The unprecedented 2015 outbreaks of highly pathogenic avian influenza (HPAI) H5N2 in the U.S. devastated its poultry industry and resulted in over $3 billion economic impacts. Today HPAI continues eroding poultry operations and disrupting animal protein supply chains around the world. Anecdotal evidence in 2015 suggested that in some cases the AI virus was aerially introduced into poultry houses, as abnormal bird mortality started near air inlets of the infected houses. This study modeled air movement trajectories and virus concentrations that were used to assess the probability or risk of airborne transmission for the 77 HPAI cases in Iowa. The results show that majority of the positive cases in Iowa might have received airborne virus, carried by fine particulate matter, from infected farms within the state  and infected farms from the neighboring states . The modeled airborne virus concentrations at the Iowa recipient sites never exceeded the minimal infective doses for poultry; however, the continuous exposure might have increased airborne infection risks. In the worst-case scenario , 33 Iowa cases had > 10% (three cases > 50%) infection probability, indicating a medium to high risk of airborne transmission for these cases. Probability of airborne HPAI infection could be affected by farm type, flock size, and distance to previously infected farms; and more importantly, it can be markedly reduced by swift depopulation and inlet air filtration. The research results provide insights into the risk of airborne transmission of HPAI virus via fine dust particles and the importance of preventative and containment strategies such as air filtration and quick depopulation of infected flocks. The outbreak was eventually under control after massive culling; but how the disease was spread among the farms remains unknown. As traditional biosecurity protocols for preventing direct contact and vector transmissions proved unsuccessful, airborne transmission – a mechanism by which virus is carried in fine particulate matter (PM) and aerially transported from infected to recipient farms – received increasing attention and concern. Anecdotal evidence in 2015 claimed that bird mortality started to increase near the air inlets of the inflected poultry houses. Recent studies showed that AI virus was airborne transmissible in a ferret model4. Some vigilant producers have installed or planned to install incoming air-treatment systems to reduce the risk of airborne transmission of AI virus, even though such transmission pathway remains scientifically explored. This is the essence and motivation of our study described in this paper – investigating the risk of AI airborne transmission by making use of the 2015 HPAI data and offering science-based guidance for implementing effective prevention or containment strategies5.The devastating outbreaks of highly pathogenic avian influenza (HPAI) in the U.S. in 2015 caused significant economic losses to the poultry industry and disruption of egg supply (shortage and price hike). In less than 7 months, 232 farms in 15 states and over 50 million birds were affected6. While air sampling is a direct method to investigate local and regional transmissions7, it is not suitable for delineating long-distance transmission because of its inherent limitations such as insensitivity of the samplers8, sampling stress imposed to target pathogens9, and difficulty in selecting sampling locations due to varying wind direction. As such, long-distance transmission of pathogens in large scale has been often examined with various meteorological models. Among the various models, the Hybrid Single Particle Lagrangian Integrated Trajectory (HYSPLIT) model is frequently used for animal pathogens like foot-and-mouth disease virus11, blue tongue virus12, etc. The major advantages of the HYSPLIT model lie in its flexibility to almost all inhomogeneous and time-varying meteorological conditions as well as non-flat terrain13, and its ability to project backward trajectories and visualize origins of viral particles14. As an incorporated module, it accounts for biological decay of pathogens under different environmental conditions that improves the modeling accuracy11. Air trajectory and concentration modelings are the core modules of the HYSPLIT model.Efforts have been made to recover airborne AI virus downwind of infected farms by using aerosol samplers, as done during the 2015 outbreak16, all dealt with cases within small areas (5–25 km radius). However, the airborne viral particles could be transported over hundreds of kilometers, thus resulting in much broader impacts18. To address the long-distance airborne transmission of HPAI, we collected information of all 232 infected cases spanning 15 states in the U.S. during the 2015 HPAI outbreak, and examined airborne transmission of AI virus from these cases to the 77 infected cases in Iowa . We modeled the air movement trajectories from the previously infected farms to the recipient farms, and estimated virus concentrations that were used to assess the probability of airborne transmission for the Iowa cases. Factors affecting the probability of airborne transmission were investigated. We also examined the effectiveness of airborne transmission risk-reduction strategies, including swift depopulation of the infected flocks (within 24 h ) and filtration of incoming ventilation air at the recipient farms. While this study focuses on airborne transmission only, it is not our intention to rule out any other possible transmission modes, e.g. direct, vector, or combined transmissions.Modeling studies of airborne transmission of AI virus among farms are scant. Of the limited published studiesInformation of 232 infected poultry farms was obtained from the Emergency Management Response System (EMRS) of USDA Animal and Plant Health Inspection Service (APHIS). Each record or infected farm was identified by farm name, physical address, county and state, infection confirmation date, depopulation date, and number of birds infected. In this study, we focused on the intra-state and inter-state airborne transmission for all 77 cases in Iowa Table . Only inBackward trajectory modeling was performed for Iowa cases to examine if they received air passing other infected farms before they became infected. Considering the incubation period could be as long as 21 days (communication with Dr. James Roth at Iowa State University), air trajectories of 21 days prior to the infection confirmation date were investigated for each case. As temporal wind speed and direction varied, modeling was done at 8-hr time intervals on each day, yielding three trajectories covering the air arrival time at 8 am, 4 pm and 12 am Local Standard Time (LST). Incoming air to the recipient farms was examined at the height of 1.5 m above ground for turkey farms and 3.0 m for hen farms, considering the height of the typical air inlets. These heights were considered because air entered most turkey growing houses through ventilation curtains surrounding side walls while air entered typical hen houses through eave inlets. Backyard farms were treated as turkey farms, and pullets  farms were treated as hen farms. The trajectory was exported as.kmz file, and loaded in Google Earth for further analysis. With backward trajectory modeling, we projected air movements showing clear patterns that viral particles could have been transported from originating infected farms to recipient farms. This finding necessitated concentration modeling to look at whether the amounts of virus released from the infected farms were high enough to cause infections at the recipient farms.10  and PM2.5  were estimated using both default and ceiling input values in the HYSPLIT model  for turkeys21 and 103.5 EID50 for laying hens22. Lung capacity and respiratory rate are 7.7 × 10−5 m3 and 2.4 × 103 times/h for turkeys, and 1.4 × 10−5 m3 and 1.6 × 103 times/h for laying hens, respectively24. The resultant MIDs were 10 EID50/m3 for turkeys and 281 EID50/m3 for laying hens over a 21-day exposure.The MIDs were calculated based on the infective dose, lung capacity, respiratory rate, and exposure time. The infective doses were 1025. The probability of farm infection (Ph) depends on the probability of individual-animal infection (Pi) at a certain dosage (d) and flock size (nf). The probability that one EID50 of AI virus will infect an animal (θ) has not been reported. By conservatively assuming 95% of birds would get infected at the aforementioned infective doses, θ was 0.00069 for turkeys and 0.00022 for laying hens.A binomial distribution was used to calculate the probability of farm infection, as described in previous studiesnd), we used the following formula.To take into account that a flock may be exposed for multiple days /m3  of PM10 was much greater than that of PM2.5 , 45 (58%) and 25 (33%) of the Iowa cases in medium, low, and extremely low risk category, respectively , 30 (39%), 33 (43%) and 11 (14%) of the Iowa cases were classified in the high, medium, low, and extremely low risk category, respectively  would further inactivate the influenza virus36. Ultimately, the trajectory modeling showed air movements from the originating infected farms to the recipient farms, which warranted concentration modeling for risk analysis.Our backward air trajectory modeling revealed the possibilities of both intra-state and inter-state airborne transmissions for the Iowa cases. The inter-state transmission was associated only with nearby states . Other states with reported positive cases were excluded from the Iowa cases because the air trajectories either did not pass the sites under consideration or the positive cases ceased to emit virus after depopulation. In this study, air trajectories were projected for 1-day periods because we assumed that the airborne AI virus would significantly lose its viability within this time frame. Though the survival of airborne AI has not been well investigated, studies on human or pig adapted influenza A virus subtypes have revealed pronounced reductions (~10 fold) in their