Rhynchobatus djiddensis), a novel virus, distinct from all known viral families in regard to particle size, morphology, genome organization, and helicase phylogeny was discovered. Large inclusion bodies containing 75-nm icosahedral viral particles were present within epithelial cell nuclei in the proliferative skin lesions. Deep metagenomic sequencing revealed a 22-kb circular dsDNA viral genome, tentatively named guitarfish “adomavirus” (GAdoV), with only distant homology to two other fish viruses, Japanese eel endothelial cell-infecting virus (JEECV) and a recently reported marbled eel virus. Phylogenetic analysis of the helicase domain places the guitarfish virus in a novel clade that is equidistant between members of the Papillomaviridae and Polyomaviridae families. Specific PCR, quantitative PCR, and in situ hybridization were used to detect, quantify, and confirm that GAdoV DNA was localized to affected epithelial cell nuclei. Changes in the viral titer, as well as the presence of a hybridization signal, coincided with the progression and then final resolution of gross and microscopic lesions. The results indicate that GAdoV is the causative agent of the proliferative skin lesions.Only eight families of double-stranded DNA (dsDNA) viruses are known to infect vertebrate animals. During an investigation of papillomatous skin disease in an elasmobranch species, the giant guitarfish , exemplifying the difficulties of discovering novel viruses within seas of unidentifiable “dark matter” associated with deep sequencing data. The novel adomavirus represents the first viral genome shown to cause clinical disease in a cartilaginous fish species, the giant guitarfish. Our findings demonstrate that emerging fish viruses are fertile ground to expand our understanding of viral evolution in vertebrates. Viruses do not leave a fossil record. Their evolutionary history is usually deduced from the molecular phylogeny of viruses found in extant hosts, except in occasional specimens where viruses are exceptionally well preserved by extremely cold or stable environments –4. Ances–Polyomaviridae, Papillomaviridae, Adenoviridae, Herpesviridae, and Alloherpesviridae, and three families in the proposed supergroup Megavirales, namely, Poxviridae, Asfarviridae, and Iridoviridae. The origins and ancient evolutionary history of these families remain elusive  endothelial cell-infecting virus (JEECV) is the cause of viral endothelial cell necrosis of eels (VECNE) disease (Anguilla marmorata) in Taiwan  viruses known to infect vertebrates, including the  elusive –10. Infeelusive –, 11–13.  disease , 15. Then Taiwan . Althougin situ hybridization findings that establish it as the etiologic agent of a proliferative skin disease in the giant guitarfish. A concurrent study focused on viral gene evolution and classification  suggests that the helicase and capsid genes of the guitarfish virus, JEECV, and marbled eel virus share a complex evolutionary history with polyomaviruses, papillomaviruses, and adenoviruses and that these three fish viruses may represent a novel viral lineage—adomavirus. Here, we aim to document the first description of disease caused by this new clade of adomavirus in an elasmobranch, the giant guitarfish.Elasmobranch fishes, including the sharks, rays, guitarfishes, and others, evolved from ancestral species at least 419 MYA during the early Devonian period. Compared to bony teleost fish, such as eels, examples of viral diseases in elasmobranchs are rare , 18. DurGrossly, pink to red, variably sized, villus-like skin lesions were widely distributed over ventral surfaces, involving the perioral area, claspers, pectoral and pelvic fins and their bases . A heavide novo assembly and alignments were used to construct the complete circular 22-kb genome of a second virus, guitarfish adomavirus (GAdoV)  . However (GAdoV) , that wa10.1128/mBio.00185-18.1FIG S1 FIG S1, PDF file, 1 MB.Coverage analysis of the next-generation sequencing of guitarfish adomavirus. Download Copyright © 2018 Dill et al.2018Dill et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the The genome organization and viral proteins of GAdoV were highly divergent compared to known viral