% probability [i.e., five bees/colony per sampling event) and our ability to detect the most common honey bee pathogens from a variable number of bees obtained from a single colony  using pathogen-specific PCR (Honey bee samples (~100 mixed aged bees obtained from the top of frames in the middle of the colony) were collected at each sampling event and five female bees from each sample were used for RNA extraction, cDNA synthesis, pathogen-specific PCR and qPCR . There ang event ,20,49,50ific PCR , we perfific PCR . Based oific PCR . This reCrithidia mellificae/Lotmaria passim, Paenibacillus larvae, ABPV, IAPV, KBV, Nosema spp., and Melissococcus plutonius  was combined with CsCl to a density of 1.1 (207.9 g/L), and then overlaid on top of a CsCl step-gradient of 1.2 (263.3 g/L), 1.25 (322.7 g/L), 1.3 (386.6 g/L), 1.4 (529.6 g/L), and 1.5 (652.7 g/L) in an Ultra-Clear 14 × 89 mm tube , as it is likely that most viruses would fall within one of these interfaces [× g  using a SW41 rotor at 10 °C (Beckman Coulter Optima L-90K ultracentrifuge). The gradient was fractioned into 1 mL aliquots and each aliquot was concentrated approximately 10-fold using Amicon Ultra-0.5 centrifugal filter units NMWL 100,000 (EMD Millipore) in PBS pH 7.4. Samples were stored at 4 °C or −20 °C. In parallel, the second half of virus-containing supernatant (4 mL) was pelleted by ultracentrifugation in an Ultra-Clear 16 × 76 mm tube (Beckman Coulter) for 3 h at 76,000× g  using a Type 50 Ti rotor at 10 °C. The pellet was suspended in 100 μL PBS pH 7.4. This sample (ultra) and all gradient samples were tested again for LSV1, LSV2, LSV3, LSV4, BQCV, DWV, SBV, ABPV, CBPV, IAPV, and KBV by PCR, and the relative abundance of LSV2 and BQCV in these samples was assessed by qPCR.Viruses were isolated from honey bees obtained from a single colony  using methods similar to those described ,49. Thisterfaces . The samSamples were diluted in water, stained with 2% uranyl acetate and imaged by transmission electron microscopy (TEM) at 15,000× and 37,000× magnification. TEM Leo 912  equipped with a Proscan CCD camera.Purified virus was mixed with an equal volume of 2× Laemmli buffer, heated at 95 °C for three minutes, and electrophoresed through a 10% polyacrylamide gel at 150 V for one hour. The gel was stained in Coomassie blue overnight at 4 °C, then destained for 2–4 h at 4 °C. Bands were imaged with a SynGene G: Box gel documentation system.The Lake Sinai virus containing phylogeny was inferred using Bayesian inference ,52 as imGenBank accession numbers for either the RdRp sequences or the genome sequences from where the RdRp sequence obtained are as follows: LSV1, Lake Sinai virus 1 (HQ871931), LSV1 MT2014 (KR021356), LSV2 (HQ888865), LSV3 (AFJ04713), LSV4 (AGN98104), LSV Belgium 2015 (KM886905), LSV Belgium 2015 (KM886903), LSV Belgium 2015 (KM886904), LSV6 MT2014 (KR021357), LSV5 JR (AGU62868), LSV5 JR (AGU62866), LSV5 JR (AGU62870), LSV5 JR (AGU62872), LSV5 JR (AGU62874), LSV5 JR (AGU62876), AACV, Anopheline-associated C virus RdRp (YP_009011225), CBPV, Chronic bee paralysis virus (YP_001911137), AlphaNV, Alphanodavirus RdRp (GU976287), MoNV, Mosinovirus RdRp (AIO11151), PaV, Pariacoto virus RdRp (NC_003691), PiRV, Pieris rapae virus RdRp (AY962576), BGANV, Bat guano associated nodavirus (HM228873), BFV, Barfin flounder nervous necrosis virus RdRp (NC_011063), SJNNV, Striped Jack nervous necrosis virus ProtA (NC_003448), RSGNNV, Redspotted grouper nervous necrosis virus (AAW32087), AMV, Drosophila melanogaster American nodavirus ProtA (GQ342965), FHV, Flock house virus RdRp (Q66929), MrNV, Macrobrachium rosenbergii nodavirus RdRp (NC_005094), PvNV, Penaeus vannamei nodavirus RdRp (NC_014978), NoV Nodamura virus RdRp , LeBNV, Le Blanc nodavirus (JQ943579), OrsNV, Orsay nodavirus RdRp (HM030970), SanNV, Santeuil nodavirus RdRp (NC_015069), MNSV, Melon necrotic spot virus RdRp (53276), CrRLV, Carrot red leaf virus RdRp (YP_077186).The putative LSV2 capsid protein band  water/acetonitrile with 0.1% formic acid, which were combined and dried, then re-solubilized in 30 µL of the same 95/5 solution for HPLC-MS analysis. Injection volumes of 2 µL were processed via Dionex Ultimate 3000 nano UHPLC, with an Acclaim PepMap100 C18 column used for trapping (100 μm × 2 cm) and an Acclaim PepMap RSLC C18  for final peptide separation.In-gel digestion was performed as described in  with min2O with 0.1% (v/v) formic acid for channel “A” and 80/20 acetonitrile/water for channel “B”. Following sample trapping for 5 min at a flow rate of 10 μL/min, the HPLC valve was switched to elution position. From 0.0 to 