viability in the air within a day37. Research has been increasingly focusing on small PM, e.g. PM10 and PM2.5, because smaller particles, as compared to larger ones, remain airborne for longer time and can deposit deeper in human and animal respiratory systems, thus inducing severer health risk. In this study, the estimated concentrations of virus carried by PM10 were much higher than those by PM2.5. Although PM2.5 may be aerially transported farther due to its lower deposition velocity as compared to PM1039, the amount of virus carried is much less. It has been reported that pathogens carried by a particle is proportional to the particle volume40, implying that a spherical particle of 2.5 µm would have only 1.6% holding/carrying capacity of a 10 µm counterpart. As a result, we noticed that the modeled impact area (with virus concentration higher than 10−7 EID50/m3) for PM10 was much greater than that for PM2.5  could still have experienced high to medium probability of airborne infection. It suggests that the infection probability model is more sensitive, thus conservative, for assessing airborne infection risk than the MID model.Two generally accepted principles govern the infection development in animals. First, infection occurs when an animal receives the amount of pathogen exceeding its MID. Second, a single virus particle is sufficient to infect an animal at a certain probability of infection22. Furthermore, turkey barns are more exposed to ambient conditions because of the open sidewalls and no air impaction along the pathway; whereas egg-type poultry barns are totally enclosed and employ eave/attic air inlets that can create impaction to incoming particles, thus reducing the amount of particles entering the animal space. These structural differences likely contributed to the facts that more turkey farms were inflected and were prone to higher infection probability than the egg-type poultry facilities.While virus concentration plays a key role in the infection probability, differences in susceptibility to virus among poultry types matter as well. The MID for turkeys is about one third of that for egg-type birds42. As distance from a source farm increases, virus/particle concentrations decreased significantly, thus reducing the probability of airborne infection.Other factors affecting probability of infection include flock size and distance to the nearest-infected premises. Larger flocks were found to have greater chance of infection, assuming all birds are equally susceptible to the HPAI virus. Therefore, albeit experiencing similar modeled virus concentrations to the turkey farms, all five infected backyard farms were at low infection risk of airborne transmission because of the small bird flock sizes Fig. . Both plAs soon as a contagious disease is confirmed at a farm, a regulatory control area will be established surrounding the infected premise. Farms within the control area need to be depopulated or quarantined, and movements of animals and animal products within this area are prohibited or restricted. The minimal size of the control area is 10 km in radius. Our findings reveal that the minimal control area would cover the cases in the high and medium risk categories of airborne infection.43. Whole-house thermally-assisted ventilation shutdown depopulation (VSD) could be used as a backup plan44. Execution of these guidelines might have played a key role in taking fast control of subsequent AI outbreaks in the U.S. Our results also suggested that 24-h depopulation could significantly reduce the probability of infection at the recipient farms. Besides the governmental guidelines for controlling airborne transmission, some egg producers installed or plan to install air filtration systems to reduce the virus particles in the incoming air to prevent AI infection. Our group has been investigating the removal efficiency of an air filtration system with a commercial laying-hen house, and our results to date indicate a reduction rate of up to 75% in incoming particles (depending on the season)33. A 75% removal efficiency would help to reduce the infection risk, but 7 cases (9%) are still in medium risk . This delayed depopulation may have increased the chance of airborne transmission because the HPAI virus kept releasing to ambient environment through ventilation. Based on lessons learned from the 2015 HPAI outbreak, the U.S. Department of Agriculture (USDA) requires that infected birds be depopulated within 24 h of confirmationisk Fig. . To redu46. It is possible that some birds breathe in more viral particles than others47. But for infection risk assessment in this study, all birds were assumed to receive or breathe in the same viral dosage. Thirdly, the HYSPLIT model is helpful to understand the possibility and risk of airborne transmission among farms, but an affirmed airborne transmission cannot be concluded without the genetic analysis, e.g. genome sequencing and molecular network analysis50.While the HYSPLIT model is a useful tool to address potential long-distance airborne transmission of AI virus, it has limitations. Firstly, the horizontal resolution of 12 km makes it less accurate to estimate the virus concentrations within 12 km of an infected farm. Our data show that 44% of the case-days  had the infected and recipient farms being <12 km apart. For these case-days, even if the infection risk of the recipient farm fell in low-risk category or even if the virus concentration at the recipient farm was lower than the MID, special attention is needed because the virus concentrations could have been underestimated. Secondly, virus is assumed to be evenly distributed in the air, like gas, in the HYSPLIT model. However, the majority of the airborne virus is actually attached to dust particles, resulting in non-uniform distribution of the virus in a microenvironment−5 EID50/m3). Such viral levels would be extremely challenging, if not impossible, to detect with currently available air samplers. Our attempts to assess airborne transmission by air sampling downwind of three infected farms in Iowa failed isolating any viable virus . The modeling in the current study suggests that some Iowa cases could have been exposed to high risk of airborne transmission. Thus, air sampling may not be a suitable or reliable method to evaluate long-distance transmission and caution should be taken when interpreting negative air samples because they may not indicate the truly bio-safe aerial environment.The modeled airborne viral concentrations in Iowa during the 2015 AI outbreak were extremely low , the deposition efficiency may vary.Because the processes of AI airborne transmission – virus shedding, aerosolization, transportation, deposition, and infection – are not fully understood, this study relied on assumptions based on the best available knowledge. We assumed that all virus was only shed through feces, although it is also present in poultry’s respiratory tract and thus may be released through respirationIn conclusion, air movement and airborne transmission of HPAI were investigated for 77 Iowa cases of the 2015 outbreak using the HYSPLIT model. During the HPAI outbreak in Iowa, air moved from the infected sites to the recipient sites that were subsequently confirmed positive. The modeled virus concentrations largely depend on the values of the input parameters, such as virus shedding rate, emission rate and half-life. The estimated virus concentrations did not exceed the MIDs for poultry. The infection probability by airborne transmission was generally low, although 33 Iowa cases were at medium to high risk of airborne transmission under the worst-case scenario. The infection risk seems affected by the house type (open sided vs. fully closed), flock size, and distance to the previously infected farm. The risk of AI airborne transmission may be considerably reduced by inlet air filtration and/or fast depopulation of inflected flocks .Supplementary InformationMovie S1Movie S2


OBJECTIVES/SPECIFIC AIMS: We sought to investigate the role of the host microbiome during severe, acute respiratory infection (ARI) to understand the drivers of both acute clinical pathogenesis. METHODS/STUDY POPULATION: Nasopharyngeal swabs comprised of mixed cell populations at the active site of infection were collected from 192 hospitalized pediatric patients with ARI. We combined comprehensive respiratory virus detection and virus genome sequencing with 16S rRNA gene sequencing to evaluate the microbial content of the airway during ARI. This data was coupled with 11 clinical parameters, which were compiled to create a clinical severity score. The microbiome profiles were assessed to determine if clinical severity of infection, and/or specific virus was associated with increased clinical severity. RESULTS/ANTICIPATED RESULTS: We identified 8 major microbiome profiles classified by dominant bacterial genus, Moraxella, Corynebacterium, Staphylococcus, Haemophilus, Streptococcus, Alloiococcus, Schlegelella, and Diverse. Increased clinical severity was significantly associated with microbiome profiles dominated by Haemophilus, Streptococcus, and Schlegelella, whereas Corynebacterium and Alloiococcus were more prevalent in children with less severe disease. Independent of the microbial community, more than 60% of patients with the highest clinical severity were infected with either respiratory syncytial virus or rhinovirus. DISCUSSION/SIGNIFICANCE OF IMPACT: Our results indicate that individually and in combination, both virus and microbial composition may drive clinical severity during acute respiratory viral infections. It is still unclear how the complex interplay between virus, bacterial community, and the host response influence long-term respiratory impacts, such as the development of asthma. Nonetheless, during ARIs therapeutic interventions such as antibiotics and probiotics may be warranted in a subset of patients that are identified to have both a virus and microbiome profile that is associated with increased pathogenesis to limit both acute and long-term phenotypes.