families . This adPapillomaviridae and Polyomaviridae (The most conserved ORF among the guitarfish and eel viruses is the EO1 helicase. The predicted EO1 protein shows a segment with distant similarity to the superfamily 3 helicase domains of both the E1 proteins of papillomaviruses (<27% protein identity) and the LT proteins of polyomaviruses (<23% protein identity). Phylogenetic analysis of the helicase domain in EO1 showed that GAdoV constitutes a novel clade, with equal distance to members of the viral families, aviridae . The twoaviridae . This peaviridae , was detaviridae  and do nin situ hybridization (FISH), using separate adomavirus-specific and polyomavirus-specific probes, demonstrated only the presence of adomavirus nucleic acid within histological lesions.Molecular findings suggest that only infection by GAdoV, not the polyomavirus, was productive in the guitarfish. Metagenomics with random amplification was performed to achieve agnostic NGS sequencing, revealing a much larger number of reads in affected tissue for the adomavirus  compared with the polyomavirus (634). Even though comparison of the number of NGS reads is only semiquantitative, a substantially larger amount of adomavirus DNA was present for sequencing than that of the polyomavirus. Both conventional and real-time PCR amplicons were reconfirmed by Sanger sequencing for the presence of the adomaviral nucleic acids. Furthermore, as detailed below, subsequent fluorescent 5 ± 2.53 × 103 copies/µl to 2.42 × 107 ± 1.2 × 107 copies/µl during the approximately 10 weeks while lesions were active , histopathology, and FISH were performed at three time points at 8- to 10-week intervals  followine active . Additioe active . Hybridi2 ± 4.26 × 101 copies/µl , tentatively named guitarfish adomavirus, and show molecular evidence of its pathogenicity. This virus and a previously described polyomavirus, GfPyV1, were discovered coinfecting proliferative skin lesions  in a giaThe 75-nm icosahedral particles of GAdoV are similar in size and shape to the two other related viruses from the Japanese and marbled eel –16 Fig.– and bearPolyomaviruses and other unique DNA viruses have been described only recently in fish, and little is known regarding their host range or disease potential. Now, in addition to the Japanese and marbled eel viruses –16, disc–Despite their similarities in size, ultrastructure, and genome organization, as well as their association with fish species, the diseases caused by the eel viruses are highly dissimilar from that produced by GAdoV. The Japanese eel virus produces epizootics of acute fatal disease characterized by systemic hemorrhage and tissue necrosis resulting from endothelial cell infection and damage , 15. In As evidenced by results of qPCR and FISH studies, in conjunction with histopathologic and electron microscopic findings, we infer that GAdoV is the cause of the proliferative skin lesions in this guitarfish. These findings warrant further investigation of adomaviruses as emerging infectious agents capable of producing varied disease signs and lesions in different fish species. Additionally, this is the first report to describe coinfection by a polyomavirus and adomavirus in skin lesions of a single host. Although they represent only distantly related viral lineages, coinfection by the two viruses could provide fertile ground for horizontal recombination events to occur, which could be a driving force for viral evolution.Rhynchobatus djiddensis caught in the wild and measuring 148 cm and 13.5 kg was transported internationally to a public aquarium. Soon after arrival, the animal developed erythematous skin lesions on its ventral skin and tail that became raised and papillary over an 8-week period. Biopsy samples of the lesion were collected, fixed in 10% neutral buffered formalin, and submitted to the Department of Pathology, University of Georgia College of Veterinary Medicine for routine processing and histological evaluation. Additional samples were frozen at −80°C, and samples were fixed in a solution of 2% glutaraldehyde, 2% paraformaldehyde, and 0.2% picric acid in 0.1 M cacodylate-HCl buffer (pH 7.0 to 7.3) and processed for transmission electron