5.0 min, the elution solvent pump composition was held at 7% B. From 5.0 to 25.0 min, the elution solvent gradient was linearly changed from 7% to 35% B. From 25 to 27 min, the gradient was ramped from 35% to 95% B. From 27 to 32 min, the solvent was held at 95% B, and from 32 to 33 min the solvent was linearly ramped from 95% B to 7% B. From 33 to 35 min, the elution solvent was held at 7% B. During the entire run, the loading pump solvent was held at 10 μL/min of 97.5% water, 2.5% acetonitrile, and 0.1% formic acid. The mass spectrometer used was a Bruker maXis Impact with CaptiveSpray ESI source: resolution is approximately 50,000 and accuracy is 1 ppm. Spectra were collected in positive mode from 100 to 2500 m/z at a maximum rate of 2 Hz for both precursor and fragment spectra, and with adaptive acquisition time for highly-abundant ions. The resulting data files were converted to mzML files with Bruker CompassXport.Chromatography was as follows: solvent consisted of HApis proteins in TrEMBL, all human proteins in SwissProt, and additional proteins including: Lake Sinai Virus 1 , Lake Sinai virus 2 , as well as from the open reading frames of two other honey bee viruses Black Queen cell virus , and DWV , and one abundant honey bee encoded protein vitellogenin  complement of the UniProtKB [Peak lists obtained from MS/MS spectra were identified using X!Tandem version X! Tandem Sledgehammer (2013.09.01.1) . The seaThe identification settings were as follows: Trypsin with a maximum of two missed cleavages; 20.0 ppm as MS1 and 0.05 Da as MS2 tolerances; variable modifications: oxidation of m (+15.994915 Da), carbamidomethyl c +57.021464 Da), acetylation of protein n-term +42.010565 Da), pyro-cmc (−17.026549 Da), pyro-glu from n-term e (−18.010565 Da) and pyro-glu from n-term q (−17.026549 Da). Peptides and proteins were inferred from the spectrum identification results using PeptideShaker version 0.37.5 . Peptide Da, acet Da, pyroApis mellifera Major royal jelly protein 2 (UnitProt/Swiss-Prot 077061) was detected. This protein has a molecular weight similar to the LSV2 capsid protein and is very abundant in bees; therefore it is likely a contaminating protein in our honey bee virus preparation. No significant non-contaminant hits were identified in the control region of the gel.Excluding known contaminants , a total of eight unique peptides identified LSV2 capsid protein in the staining band . The eig


Apis mellifera L., approximately 20% of the honey bee subspecies in the world. We use deep ILLUMINA-based RNA sequencing to capture RNA species for the honey bee and a sampling of all non-endogenous species carried by bees. After trimming and mapping these reads to the honey bee genome, approximately 10% of the sequences (9–10 million reads per library) remained. These were then mapped to a curated set of public sequences containing ca. Sixty megabase-pairs of sequence representing known microbial species associated with honey bees. Levels of key honey bee pathogens were confirmed using quantitative PCR screens. We contrast microbial matches across different sites in Turkey, showing new country recordings of Lake Sinai virus, two Spiroplasma bacterium species, symbionts Candidatus Schmidhempelia bombi, Frischella perrara, Snodgrassella alvi, Gilliamella apicola, Lactobacillus spp.), neogregarines, and a trypanosome species. By using metagenomic analysis, this study also reveals deep molecular evidence for the presence of bacterial pathogens , Varroa destructor-1 virus, Sacbrood virus, and fungi. Despite this effort we did not detect KBV, SBPV, Tobacco ringspot virus, VdMLV (Varroa Macula like virus), Acarapis spp., Tropilaeleps spp. and Apocephalus (phorid fly). We discuss possible impacts of management practices and honey bee subspecies on microbial retinues. The described workflow and curated microbial database will be generally useful for microbial surveys of healthy and declining honey bees.Honey bees face numerous biotic threats from viruses to bacteria, fungi, protists, and mites. Here we describe a thorough analysis of microbes harbored by worker honey bees collected from field colonies in geographically distinct regions of Turkey. Turkey is one of the World's most important centers of apiculture, harboring five subspecies of  Apis mellifera L.) has ecological importance as a natural pollinator of wild flora and crops. Moreover, managed honey bees have economical importance with hive products including honey, pollen, wax, propolis, and royal jelly ; pathogens (Nosema spp. and bee viruses); pesticides, contaminated water, use of antibiotics, poor