Red-skinned pears are attractive to consumers because of their aesthetic appeal and the antioxidant-associated health benefits provided by the anthocyanins in their red skin. In China, the ‘Red Zaosu’ (RZS) red bud mutation of the Zaosu (ZS) pear has been used as a parent in Asian pear breeding to generate new cultivars with crispy red fruit and red tender shoots resembling those of the ‘Max Red Bartlett’ (MRB) pears.In this study, a segregation ratio of 1:1 was observed between plants with red or green shoots in four families with RZS as the only red shoot gene donor parent, suggesting that the red shoot trait of RZS is associated with a dominant gene. Three markers, In1400–1, In1579–1 and In1579–3, were chosen from 22 pairs of indel primers targeting regions in the vicinity of the previously identified red fruit skin locus of MRB and were able to effectively distinguish the eight red shoot plants from the eight green shoot plants. Linkage analysis indicated that the genetic distance between the two marker loci (In1579–1 and In1579–3) and the red shoot locus of RZS were both 1.4 cM, while the genetic distance between the In1400–1 marker and the red shoot locus was 2.1 cM. The physical position of the red locus in RZS should be in the 368.6 kb candidate interval at the bottom of LG4.The genetic locus responsible for the red tender shoots of RZS was located in the same interval of the red fruit skin gene of MRB, meaning that the bud mutation loci of RZS and MRB may be the same or adjacent to each other, and the red shoot trait and the red fruit skin trait in RZS may be controlled by the same, or a closely linked locus. As a result, breeders could use red shoots as a morphological marker to select for the red-skinned hybrids from RZS families.The online version of this article (10.1186/s41065-018-0063-7) contains supplementary material, which is available to authorized users. Pyrus spp.) is one of the main fruit trees in China and a popular fruit worldwide as a result of being nourishing, delicious and juicy. Pears are usually diploid with a genome size ranging from ~ 527 Mb [The pear  [The ‘Max Red Bartlett’ (MRB) pear, which has red tender shoots and fruits, is a red mutant cultivar of the famous European ‘Bartlett’ pear and is often used as a parent to breed new cultivars because of the strong inheritance of its bright red fruit skin , 16. Theoup (LG) . The traoup (LG) .‘Zaosu’ (ZS) is a famous pear cultivar in China with green fruit skin and green shoots. ‘Red Zaosu’ (RZS), which has red tender shoots and fruits such as MRB, was a mutant of ZS found in Weibei, Shanxi province in China in 2004 . We crosIn pears, most of the structural and regulatory genes responsible for anthocyanin biosynthesis have been cloned and functionally characterized , 23. In PyMADS18 was found to participate in the regulation of anthocyanin synthesis during the early stages of fruit development [MYB10 was significantly higher in the full-red mutants (MRB and ‘Early Red Doyenne du Comice’compared) than in their green parents (‘Bartlett’ and ‘Green Doyenne du Comice’) at early stages of development [MYB10 (PcMYB10 and PyMYB10) promoter might be associated with red-/green-skinned mutants [PcMYB10 was located on LG9, but not LG4, of the Red locus and is unlikely to be directly responsible for the mutation of MRB [In the European pear ‘Early Red Doyenne du Comice’ and ‘Green Doyenne du Comice’, elopment . MYB10 ielopment , 27. Furelopment , 29. The mutants , 21. Hown of MRB .There had been several reports regarding the transcriptomic and proteomic level describing the bud mutation mechanisms of RZS , 30, 31,2 and (Kousui×Huoba)3. Eight red shoot F1 individuals and eight green shoot plants were randomly selected from the progeny of a family of ‘PremP109’ × RZS cross and used to screen polymorphic primer pairs between red and green shoot plants. From the ‘PremP109’ and RZS cross, 144 progeny  were collected to be analyzed with the two parents to map the red shoot trait.Four hybrid populations were used in this study to analyze the genetic regulation of the red tender shoot trait of RZS Table . The seeYoung leaves of two parents and 144 seedlings from the ‘PremP109’ × RZS cross were collected in April 2017. The leaves were immediately frozen in liquid nitrogen and transferred to a − 80 °C freezer. Genomic DNA of the young leaves was extracted using a combination of the cetyltrimethylammonium bromide (CTAB) method and a centrifugal column . DNA intRed [Red locus in the genetic map of ‘Bayuehong’ × ‘Dangshansu’ could be estimated according to its relative position to the two common SSR markers on the two LGs. Additionally, the physical interval of the Red locus could also be estimated according to physical positions of the SNP markers (CTG1064355 and CN900214) flanking the locus.The previously published SNP and SSR high density linkage map of ‘Bayuehong’ × ‘Dangshansu’  was usedRed , was aliRed . The genRed gene was estimated, and the nearby corresponding SNP loci (CTG1064355 and CN900214) on LG4 of the ‘Bayuehong’ × ‘Dangshansuli’ map were matched to the corresponding genomic intervals. The indel markers in the nearby interval of the Red gene locus were selected from the previously constructed indel marker database (http://genedenovoweb.ticp.net:81/pear/) for subsequent use. The indel database, which covers the whole pear genome, was previously developed by re-sequencing 17 red and non-red pear varieties (data not published).According to the results of the linkage group comparison, the general physical interval of the 2+ plus), 0.2 mM dNTPs, 0.5 U of Taq polymerase , 0.3 μM each primer, and 20 ng of genomic DNA. The PCR conditions consisted of the following: pre-denaturation (98 °C for 20 s) followed by 29 cycles of denaturation (98 °C for 10 s), annealing (55 °C for 30 s), extension (72 °C for 45 s), and a final extension cycle (72 °C for 10 min).Primers were preliminarily screened using 8 red shoot individuals and 8 green shoot individuals from the ‘PremP109’ × RZS cross. Each sample was run in 20 μL of PCR reaction mixture containing 1× reaction buffer   and usinP < 5%). The 1:1 segregation ratio of the red shoot trait in three RZS families indicated that the red shoot trait in RZS was controlled by one dominant gene.Four families derived from crosses between RZS and a green shoot parent were used in segregation analyses Table  & Fig. 1Red locus on the two LGs represent a geometric sequence, the Red locus was estimated to be located at around the 57.8 cM , which was built by our team for subsequent use. The specific primer information is shown in Table SNP loci within the 51.7 cM ~ 70.3 cM interval on the LG4 of ‘Bayuehong’ × ‘Dangshansuli’ Fig.  were mapTwenty-two indel primers were used in preliminary screening of the 8 red shoot and 8 green shoot seedlings of ‘PremP109’ × RZS. Three markers, In1400–1, In1579–1 and In1579–3, could distinguish red from green hybrid plants. This result suggested that these three markers and the red shoot trait in RZS were in linkage disequilibrium, and the three markers were further used to test 144 hybrids  was located at 1.4 cM in the linkage map, and the rest of the markers were located at the opposite ends of the linkage group. The markers In1579–1 and In1579–3 were closest to Red at a distance of 1.4 cM, while the marker In1400–1 was farthest from Red at a distance of 2.1 cM.A 3.5 cM linkage group, including the molecular markers and the red shoot trait locus in RZS, was identified, as shown in Fig. Red locus in RZS was located between marker In1400–1, which was identified in scaffolds NW_008988400.1 (scaffold370.0), while markers In1579–3 and In1579–1, both belonged to NW_008988579.1 (scaffold545.0). These two scaffolds neighbored each other on the bottom of LG4. Therefore, the Red loci of RZS should be in the 368.6 kb candidate interval at the bottom of LG4, and the target candidate interval can be further divided into two subintervals: a 207.1 kb interval from 100,223 bp to 307,311 bp in scaffold NW_008988579.1, and a 161.5 kb interval from 1 bp to 161,533 bp in scaffold NW_008988400.1. It is worth noting that there is a gap between scaffolds NW_008988579.1 and NW_008988400.1 and that the size of the gap is unknown, which could change the size of the map intervals for the Red locus , ANS and UFGT or the regulatory gene MYB10 transcription factor were not found within the candidate region.Based on the reference pear genome, 28 gene models were predicted in the candidate 368.6 kb genomic region  segregation ratio. Three indel markers that were strongly linked to the previously published red fruit skin gene locus of MRB were also closely linked to the red shoot locus in RZS. The locus was mapped to a 368.6 kb candidate interval (with a gap of unknown size) on LG4 of RZS. These results indicate that the bud mutation of RZS and MRB may result from the same locus or from adjacent loci, and these regions have a decisive role in the fruit skin color and shoot color. Breeders could use the red shoots as a phenotypic marker to select for the red-skinned hybrids from RZS families.Additional file 1:Table S1. 28 gene models predicted in the candidate 368.6 kb genomic region (XLS 25 kb).