microscopy  with a previously described touchdown thermocycling profile  would cover the dynamic (linear) range of the assay (R2 ≥ 0.95). qPCRs for the tissue samples and standards were performed on a Bio-Rad IQ5 iCycler using iQ5 system software for analysis. One microliter of extracted DNA was added to each 25-µl reaction mix containing iQ SYBR green supermix (Bio-Rad) and 100 nmol each of the indicated primers. A two-step cycling program used an initial step of 3 min at 95°C, followed by 35 cycles, with 1 cycle consisting of 10 s at 95°C and 30 s at 60°C. Initial screening of all samples was done twice using one PCR well/sample. The final assessment of the presence of viral DNA was made on samples run in triplicate.SYBR green-based quantitative PCR (qPCR) was used to assess the presence and quantity of viral DNA in skin lesions. Primers targeting the adomavirus LO7 gene  were selin situ hybridization (FISH) assays using a digoxigenin-labeled probe and a biotin-streptavidin method were adapted from previously described protocols (Fluorescent rotocols , 23 usinrotocols . BrieflyMF946548.The genome of the sequenced guitarfish virus was deposited in GenBank under accession number


Three outbreaks of acute respiratory disease occurred at military camps in 2016 at Tibet, Sichuan and Yunnan province, China. The pathogen induced these three outbreaks were all confirmed as HAdV-55 by genotype-specific PCR. The outbreak in Tibet was the first report that HAdV-55 occurred in the high altitude . This study aims to determine the gene variation and evolution characteristics of these viral strains. Three strains of adenoviruses, LS89/Tibet/2016 (GenBank accession no. KY002683), SF04/SC/2016 (GenBank accession no. KY002684) and KM03/YN/2016 (GenBank accession no. KY002685) were obtained and confirmed by wholegenome sequencing. No multi-gene fragments recombination were found in these isolated HAdV-55 virus compared with previous reported HAdV-55 strains in China. The outbreaks in Tibet and in Sichuan continuously occurred. Virus isolated from Tibet (LS89/Tibet/2016) and Sichuan (SF04/SC/2016) had a similar mutation pattern and had a closer genetic evolutionary distance than KM03/YN/2016 strain, which indicates that the pathogens causing these two outbreaks may be of the same origin. Moreover, we found that heating was an effective way to inactive these viruses, which provide valuable information for the development of HAdV-55 vaccines. Our data provide new information for genetic evolution of HAdV-55, and contribute to the prevention and control of HAdV-55 infection in the future. Human adenoviruses (HAdV) is a non-enveloped, double-stranded linear DNA virus, with a diameter of 70~90 nm. HAdV usually causes acute respiratory diseases (ARD), which had become a leading cause of outbreaks in community, school and military camps ,2. To daTwo serotypes from species B, HAdV-3 and HAdV-7, are the common pathogens causing acute respiratory infection outbreaks in China previously . HoweverThree outbreaks of acute respiratory disease occurred at military camps in Tibet (January 2016 to March 2016), Sichuan (February 2016 to March 2016) and Yunnan (Jun 2016) respectively, in China. The outbreaks in Tibet and in Sichuan were continuously happened. Pathogens induced these three outbreaks were all confirmed as HAdV-55 by qPCR and sequencing. The outbreak in Tibet was the first report that HAdV-55 occurred in the high altitude . To analyze the mutation features of these viral strains, HAdV-55 virus caused these three outbreaks were isolated from swabs. Three strains of HAdV-55 virus, LS89/Tibet/2016, SF04/SC/2016 and KM03/YN/2016, were obtained and wholegenome sequences were submitted to GenBank. Sequence analysis was performed between these HAdV-55 strains and previous reported HAdV-55 virus in China. The gene variation of these three strains of HAdV-55 virus were not significant and no multi-gene fragments recombination were found. Laboratory characteristics, including thermostability and ultraviolet inactivation of these virus were tested.This study was approved by the Medical Ethics Committee of Center for Disease Control and Prevention of Chengdu Military