nutrition, and migratory beekeeping practices  and the gamma-proteobacterium Gilliamella apicola (family Orbaceae), the dominant Gram-negative members of the gut community, with each comprising up to 30–39% of the microbiota  survey, and was conducted to determine the colony losses in many regions representing honey bee diversity in Turkey. By using metagenomic analysis, our results provide new incidence records of the virome and microbiome in search of etiologically unexpected or previously unknown agents among 10 distinct provinces in Turkey and suggest a higher viral prevalence, and increased losses, in migratory beekeeping operations. Apis mellifera is an economically and ecologically important model organism and identification of pathogens and other microbes can have extensive implications for current practices in apiculture and agriculture.The surveillance and discovery of novel pathogens via high-throughput sequencing can provide a relatively unbiased view of pathogens and microbes associated with insects and other arthropods , as described in vanEngelsdorp et al. . PathogeL. passim) primers. Positive and negative control reactions were run on each 96-well plate. Pathogen loads (ΔΔCT) were determined as the difference between the CT of RPS5 and the CT of each target (ΔCT), scaled up from the minimal ΔCT across all samples.We used a thermal profile of 95°C for 30 s followed by 95°C for 5 s and 60°C for 30 s. Steps two and three were repeated for a total of 50 cycles and included plate reads for florescence during each 60°C step. Following the cycle program, products were denatured for 10 s at 95°C., reannealed and then a dissociation profile was measured between 69 and 95°C at an increment of 0.5°C to provide evidence for reaction fidelity  DNA sequence similarity with trypanosomes, S. apis and S. melliferum, was confirmed using the BLAST search tool . Libraries were run on paired-end 100-cycle reactions and flow cells using Illumina Hi-Seq 2000 machines at the University of Maryland Institute of Genome Sciences. Sequences for all six libraries have been deposited at the US National Institutes of Health NCBI “Honey Bee Disease Database” Bioproject (PRJNA52851).High throughput sequencing was performed with the pooled samples from stationary colonies of each region and all RNA samples were pooled before sequencing  by mapping sequencing reads and counting and distributing the reads across genes and transcripts based on annotated reference genes Figure . During Hochberg  to deter™ . A One-Way analysis of variance was used to test differences between group means. The total variability in the response was divided into two parts; within-group variability and between-group variability. The differences between the group means were considered to be significant if the between-group variability was broader relative to the within-group variability. Multiple comparisons of group means were done by using pooled variance estimates for these means. Student's t-tests were computed for each pair of group levels and individual pairwise comparisons. Matrix of correlation coefficients that summarized the strength of the linear relationships between each pair of response variables was calculated and Pearson product-moment correlations for each pair of variables were listed. Correlations and the significance probabilities were calculated by the pairwise deletion method and the count values differed if any pair had a missing value for either variable.Real time q-PCR data statistics were performed by using JMPN. apis (except for only three colonies in 2011) by RT-PCR. Present in these samples were Acute bee paralysis virus (ABPV), Deformed wing virus (DWV), Black queen cell virus (BQCV), and N. ceranae. Mixed infections of ABPV, DWV, BQCV, and N. ceranae were detected (Tables Kashmir bee virus (KBV), Israeli acute paralysis virus (IAPV), American foulbrood (AFB) and Sacbrood virus (SBV) were not detected by PCR in any of the samples, nor was the microsporidian pathogen N. ceranae loads were significantly different among the regions .The distribution of bee pathogens was significantly different among provinces and with beekeeping practices in 2010 . Specifically, trypanosome loads were highest in spring as opposed to fall. In Ardahan and Hatay, trypanosomes were not detected among any of the fall samples. Among the samples with trypanosome infection, thirteen 2010 samples for which products were assayed by DNA sequencing were all confirmed as being part of the “SF” strain of L. passim (identified as C. mellificae) . ABPV loads were correlated with both DWV  and N. ceranae . BQCV