Evidence on the economic impact of chronic obstructive pulmonary disease (COPD) for third-party payers and society based on large real world datasets are still scarce. Therefore, the aim of this study was to estimate the economic impact of COPD severity and its comorbidities, stratified by GOLD grade, on direct and indirect costs for an unselected population enrolled in the structured German Disease Management Program (DMP) for COPD.1) measurement or other missing values in covariates. The final dataset included 39,307 patients in GOLD grade 1 to 4. We used multiple generalized linear models to analyze the association of COPD severity with direct and indirect costs, while adjusting for sex, age, income, smoking status, body mass index, and comorbidities.All individuals enrolled in the DMP COPD were included in the analysis. Patients were only excluded if they were not insured or not enrolled in the DMP COPD the complete year before the last DMP documentation (at physician visit), had a missing forced expiratory volume in 1 s  to 1.51  in direct and from 1.16  to 1.27 (cancer) in indirect costs.The findings indicate that more severe GOLD grades in an unselected COPD population enrolled in a structured DMP are associated with tremendous additional direct and indirect costs, with comorbidities significantly increase costs. In direct cost category hospitalization and in indirect cost category premature retirement were the main cost driver. From a societal perspective prevention and interventions focusing on disease control, and slowing down disease progression and strengthening the ability to work would be beneficial in order to realize cost savings in COPD. Chronic Obstructive Pulmonary Disease (COPD) is characterized by persistent respiratory symptoms and airflow limitation, which is slowly progressive and not fully reversible. There is no known cure for COPD, but the symptoms are treatable and disease progression can be delayed. The main risk factor for COPD is tobacco smoking but other environmental exposures such as biomass fuel exposure and air pollution may contribute . COPD isFor substantial share of COPD patients, COPD is associated with exacerbations, reduced or even insufficient lung function, and further concomitant chronic diseases such as cardiovascular diseases, skeletal muscle dysfunction, metabolic syndrome, osteoporosis, depression, anxiety, lung cancer and alcohol addiction thus contributing to overall disease severity and reducing health-related quality of life , 3–5.Besides the disease burden, COPD is also associated with substantial economic costs. In the European Union, the total direct costs for COPD are estimated to be about 3% €38.6 billion) of the total health care budget  billion . ResultsFrom a decision makers perspective, there is a substantial need on detailed knowledge about disease severity related to health care expenditures and cost-driving effects in patients with COPD in a real world setting to provide a rational basis for investments and resource allocation in a health care system. Therefore, the aim of this study was to estimate the costs of COPD severity, stratified by GOLD grades, and its comorbidities, with a large real-world dataset from a statutory health insurance fund, for patients enrolled in the structured German Disease Management Program (DMP) for COPD.The analysis was based on pseudonymized claims data routinely documented for participants of the structured German DMP for COPD, offered by AOK Bayern, a large regional health insurance fund. In Germany, DMPs were introduced by a legal framework nationwide compatible with the solidarity principle including quality-of-care requirements, a strict accreditation process, and initially strong financial incentives for statutory health insurance funds to set up programs , 9. Due 1) measurement or other missing values or absurd measures in covariates e.g. height or weight. Enrollment in the DMP COPD ensures that patients had COPD, as diagnostic confirmation is necessary. Physicians were only allowed to enroll patients if the Tiffeneau-Index ) was ≤70% and a) the reduction in FEV1 was < 80% of the target value or b) an increase in FEV1 by less than 15% and/or by less than 200 ml in 10 min after inhalation of a short-acting beta-2-sympathomimetic or 30 min after inhalation of a short-acting anticholinergic or c) an increase in FEV1 by less than 15% and/or by less than 200 ml after at least 14 days of systematic glucocorticosteroids or at least 28 days of inhaled glucocorticosteroids in a stable disease episode and evidence of airway resistance elevation or pulmonary hyperinflation or gas exchange disorder in patients with FEV1/FVC > 70% and a radiographic examination of the thoracic organs that has ruled out another symptom-explaining disease. For the grading of COPD GOLD grades in the patients we used percentage predicted values based on reference values from the Global Lung Function Initiative  [All individuals enrolled in the DMP COPD (AOK curaplan) were included in the study. Persons were only excluded if they were not insured or not enrolled in the DMP COPD the complete year before the last DMP documentation (at physician visit), had a missing forced expiratory volume in 1 s  visit. The number of hospital days was based on hospital invoices, and for medication usage the number of prescribed medications were summed up. Over-the-counter pharmaceuticals, non-pharmacy medicines, and dietary supplements were not included as they are not available in claims data.Claims data include information on inpatient treatment, outpatient-physician treatment , prescribed medication, prescribed medical aids, prescribed remedies and rehabilitation. Furthermore absence from work with sickness notification is documented. Health care utilization was based on individual claims data for the year before the most recent DMP documentation with FEVInformation on retirement/employment status was derived from the insurance status of the patients. Work absence in the last 12 months was based on summed up days with sickness notification handed into the statutory health insurance fund.Overall health care expenditures included direct and indirect health care costs. Direct costs reflect costs out of the perspective of a third party payer and were calculated by summing up all cost categories in the last year. This included costs for hospitalization based on Diagnoses Related Groups (DRGs), outpatient care based on EBM (einheitlicher Bewertungsmaßstab), family doctor-centred health care (hausarztzentrierte Versorgung), and integrated care (integrierte Versorgung), medication based on pharmacy sales prices (including discount agreements), rehabilitation (if covered by AOK Bayern) based on rehabilitation invoices, aids and remedies based on invoices of health care supply stores, and travel expenses based on public transport tickets, invoices of taxis and a fixed fee per kilometer (by usage of private car). Lump-sum payments for physicians were considered in the period they were paid, even when the partially covered the period before or after. All direct costs were inflated to the year 2018, using the inflation rate as reported for Germany by the Organisation for Economic Co-operation and Development (OECD).Indirect costs were considered only for patients under the age of 65 years and out of a societal perspective. Costs of sick days were calculated for participants with an insurance status, which indicated that the person was full- or part-time employed or self-employed. Costs per day of work loss were calculated by dividing 2018 German mean annual labor costs  by 365 days, as sickness notification also include weekends and public holidays. Premature retirement before the regular retirement age of 65 years was based on insurance status and valued by mean annual German labor costs .In this analysis the human capital approach was preferred over the friction cost approach to calculate productivity losses in paid work for society, following current recommendations .2). The socio-economic variable yearly income was based on the information the claims data provided for annual gross pay (Bruttojahresentgelt), retirement income, and pensions (Versorgungsbezüge). We considered the comorbidities diabetes, stroke, myocardial infarction, cancer and arthritis as they have been shown to be important in terms of prevalence and clinical relevance in COPD [Patients’ characteristics, socio-economic variables, and data on comorbidities were assessed based on claims data and DMP documentation. Characteristics were patients’ age group, sex, smoking status, and body mass index  for continuous variables and Chip-values for cost differences were derived by bootstrapping the original data set using 5000 replications [Generalized linear models (GLM) were conducted to quantify the association between GOLD in grades 1 to 4 and direct and indirect costs. As the distribution of costs is typically highly skewed, a gamma model with log-link was used , 14. Theications . The sinAll analyses were performed using the SAS statistical package version 9.4 .1-value was available. Further, 27 patients were excluded as their height was below 1.20 m and for 2 patients the BMI could not be calculated due to missing body weight. Finally, 2606 patients were excluded as they were not insured or not enrolled in the DMP COPD the complete year before the last DMP documentation with FEV1 measurement. The final dataset contained 39,307 patients. Differences in patient characteristics . The percentage of women decreased over GOLD grades from 52.4% in grade 1 to 32.9% in grade 4 (p < 0.0001). More patients in higher disease grades were in the lower income groups (p < 0.0001). The percentage of current smokers and former smokers was found to be higher in more severe grades (p < 0.0001). There was also a decline in BMI from 29.3 in grade 1 to 26.0 in grade 4 (p < 0.0001). The prevalence of considered comorbidities was more pronounced in the less severe GOLD grades 1 and 2 than in the more severe GOLD grades 3 and 4, except in case of myocardial infarction.Table Table In Table Table In the extended model the effects of GOLD grades hardly changed or even increased slightly, compared to the basic model, as shown in Table Adjusted mean annual costs for GOLD grades 1 to 4 from the basic model are illustrated in Table This study estimated overall health care expenditures of unselected COPD patients