Region. The patients’ identification information had been removed. This study aims to analysis the gene variation of HAdV-B55 virus isolated from swab samples and does not involve patients’ identification information. Therefore, the need for informed consent was waived by the Medical Ethics Committee.2.The human laryngeal cancer cell line HEp-2  was purchased from BioVector NTCC Inc, Beijing, China. and cultured under standard conditions in DMEM  supplemented with 10% fetal bovine serum (Life Technologies) plus 100 μg/mL of penicillin-streptomycin (Life Technologies) at 37°C in the presence of 5% CO2. The cytopathic effect (CPE) of inoculated cells were observed every day. Cell cultrues were harvested when the morphology of the cells corresponding to mid to full CPE (approximately 80% CPE). The harvested cell cultures were pooled and pelleted by centrifugation at 1000g for 5 min, which is the 1st generation virus stocks. The 2nd generation virus stocks were produced with 1st generation virus stocks infected Hep-2 cells, and so as the 3rd generation virus stocks. The 3rd generation virus stocks were used for whole-genome analyses and in vitro infection experiments.Swab samples were collected from patients with febrile respiratory infectious illness. HEp-2 cells were cultured in 24-well plates at 70% density and incubated with 100 μL swab sample for 6 hours, followed by the addition of fresh maintenance medium . The culture plates were incubated at 37°C in 5% COA wild type (WT) of HAdV-55 strain Y16/SX/2011 was obtained from Beijing Institute of Microbiology and Epidemiology [Twelve PCR products covering the entire genome of HAdV-B55 were obtained. Whole genome sequences were obtained using sequencing. Primers used were shown in 4 cells per well) were seeded overnight in 96-well plates. Frozen virus stocks were diluted (10−1) in sterile 1% aqueous sodium deoxycholate (Sigma Aldrich) and mixed for 15 min to disaggregate virus. The virus was then subjected to 10-fold serial dilution in maintenance medium  and 100 μL of the diluted virus from the 10−3 dilution onwards was added into each well of HEp-2 cells. Plates were incubated at 37°C and observed daily using an inverted phase contrast microscope for the appearance of distinct CPE. Virus titers were reported as 50% cell culture-infectious doses per volume (CCID50/mL).The virus titers were enumerated using the Reed and Muench method . Briefly50 and incubated at 37°C. Supernatants and cells were harvested at 1, 3, 5 and 7 d post-infection. The intracellular and extracellular viral levels were determined by real-time quantitative PCR (qPCR) assays.HEp-2 cells in DMEM containing 2% FBS were infected with viruses at an 100 CCID4 cells were seeded into each well of a standard 96-well plate and incubated at 37°C with 5% CO2. After overnight culture, the media was removed, and the cells were infected with different virus strains. Infected and uninfected cells were assayed at 12, 24 and 48 h post-infection using CCK8 kit. The absorbance was determined at 490 nm using a microplate spectrophotometer .Cell viability was determined using an Cell Counting Kit  according to the manufacturer’s protocol. Briefly, 1.5×10The viruses were incubated at 56°C for 30 minutes. HEp-2 cells were infected with normal and thermo-inactive viruses for 6 hours. Viruses were removed form the cells and fresh DMEM were added. Intracellular viral DNA levels were determined using real-time qPCR method at 24 hours post infection.HAdV-55 virus were inactive by ultraviolet (UV) irradiation at a wavelength of 254 nm. Specifically, viruses were placed in a 48-well plate and formed thin films which are necessary to ensure complete penetration of the UV light. Viruses were treated with UV irradiation for 30 minutes. The control viruses were equally placed in a 48-well plates without ultraviolet irradiation. HEp-2 cells were infected with UV-inactive and control viruses. Viral DNA levels were determined using real-time qPCR method at 24 hours after infection.. HAdV-55-F: 5’-AGATGAAGAAAGTAAACCGATTT-3’, HAdV-55-R: 5’-CCATCAAGGTCAGTCCAA-3’, HAdV-55-probe 5’-FAM-ATCAGCCAGAACCT CAGCT-BHQ1-3’. The 10-fold serial dilutions of viruses that had been