correlated with DWV within samples from Ankara, Bitlis, Edirne, and Muğla. In Bitlis, ABPV levels were positively correlated with DWV . In Hatay, BQCV correlated with N. ceranae . Among 2011 samples ABPV showed correlations with DWV  and in some regions with N. ceranae. A positive correlation was observed between trypanosome load and N. ceranae  and between trypanosomes and BQCV .Dual infections of DWV and BQCV were detected in both migratory and stationary beekeepers in 2010 (50–100% of honey bees in six provinces sampled). Dual infections with these two viruses were less commonly observed in 2011 (18–40% of honey bees in four of the provinces sampled). Triple infections of ABPV, BQCV, and DWV were seen in three regions (50–100% of honeybee samples) in 2010, whereas occurred less (18–40% of honey bee samples) at four regions in 2011. Among 2010 samples, According to the survey results, there was an increase in colony losses in 2011 when compared with 2010 losses Figure . KırklarVarroa destructor virus-1 as well, a first for these regions. Overall, the DWV/VDV group of iflaviruses was the predominant viral taxon  and Israel Acute Bee Paralysis Virus (IAPV) were not detected in sampled colonies by RT-qPCR, while the related virus Acute Bee Paralysis Virus (ABPV) was abundant in most of the samples and was present in the regions where colony declines were observed. Historically, KBV has been found most frequently in the United States and Australia  were fairly abundant in Hatay, Yığılca, and Muğla. CBPV reads were relatively rare in most of the regions and were not detected in Yığılca and Ardahan. CBPV was reported in seven of 28 (25%) samples from six provinces of Black Sea region in Turkey  were especially prevalent. While DWV amounts were higher, reads for the closely related In this study, despite tens of millions of microbial gene reads, few reads matching IAPV, KBV, and SBV were seen. IAPV, KBV, and SBV were not detected using RT-qPCR, indicating that RNA-seq sensitivity was higher than that of qPCR, or that current qPCR primers for this group must be redesigned to capture all strains. Given the RNA-Seq data, we can consider that these pathogens are quite rare in our samples. KBV has been absent in some other European surveys  was not detected within our samples, nor was VdMLV (Varroa Macula-like virus). A plant-pathogenic RNA virus, tobacco ringspot virus (TRSV) was not detected in any of the RNA samples in our study.Apis mellifera from several continents  persisted at extremely low levels among regions in this study. Among arthropod parasites of honey bees, we found no genetic evidence for the presence of the tarsonemid tracheal mite, Acarapis woodi within our samples. The Asian parasitic mite Tropilaelaps is considered more dangerous to A. mellifera than the parasitic mite Varroa destructor  in the study of Ravoet et al. , protists , viruses , symbionts , microsporidia and fungi in A. mellifera colonies in distinct regions of Turkey. The presence of KBV, SBPV, Tobacco ringspot virus, VdMLV (Varroa Macula like), Acarapis spp., Tropilaelaps claerae and Apocephalus (phorid fly) were also examined. As in other countries, bee viruses were correlated with colony losses in Turkey. In comparison with 2010, the increase in pathogen loads in 2011 might be a factor for increased colony losses observed in this study. It seems likely that migratory beekeeping practices enable the spread of disease factors among honey bees in places where they visit and causing an important threat to the honey bee colonies. In addition, the impacts of parasites and pathogens varies between regions, perhaps reflecting different honey bee genetic traits. Migratory beekeeping was correlated with both higher disease loads and a potential risk of dispersing regional parasites and pathogens across the country. This practice also allows for greater gene flow between migratory honey bee populations and local populations. Current diversity and local genetic structure can be preserved with selection strategies and establishing broad areas of isolation to reduce the risks of migratory beekeeping practices. While experimental work and longitudinal analyses will be needed to confirm causes of bee declines, our analyses, reference sequences, and strategy will help reduce the set of likely causes.In conclusion, we screened bee-derived RNA against the most complete sequence set for honey bee associates used to date. We assessed levels of known and novel parasites, pathogens, and symbionts. We present quantitative data for bacterial pathogens (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.