enrolled in a structured German DMP for COPD in different severity grades using real world data. Progression of COPD is associated with tremendous costs for third party payers, but even more for society as indirect costs exceed direct costs almost 2 times in grade 3 and 4 and more than 3 times in grade 1 and 2. Adjusting for major comorbidities did not decrease the effect of more pronounced disease severity of COPD on costs, so that comorbidities independently added to the direct as well as indirect costs.When comparing our results to previously published work, we observe a huge variance of reported costs for COPD.A systematic review  of 7 COPA similar trend of increasing costs per patient for COPD could be found in most international studies. Dal Negro  found meContrarily Tsai et al.  found a COPD and Systematic Consequences – Comorbidities Network). COSYCONET is a prospective, observational, multicenter study that included 2741 patients aged 40 years or older with physician-diagnosed COPD. The LQ-DMP study also includes patients younger than 40 years, but only 130 individuals (0.33%) were aged younger than 40. Thus we think that including the entire range does not crucially affect comparability. The COSYCONET study might be prone to a specific selection of patients, as patients with a previous lung transplantation or lung volume reduction surgery and lung malignancies were excluded. Furthermore, only clinical stable patients, defined as having no moderate or severe exacerbations in the last four weeks before enrollment were included. Besides that, patients had to visit a study center for several hours for medical examination and data collection, which might exclude patients in bad overall conditions. This might lead to overall more healthy patients in each GOLD grade compared to the LQ-DMP study.The most recently published German study from Wacker et al.  calculatAll in all, the COSYCONET cohort is on average more than 4 years younger and the BMI is two points lower. The prevalence of comorbidities was higher in the LQ-DMP cohort for all considered diseases, for diabetes, stroke myocardial infarction, cancer and arthritis by 22.6, 24.6, 4.1, 19.9, and 5.13%, respectively. The much higher prevalence of never smokers 54.9% vs. 5.4% and the much lower prevalence of former smokers 68.1% vs. 14.1% in LQ-DMP is due to the fact that we only had data on smoking status for the last 8 years, and if patients haven’t smoked during that period we classified them as never smokers. Despite these differences the factors regarding direct and indirect costs for current smokers of 0.93 (0.79–1.09) and 0.96 (0.68–1.36) in Wacker et al. and 0.91 (0.89–0.94) and 0.94 (0.86–1.03) in our analysis and for former smokers and 1.04 (0–91-1.18) and 1.24 (0.93–1.67) in Wacker et al. and 1.12 (1.08–1.16) and 1.03 (0.91–1.15) in our analysis are quite similar. The reduced costs for smokers and the higher costs for former smokers might be prone to reverse causation, since former smokers might quit smoking because of a recent adverse event .The reported annual direct costs in COSYCONET were higher than in LQ-DMP by a factor of 1.17, 1.24, 1.41, and 1.29 for GOLD grade 1, 2, 3, and 4, respectively. Similarly, higher costs arise in COSYCONET for annual indirect costs. The factors were of 1.25 in grade 1, 1.23 in grade 2, 1.44 in grade 3, and 1.74 in grade 4 and exceed the factors for direct costs. The higher reported direct costs in COSYCONET might be caused by the use of a weighted average costs catalogue  to estimFurther, both studies conducted models estimating the effect of COPD GOLD grade in the basic model, and GOLD grade plus additional comorbidities in the extended model, on direct and indirect costs. The only difference in covariates was that Wacker et al.  used eduIn a review and three recently published articles the influence of different comorbidities on costs in COPD patients were considered. The review conducted by Huber et al.  estimateOur analysis is also subject to several limitations. The results are based on claims data of a large single statutory health insurance fund (AOK Bayern). Therefore the transferability of the findings might be limited, and claims data are collected for administrative and billing rather than for research purposes, and as characteristics of the insured can differ across statutory health insurance funds. Further, the population consists of patients enrolled in the DMP COPD which is voluntary that could cause self-selection effects, for example, that persons who are more health-conscious or more active overall would more often decide to take part in a DMP. While patients who are not adherent to the requirements of the DMP (continuous physician care), which might be predominantly patients in more severe GOLD grades, are excluded from the DMP. Further, physicians might enroll patients with special characteristics with a higher likelihood in a DMP . TherefoThe determination of the contact frequency with physicians in claims data is associated with methodological limitations. In recent year’s physician reimbursement was strongly shifted from fee-for service to lump-sum that means more contacts might be covered by one lump-sum fee, which tend to lead to an underestimation of the contact incidence in our study. Despite the presumption, the contact frequencies are quite in line with the rates reported by Wacker et al. .The rather restrictive inclusion of comorbidities in the statistical analyzes may have led to an overestimation of the estimates of the COPD severity grades as well as the included comorbidities on costs, but it also limits the problem of multicollinearity between comorbidities, that affects individual predictors negatively. Further, prevalence of comorbidities might be overestimated in claims data, as there are financial incentives for hospitals for up coding. We best possibly mitigated this potential source of bias by requiring multiple diagnoses over an extended period of time.We only had cost data for rehabilitation if the statutory health insurance was the third party payer, this was the case in most of the rehabilitations . Interestingly, the length of rehabilitation stay differs tremendously according to the payer, so the mean is 23.4 days if the health insurance, and 27.8 days if the pension fund, is the third party payer. This adds up to a potential underestimation of approximately 30 to 40% of the reported rehabilitation costs in our analysis.Direct health care costs were estimated form the perspective of a third-party payer, while the perspective for indirect costs were a societal. This methodological inconsistency is caused by the data basis. Direct costs would likely have been higher in a societal perspective, too, as we did not consider aspects such as out-of-pocket payments or support provided by family members.Also the calculation of indirect costs with claims data is associated with some methodological difficulties. Premature retirement is based on insurance status of the patients, which is not further specified, therefore we were not aware of the real reason of premature retirement. Further, the costs of incapacity for work are underestimated in our results for two reasons. Firstly, a sickness notification issued by a physician has only be presented the employer for three or more days. Secondly, not all sickness notifications are handed in to the statutory health insurance, as only incapacity for work longer than 6 weeks is relevant to disbursement of sickness benefit. Thirdly, we had to use mean annual labor costs instead of the patient specific annual income, as for premature retired persons we only had actual retirement earnings, which are way lower than the potential earnings on the job market. Fourthly, in our analysis we used the human capital approach, which might overestimate the costs, instead of the friction costs approach. However, the discussion about the most appropriate approach is still on-going –52, and In our analyses we used yearly income, as information on education are not available in claims data, as a control variable for socioeconomic status. This might be problematic as yearly income correlates with insurance status (e.g. working or retired) which is directly associated with indirect costs.1, weight and height allowing a FEV1 percentage prediction to classify the patients according to GOLD grades. The biggest strength is, besides the unselected patient population, that our direct cost calculations are based on real expenditures of a large regional statutory health insurance fund and not on weighted average costs as used in questionnaire based observational cohort studies, which also prone to recall bias.Despite these limitations in data and methodology, we are strongly convinced that a claims data-based approach is well suited to provide comprehensive insights into current costs structures of patients enrolled in a structured DMP for COPD. We analyzed a large unselected data set, with almost 40,000 COPD patients, which allowed us a stratification for GOLD grades while remaining with a sound sample size for the single strata. Besides claims data of a statutory health insurance fund we also used DMP COPD documentation data including clinical measures such as FEVIn conclusion, our results, based on a large unselected real-world dataset of patients enrolled in a structured DMP for COPD, demonstrate that more severe GOLD grades are associated with substantial additional direct and indirect costs, and with comorbidities independently significantly increase costs. In direct cost categories hospitalization was the main cost driver and in indirect costs premature retirement. All in all, COPD causes tremendous costs for third party payers, but even more for society as indirect costs exceed direct costs almost 2 times in grade 3 and 4 and more than 3 times in grade 1 and 2. Based on the results of international cost-of-illness studies, there is a continuous annual increase in average costs per COPD patient in each GOLD grade. From a statutory health insurance fund perspective as well as from a societal perspective prevention and interventions focusing on a better disease control, to avoid hospitalizations, and slowing down disease progression and strengthening the ability to work would be beneficial not only in terms of patient-relevant endpoints such as survival and health-related quality of life, but they also carry the potential to reduce costs associated with COPD.Additional file 1: Table S1. Characteristics included vs. excluded patients. Table S2. Unadjusted healthcare utilization, work absenteeism and resulting costs included vs. excluded patients. (DOCX 22 kb)


This case series of right unilateral diaphragmatic paralysis suggests peripheral nervous system involvement. Since the first identification of neonatal microcephaly cases associated with congenital Zika virus infection in Brazil in 2015, a distinctive constellation of clinical features of congenital Zika syndrome has been described. Fetal brain disruption sequence is hypothesized to underlie the devastating effects of the virus on the central nervous system. However, little is known about the effects of congenital Zika virus infection on the peripheral nervous system. We describe a series of 4 cases of right unilateral diaphragmatic paralysis in infants with congenital Zika syndrome suggesting peripheral nervous system involvement and Zika virus as a unique congenital infectious cause of this finding. All the patients described also had arthrogryposis  and died from complications related to progressive respiratory failure. Aedes spp. mosquitoes  that had received US Food and Drug Administration emergency use authorization; we tested samples from patient 1 by the InBios ZIKV Detect MAC-ELISA  and tested samples from patients 2–4 by the CDC Zika MAC-ELISA  samples, or both. We tested for Zika virus RNA using the Zika Virus RNA Qualitative Real-Time RT-PCR test  on serum and urine in patient 1 and by the CDC Trioplex RT-PCR assay in patients 2–4 , severe microcephaly , and diffuse intracranial calcifications. The mother declined amniocentesis; noninvasive prenatal screening for aneuploidy was negative. Maternal serologic testing was negative for dengue virus (DENV) and Zika virus IgM and negative for evidence of recent infection with cytomegalovirus, rubella, varicella-zoster virus, syphilis, The infant had a birthweight of 2,020 g and a head circumference of 27.5 cm (z-score −4.3), in keeping with severe microcephaly. Arthrogryposis involving the hips, knees, ankles, and elbows was observed, along with bilateral talipes equinovarus. The infant was intubated shortly after birth because of poor respiratory effort. A computed tomography (CT) scan of the head showed severe microcephaly with intracranial volume loss, including thinning of the cortical mantle, and callosal and pontocerebellar hypoplasia with ex vacuo ventriculomegaly. Multiple dystrophic bandlike calcifications were seen along the corticomedullary junction and periventricular white matter, as well as within the basal ganglia and brainstem. Optic nerves were diminutive. Overriding cranial sutures, prominent occipital shelf, and scalp rugae were confirmatory for fetal brain disruption sequence. Chest radiography revealed a markedly and persistently elevated right hemidiaphragm , panel AA female infant was born at 40 weeks’ gestation to a 21-year-old primigravida mother from Brazil via cesarean section for breech presentation. The mother reported having an illness characterized by rash but no fever during the first trimester of pregnancy . Fetal uA female infant was born at 39 weeks’ gestation to a 17-year-old primigravida mother from Brazil via spontaneous vaginal delivery . Fetal uA female infant was born at 41 weeks’ gestation to a 21-year-old primigravida mother from Brazil via cesarean section for breech presentation. The mother reported having an afebrile illness with rash during the first trimester of pregnancy . Fetal uWe report a series of 4 patients with CZS and right unilateral diaphragmatic paralysis suggesting PNS involvement in CZS and Zika virus as a unique congenital infectious cause of this finding. All the patients were female infants born at term to primiparous mothers who reported symptoms suggestive of Zika virus infection during the first trimester of pregnancy. All infants in this case series had severe microcephaly and arthrogryposis and died from progressive respiratory failure.,A recent study of Zika virus–infected macaques reported evidence of viral tropism to the peripheral nerves . In a previous report of a large cohort (n = 87) of neonates with CZS, only infants with unilateral diaphragmatic paralysis  died before hospital discharge, and all required respiratory support following birth  because of patient instability and the risks associated with patient transport to a radiology suite in all the cases presented. Electrodiagnostic studies (electromyography/nerve conduction studies) and nerve biopsies to further investigate the underlying mechanism of the diaphragmatic paralysis also could not be obtained, for similar reasons. The differential diagnosis of right unilateral hemidiaphragm elevation includes diaphragmatic eventration, a hepatic mass, or decreased lung volume ; however, there was no evidence of these abnormalities on examination or other imaging studies and, aside from some reports of pulmonary hypoplasia, they have not been previously reported in patients with CZS (This case series of infants with CZS, arthrogryposis, and unilateral diaphragmatic paralysis describes a unique constellation of clinical findings that has not been described with other congenital infections. The presence of both arthrogryposis and unilateral diaphragmatic paralysis suggests likely involvement of the PNS, spinal cord, or both in these severely affected infants. The presence of diaphragmatic paralysis may also represent a risk factor for early death in infants born with CZS. Further studies to elucidate the precise mechanism leading to diaphragmatic paralysis in these patients are required.


Pothos latent virus. These two values, below the species demarcation criteria for the genus, indicate that the detected virus is a new member of genus Aureusvirus, family Tombusviridae, with the proposed name Elderberry aureusvirus 1 (ElAV1). A survey confirmed the wide distribution of ElAV1 in elderberry in the Czech Republic. Phylogenetic analyses of RdRp and CP sequences showed distinct microevolution of geographically separated isolates, with a tendency for isolates coming from close localities or from the same region to cluster together but heterogeneity of viral populations down to a local scale was also observed. The symptomatology of the new virus is not fully clear, but many infected trees were either asymptomatic or showed mild chlorotic mosaics. More severe symptoms, potentially impacting yields of flowers or berries, were observed in plants with mixed infections of ElAV1 and other elderberry viruses. Further efforts are now needed to determine ElAV1 prevalence outside the Czech Republic and to unravel its epidemiology.A novel virus infecting elderberry was identified by high-throughput Illumina sequencing of double strand RNAs isolated form elderberry leaves. The complete genome sequence obtained (4512 nucleotides in length) shows an organization typical for aureusviruses, with five open reading frames (ORFs) and the typical ORF1-RT expression by the readthrough of an amber stop codon. The analysis of the RNA-dependent RNA polymerase (RdRp) and coat protein (CP) sequences showed the highest identity (respectively 75.7% and 55%) with the corresponding amino acid sequences of  Aureusvirus is a genus of single-stranded, positive strand RNA viruses of the family Tombusviridae. It was originally established with two members, Pothos latent virus (PoLV) as a type species and Cucumber leaf spot virus (CLSV) [Maize white line mosaic virus (MWLMV), Johnsongrass chlorotic stripe mosaic virus (JCSMV) and Yam spherical virus (YSV), as well as the related Sesame necrotic mosaic virus (SNMV), which is not yet formally recognized [Poaceae species, mainly maize  have also been reported [s (CLSV) ; currentcognized . Aureusvcognized –5, yam fcognized , sesame cognized , cucumbecognized , 8 and v MWLMV, , 10) or  or Aureu MWLMV, , 11). Au. AuAureureported .Tombusviridae members encoding five ORFs, with an uncapped 5´ end and a non-polyadenylated 3´ end. ORF1 encodes a replication-associated protein. Readthrough of an amber stop codon results in the translation of ORF1-RT, which contains an RNA dependent RNA polymerase domain; both proteins are expressed directly and contribute to the replicase complex. The coat protein (CP), encoded by ORF2, and movement protein (MP) encoded by ORF3 are expressed from two distinct subgenomic messenger RNAs (sgRNA1 of 2.0 kDa and sgRNA2 of 0.8 kDa). ORF4, encoding a viral suppressor of silencing, is translated by the leaky scanning of sgRNA2 [The genomes of aureusviruses are 4.29−4.46 kb long, with an organization typical for Sambucus nigra L.) is a deciduous tree native to Europe and North America. Its flowers and berries are used to prepare infusions, syrups and jellies and in traditional medicine. The popularity of this plant has increased in recent years in the pharmaceutical and food industries, due to its antiseptic and antiviral properties as well as to the interest in the colour compounds present in the berries [Betaflexiviridae) such as Elderberry carlavirus (ECV) or Blueberry scorch virus (BlScV) [Secoviridae) such as Cherry leaf roll virus (CLRV), Arabis mosaic virus (ArMV), Tobacco ringspot virus (TRSV), Tomato black ring virus (TBRV) or Cherry rasp leaf virus [Elderberry latent virus (ELV), Tobacco necrosis virus (TNV) or Tomato bushy stunt virus (TBSV) in the Tombusviridae family [Sambucus plants, and described through next generation sequencing approaches, include elderberry carlaviruses with the proposed name Elderberry virus A–E (Betaflexiviridae) [Bromoviridae) [Elderberry  –19; nepoaf virus –23; and e family , 24, 25.viridae)  and the viridae) .Sambucus nigra L. is a common tree, wildly growing in the Czech Republic. The plant is not protected being classified as B (common) taxon according IUCN and it is not listed in The Red List of the Czech Republic. The all samples were collected in the freely accessible public area and none private land, at any of sampled locations, was visited. None specific permissions were required to perform present study.The Sambucus nigra L.) during surveys carried out in 2015–2017 in 12 localities of Central Moravia, South Moravia and South Bohemia (Czech Republic). Sampled plants were either asymptomatic or showed various virus-like symptoms, such as leaf chlorosis, chlorotic mosaics and chlorotic spots on leaves, vein clearing, leaf