Ebola virus disease (EVD) has mostly affected economically deprived countries as limited resources adversely affect a country’s infrastructure and administration. Probing into the factors that led to the widespread outbreak, setting forth plans to counter EVD cases in developing countries, and devising definitive measures to limit the spread of the disease are essential steps that must be immediately taken. In this review we summarize the pathogenesis of EVD and the factors that led to its spread. We also highlight interventions employed by certain countries that have successfully limited the epidemic, and add a few preventive measures after studying the current data. According to the available data, barriers to prevent and control the disease in affected countries include irresolute and disorganized health systems, substandard sanitary conditions, poor personal hygiene practices, and false beliefs and stigma related to EVD. The public health sector along with the respective chief authorities in developing countries must devise strategies, keeping the available resources in mind, to deal with the outbreak before it occurs. As a first step, communities should be educated on EVD’s symptoms, history, mode of transmission, and methods of protection, including the importance of personal hygiene practices, via seminars, newspapers, and other social media. A popular opinion leader (POL) giving this information would further help to remove the misconception about the nature of the disease and indirectly improve the quality of life of affected patients and their families.The online version of this article (doi:10.1186/s40249-015-0048-y) contains supplementary material, which is available to authorized users. Please see Additional file The first Ebola virus disease (EVD) outbreak occurred simultaneously in Nzara, Sudan (involving 281 patients out of which 151 died 54%]) . The fir%) . The We conducted a narrative clinical review on the epidemiology, transmission, clinical manifestation, diagnosis, and prevention of Ebola virus. We searched for online articles using MEDLINE® and Google Scholar. The keywords/phrases used were: “Ebola virus disease”, “Epidemiology of Ebola virus disease”, “Mortality and morbidity associated with Ebola virus disease”, “Pathogenesis of Ebola virus disease”, “Clinical manifestation of Ebola virus disease”, and “Prevention of Ebola virus disease”. Articles dated from 1976 to 2015 were included in the study. Articles were reviewed from November 2014 to March 2015. Search results yielded a total of 174 articles out of which 52 articles were included in this study.Filoviridae, which compromises three genera: Cuevavirus, Marburgvirus, and Ebolavirus. This family belongs to the order Mononegavirales. Five species of Ebola virus have been identified: Zaire, Sudan, Bundibugyo, Reston, and Taï Forest. Bundibugyo ebolavirus, Zaire ebolavirus, and Sudan ebolavirus have been associated with large outbreaks in Africa. The current West African outbreak was caused by Zaire ebolavirus, which is also the most virulent among the five species. Since 1976 it has caused multiple outbreaks in Central Africa, with a mortality rate of 55%–88% [Ebola virus belongs to the family  55%–88% -14. The  55%–88% .Pteropodidae families of fruit bats are thought to be the natural reservoirs of Ebola virus . Humans The Center for Disease Control and Prevention (CDC-US) and the WHO have recommended that infected individuals should be quarantined for 21 days. The incubation period of Ebola virus is 2–21 days after infection. Latest studies have shown that Ebola virus transmission occurs when there is a high viral load of bodily fluids . The perThe initial symptoms of EVD include fever, headache, fatigue, sore throat, and muscle pain, which are followed by anorexia, nausea, diarrhea, vomiting, rash, abdominal pain, cough, shortness of breath, postural hypotension, edema, headache, confusion, and coma . In someZaire ebolavirus is most likely due to lymphocyte apoptosis via Fas/FasL interaction [Ebola virus usually reaches detectable levels in blood after three days of symptoms . A negateraction . The exceraction . Accordieraction .According to an estimate, 2–8 patients are exported from the three major Ebola-inflicted countries in Africa monthly, with 64% of the destinations being economically deprived countries . This poAccording to the available data, barriers to prevent and control the disease in affected countries include irresolute and disorganized health systems, substandard sanitary conditions, poor personal hygiene practices, and false beliefs and stigma related to EVD . There aAs a first step, communities should be educated on EVD’s symptoms, history, mode of transmission, and methods of protection, including the importance of personal hygiene practices, via seminars, newspapers, and other social media. A popular opinion leader (POL) giving this information would further help to remove the misconception about the nature of the disease and indirectly improve the quality of life of affected patients and their families ,38. In aAt state level, funds for proper sanitation, especially in rural areas, should be reserved. Measures should be taken to ensure the availability of water and electricity in all areas. Internet and telephone services should be made available round the clock in all hospitals and healthcare facilities for effective communication, and for reporting and enrolling EVD cases. Home protective kits can further enhance control measures, thus their distribution at household-level should be considered .Incident management systems (IMSs), such as the one adopted by the CDC for the control of the current epidemic, have proven efficacious in preventing the spread and adequately controlling the disease ,43. A reMany drugs are being probed as preventive medications for EVD, such as amiodarone, chloroquine, and clomiphene . An effeThe public health sector along with the respective chief authorities in developing countries must devise strategies, keeping the available resources in mind, to deal with the outbreak before it occurs.