crinkling, rolling and dwarfing, or non-uniform fruit development and ripening. Some trees also showed evidence of early leaf senescence. About 20 g of leaves were collected separately from each plant and subsequently split up into 0.1 and 10 g aliquots that were stored at -80°C until used.Leaf samples were randomly collected from July to September from wild elderberry trees (Double-stranded RNAs (dsRNAs) were purified from 10 g of leaves of the B15 tree using a double CF11 cellulose batch chromatography procedure , 29. TotPurified dsRNAs were used for the preparation of a cDNA library using the True Seq Stranded mRNA Library preparation kit (Illumina) and sequenced in a multiplexed run of 100 bp single reads using a HiSeq2500, together with other banks prepared from unrelated plant samples. Both library construction and sequencing were performed by SEQMe s.r.o. (Czech Republic).For targeted Sanger sequencing of coat protein and RdRp genes or RACE-PCR fragments (at least five clones) all nucleic acids were isolated using the QIAquick Gel Extraction kit (Qiagen). Isolated DNA was sequenced using the BigDye Terminator v3.1 Cycle Sequencing kit (Applied Biosystems) and an ABI PRISM 3730 Genetic analyser.Total RNAs were used for the detection of the aureusvirus and for the amplification of targeted viral genome parts using RT-PCR. Complementary DNAs were synthetized using random hexamers and the BioScript reverse transcriptase (Bioline). PCR amplification involved the ElAur1F/ElAur1R primer pair targeting the RdRp gene for virus detection ; the ElAur1-RdRpF/ElAur1-RdRpR primer pair targeting the RdRp domain ; or the ElAur1-CPF/ElAur1-CPR pair targeting the full CP gene  (for list of primers see The sequences of the 5´- and 3´-ends of the genome obtained by NGS were verified and completed using the SMARTer RACE 5´/3´ kit (Clontech) and the supplied nested PCR protocol. The 5´-end was obtained using primers 5RPAur-dir followed by 5RPAur-nt, the 3´-end using the 3RPAur-dir and 3RPAur-nt primers, respectively . The obtApple mosaic virus (ApMV), Arabis mosaic virus (ArMV), Cherry leaf roll virus (CLRV), Cherry rasp leaf virus (CRLV), elderberry viruses A-E (ElVA-ElVE), Sambucus virus S (SVS), Strawberry latent ringspot virus (SLRSV) and Tomato black ring virus (TBRV) was evaluated by RT-PCR reactions performed on cDNAs obtained as described above. The following specific primer pairs and original protocols were used: APMV-s and APMV-a for ApMV [The potential co-infection of elderberry plants by for ApMV , AP1 andfor ApMV , CLRVi1 for ApMV , JQ3D3FFfor ApMV , Unicarlfor ApMV , 1FCPBrofor ApMV , SLRSViFfor ApMV  and TBRVDe novo assembly of high quality reads was performed with the CLC Genomics Workbench 9, using default parameters and a minimum contig length of 200 bp. The obtained contigs were annotated by BLASTN and BLASTX analyses [analyses  against analyses . Sanger Tombusviridae sequences using the ClustalW algorithm [Multiple alignments, pairwise genetic distance computations, model selections, all phylogenetic analyses and tree visualisations were performed using Mega v. 7.0.14 . The obtlgorithm . Phylogelgorithm , 40 and lgorithm  for nucllgorithm  for aminlgorithm . Conservlgorithm , 44. Thelgorithm  under acPothos latent virus ; contig B15-40, 2436 nt in length  showed 83.2% identity with the partial coat protein sequence of PoLV-pigeonpea (AJ243370). The scaffolding of the overlapping ends these contigs  showing virus-like symptoms such as leaf chlorosis, leaf dwarfing and reduced, non-uniform fruit development, was selected for detailed NGS analysis. Highly purified double-stranded RNAs were sequenced using the Illumina HiSeq2500 platform. In total, 27,747,697 reads (average length 100 nt) were obtained and were assembled in 4638 contigs, two of which showed high identity values with aureusvirus sequences available in GenBank database. Contig B15-325, 2137 nt in length  showed 78.2% identity with the RNA-dependent RNA polymerase gene of  contigs , togetheTombusviridae family  and comprises five ORFs with the typical organisation of aureusviruses in the e family . The firPothos latent virus isolate pigeonpea (AJ243370) and Yam spherical virus (NC_022895). Comparisons of the deduced amino acid sequence showed respectively 75.7% and 75.4% identity with the RdRps of PoLV-pigeonpea and YSV. The highest aa identity (78.7%) was found with the partial RdRp of Sesame necrotic mosaic virus (DQ367845). A similar analysis of CP gene sequences confirmed the genetic differentiation of the B15 virus, with highest nucleotide identity levels of respectively 60.3% and 58.4% (55% and 54.2% aa-identity) with PoLV isolates lisianthus and pigeonpea   of the RdRp-encoding region showed highest identity (70.6%) with the aureusviruses Aureusvirus, i.e. less than 60% aa sequence identity for the CP and less than 80% aa sequence identity for the polymerase. This clearly indicates that isolate B15 represents a new aureusvirus species, tentatively named Elderberry aureusvirus 1 (ElAV1). Comparison of the available complete genomic sequences of aureusviruses and reference species within the family Tombusviridae and their phylogenetic analysis confirmed the position of the new virus in the Aureusvirus genus, with the B15 isolate forming a separate branch in the close relation to PoLV and YSV . The expected product of 457 bp  was amplThe trees infected by Elderberry aureus virus 1 were either asymptomatic or showed mild symptoms of virus infection, mainly mild chlorosis or diffuse chlorotic mosaics on the leaves. In a few cases a non-uniform flowering or fruit ripening was also observed . In someSequence analysis of the ElAur1F/R amplicons confirmed the specificity and robustness of the detection assay, as no non-specific PCR products were identified. All isolates, including the ElAV1-B15 reference isolate, shared 91.2–99.8% nt identity (93.3–100% amino acid identity of the encoded protein). A phylogenetic analysis of these nucleotide sequences confirmed the significant variability of the virus and showed the existence of several well supported clusters but also of isolated sequences at the end of long branches . The isoTo further analyse the genetic variability of the new aureusvirus, the sequence of two genomic regions, the RdRp and the CP were analysed. For the first one, a sequence covering almost the full RdRp of ORF1-RT (1258 bp) was assembled from two overlapping PCR fragments amplified using the ElAur1F/ElAur1R and ElAur1-RdRpF/R primer pairs. For the second one, the complete sequence of the coat protein gene (1134 bp) was obtained from the ElAur1-CPF/R amplicon. Despite the fact, that the primers used were designed based on the B15 isolate sequence only, both sequences were successfully obtained for 14 isolates coming from all Czech regions. ElAV1 isolates showed higher variability in the RdRp domain, with 90.6–99.8% nucleotide identity (95.5–100% aa identity) to be compared to 95.5–100% nucleotide identity (96.8–100% aa identity) for the CP gene.Phylogenetic analyses confirmed the diversity of the virus and the tendency for isolates from the same region to cluster together as one or two main groups . In partElderberry is a widely growing shrub or tree, popular in the central Europe and in other areas due to the efficacious or useful compounds present in its fruits and flowers. Not surprisingly, in agreement with its use in traditional medicine, elderberry was a center of interest for virologists in the former Czechoslovakia, and in other countries too, resulting in the description of various diseases and virus-like infections based only on the observation of symptomatology and/or supported by transmission experiments to differential or indicator hosts and by electron microscopy , 45, 46.Next generation sequencing is proving a very powerful tool for the description of new viruses and for solving etiological issues. Until recently various elderberry carlaviruses described based on viral particle morphology and on detection assays using polyclonal antibodies could be found in the virological literature, named as Elderberry virus A, Elderberry carlavirus, and finally re-named as Elderberry symptomless virus , 46, 47.Tombusviridae, detected and characterized here, shows that the elderberry virome is wider than was expected. The family Tombusviridae comprises 16 genera, and three viral species belonging to different Tombusviridae genera, ELV (Pelarspovirus), TNV (Necrovirus) and ToBSV (Tombusvirus), have been reported to unfrequently infect elderberry [Tombusviridae species, representing the sixth member of Aureusvirus genus.Elderberry aureusvirus 1, a novel aureusvirus in the family derberry , 24, 25.The presence of virus in various randomly sampled trees detected using RT-PCR and confirmed by subsequent sequencing shows that ElAV1 is widely distributed, at least in the Czech Republic. The detected genetic diversity suggests that the virus has been present in these regions for a long time. Aureusviruses generally have a limited natural host range  and our The symptomatology of the new aureusvirus in infected elderberry trees is not fully clear. Despite the mild chlorosis and mild or diffuse mosaics observed on the leaves of many infected trees, these symptoms cannot be unambiguously connected with the virus. Elderberry trees prefer humid soils rich in nitrogen and phosphorus, but they tolerate a wide range of soil types and often grow in villages and towns at ruderal sites, around roads or streams , 48. It Nicotiana benthamiana and Chenopodium quinoa by mechanical inoculation have been unsuccessful so far, although the closely related Pothos latent virus is reported to be mechanically transmissible to these herbaceous hosts [The epidemiology of ElAV1 still remains unclear. Efforts at experimental transmission of ElAV1 to us hosts , 12. TheS1 Table(XLSX)Click here for additional data file.S1 FigLanes: L, GeneRuler 100 bp DNA ladder (Thermo Scientific); 1–15, elderberry samples.(PDF)Click here for additional data file.S2 FigPhylogenetic tree of Elderberry aureusvirus 1 isolates reconstructed using neighbor-joining method based on the RdRp (A) and complete coat (B) proteins. PoLV used as an outgroup to root the tree. Bootstrap values ≥70% obtained from 500 replicates are shown. The scale bars represent 0.5% sequence divergence. Isolates are marked by name and GenBank Accession number.(PDF)Click here for additional data file.


Schizophrenia is one of the leading public health issues in psychiatry and imposes a heavy financial burden on the healthcare systems. This study aims to report the direct medical costs and the associated factors for patients with schizophrenia in Guangzhou city, Southern China.This was a retrospective 4-year cohort study. Data were obtained from urban health insurance claims databases of Guangzhou city, which contains patients’ sociodemographic characteristics, direct medical costs of inpatient and outpatient care. The study cohort  was identified using the International Classification of Diseases Tenth version (F20). Their outpatient care information was merged from outpatient claims database. Descriptive analysis and the multivariate regression analysis based on Generalized Estimating Equations model were conducted.A total of 2971 patients were identified in the baseline. The cohort had a mean age of 50.3 years old, 60.6% were male, and 67.0% received medical treatment in the tertiary hospitals. The average annual length of stay was 254.7 days. The average annual total direct medical costs per patient was 41,972.4 Chinese Yuan (CNY) ($6852.5). The inpatient costs remained as the key component of total medical costs. The Urban Employee Basic Medical Insurance enrollees with schizophrenia had higher average costs for hospitalization  than the Urban Resident Basic Medical Insurance enrollees , and had higher reimbursement rate (85.8% and 61.5%). The non-medication treatment costs accounted for the biggest proportion of inpatient costs for both schemes (55.8% and 64.7%). Regression analysis suggested that insurance type, age, hospital levels, and length of stay were significantly associated with inpatient costs of schizophrenia.The direct annual medical costs of schizophrenia were high and varied by types of insurance in urban China. The findings of this study provide vital information to understand the burden of schizophrenia in China. Results of this study can help decision-makers assess the financial impact of schizophrenia. Schizophrenia is one of the leading public health issues in psychiatry and is ranked among the top 25 leading causes of disability worldwide , 2. A meSchizophrenia imposed a heavy financial burden on the healthcare systems. A review of cost-of-illness studies for schizophrenia worldwide reported that the direct medical costs of treating schizophrenia accounted for between 1.4 and 3.0% of total national health expenditures . The ecoChina expands health insurance coverage to all urban residents with two social health insurance schemes—the Urban Employee Basic Medical Insurance  and the Urban Resident Basic Medical Insurance   were calculated for demographic information and costs. Since the medical cost data usually has a skewed distribution, the Kruskal–Wallis test was used to investigate whether the differences in patients’ characteristics of two health insurance schemes were statistically significant. To identify the predictors of total inpatient costs, a popular statistical approach to fit a marginal model for longitudinal data, the Generalized Estimating Equations (GEE) model , was performed in this study . All staA total of 2971 patients were identified in the baseline period  and the vast majority was inpatient costs: mean inpatient costs  versus mean outpatient costs  . Furthermore, in the baseline period the per capita inpatient costs of patients with the UEBMI were higher than those of patients with the URBMI scheme, while the per capita outpatient costs of the UEBMI patients were lower than those of the URBMI patients (P < 0.05). The percentage of reimbursement expenses out of inpatient costs for the UEBMI scheme patients (85.8%) was much higher than the patients with the URBMI scheme 61.5%), because these two insurance schemes had different benefit packages as indicated in Table .5%, becaAmong 2971 total patients, there were 2642 schizophrenia patients who used inpatient care only and 329 patients who used both inpatient and outpatient care in the baseline period   per capita in the baseline period, and increased to CNY56,544.1 (US$9231.5) at the end of the 3-year follow-up period. The inpatient costs remained as the key component of the total medical costs in 4 years. The type of insurance, age, higher hospital levels and longer LOS were significantly associated with inpatient costs. This is the first cohort study using sample from the claims database of an entire city to examine the direct medical costs of schizophrenia patients and compare the healthcare costs under two different urban insurance schemes in China.When comparing our results with findings in other countries, we observed a large variation in costs. Our total direct medical cost  was much lower than what have been reported in the United States (US$18,090, 2013 price ; US$21,6In this study, the inpatient cost was the largest contributor to the total direct medical costs for patients with schizophrenia, which was consistent with previous studies in other countries , 24, 25.It is difficult to compare the average annual direct medical costs of schizophrenia per patient reported in this study to the other three China-based studies: Wu et al.  reportedIt is the first time to evaluate the differences in direct medical costs between two urban health insurance schemes. We found that the total direct medical costs and the percentage of reimbursement expenses out of inpatient costs for those covered by the UEBMI scheme were higher than those covered by the URBMI scheme, mostly because the UEBMI had a higher benefit level for its beneficiaries . The panResults from the regression analysis found that after controlling for other confounding variables, the association between age and total annual inpatient costs was non-linear, with the 30–40 years old patients had the highest costs. Particularly, the pattern in the differences in medical costs across age categories was quite different between UEBMI patients and URBMI patients. This suggested that the management of costs for schizophrenia patients depending on a category of age might be implemented differently across different insurance schemes. Variations in the costs of schizophrenia tend to be driven by the level of hospital, with patients having medical treatment in tertiary hospitals, being more likely to incur higher inpatient costs , 36. TerThe positive association between the LOS and the hospitalization costs of schizophrenia is consistent with previous studies , 37. In In addition, the longer LOS for schizophrenia patients might be due to the reimbursement fee schedule instituted by two urban health insurance schemes in China. In the case of psychiatric hospitalization for the UEBMI and URBMI enrollees, a fixed per diem cost of around US$30 from the insurance fund is paid for the supplies without limitation of LOS . Thus, tThere were some limitations in this study. First, this study only analyzed direct medial costs. The indirect costs due to loss of productivity and family members’ informal care were not examined. Thus, we likely underestimated the total medical expenditures of schizophrenia in China. Second, clinical severity of the disease, disease duration, comorbidities and personal income levels, which were important predictors of costs, were omitted from the analysis because such data were not available in the claims dataset. Third, the study population was limited to urban enrollees under two insurance schemes in one city of China, which cannot generalize to the whole Chinese population. Further studies could consider more associated factors, indirect costs for a more comprehensive evaluation of schizophrenia costs and compare the schizophrenia costs to non-schizophrenia costs to evaluate the impact of health insurance.The direct medical costs of schizophrenia were high and varied by types of insurance in China. The findings of this study provide vital information to understand the burden of schizophrenia in China. Such information can also be used by decision makers in program evaluation and health resources allocation.