quantitated by the Reed-Muench assay were used as standard samples for standard curve analysis.Cells were homogenized, and then viral DNA was extracted using a PureLink™ Viral RNA/DNA Mini Kit . The viral loads were quantified by using HAdV type-specific real-time qPCR assays. The following cycling conditions were used in the LightCycler 2.0 system : an initial denaturing cycle at 94°C for 10 s, followed by 40 cycles of amplification . The primers and probe used in this study are as followsp < 0.05.Statistical tests were performed using the SPSS 18.0 computer program . Data are presented as means ± standard error (SD). All data were analyzed using one-way ANOVA and Dunnett’s test. Significant difference was defined as Three outbreaks of acute respiratory disease occurred at military camps in Lasa city/Tibet (January 2016 to March 2016), Shifang city/Sichuan (February 2016 to March 2016) and Kunming city/Yunnan (June 2016 to July 2016) respectively, in China. Overall, more than 300 soldiers were affected in three outbreaks. There were no died cases during the outbreaks. Bioinformatics analysis based on PCR demonstrated that these three outbreaks were all caused by human adenovirus type B55 (HAdV-55). The outbreaks in Tibet and Sichuan continuously occurred. According to the results of epidemiological survey, there are personnel exchanges between military camps in Tibet and Sichuan.Adenoviruses were isolated from swab samples of patients from Tibet, Sichuan and Yunnan. Three strains of adenoviruses, LS89/Tibet/2016 (GenBank accession no. KY002683), SF04/SC/2016 (GenBank accession no. KY002684) and KM03/YN/2016 (GenBank accession no. KY002685) were obtained and confirmed by wholegenome sequencing. Phylogenetic tree for the whole genome sequences of these virus strains demonstrated that three adenoviruses isolates in this study were all subtype B55 . All preThe whole genome sequences of LS89/Tibet/2016, SF04/SC/2016 and KM03/YN/2016 had a homology of 99.8%. The comparisons of variations for all the HAdV-55 whole genome sequences were shown in The hexon and fiber protein are the major structural capsid proteins on adenoviruses . Hexon cThe phylogenetic trees based on hexon and fiber amino acid sequence were consistent with the result of nucleic acid sequences. Moreover, amino acid sequences of hexon and fiber protiens were highly conserved among these HAdV-55 strains. Only one mutation site  was found in hexon protein of KM03/YN/2016 strain compared with other eleven HAdV-55 strains listed in To further observe the growth kinetics of the three adenoviruses isolates in this study, HEp-2 cells were infected with LS89/Tibet/2016, SF04/SC/2016 and KM03/YN/2016 in the same titers. Supernatants and cells were harvested at 1, 2, 3, 5 and 6 days post-infection, viral DNA levels were determined through real-time qPCR method. As shown in We further detect the stability of adenoviruses under hot inactivation and ultraviolet irradiation. The viral DNA levels were decreased by 5% and 50% after treated with hot inactivation and ultraviolet irradiation respectively . HEp-2 cHuman adenovirus has been recognized as a common cause of acute respiratory disease. Life-threatening adenoviral pneumonia has previously been documented among military trainees, patients with AIDS and transplant recipients. To date, there are at least 69 HAdV genotypes reported, which are classified within seven species (A-G), among which viruses in group B are the common cause of ARD associated outbreaks ,3.In the United States, HAdV-3 and HAdV-7 are highly prevalent among children . The preHere, we report three outbreaks caused by HAdV-55 occurred in China in 2016, among which two outbreaks were occurred continuously. One of the outbreaks occurred from January to March 2016 at Lasa city, Tibet, with a sea level above 3658 meters, which is the first report of HAdV-55 outbreak in plateau area. Another outbreak occurred at Shifang city, Sichuan province, from February to March 2016. Epidemiological investigation was carried out immediately after the occurrence of ARD outbreak at Shifang city, and found that three are people coming from the affected areas of the outbreak in Tibet, which is supposed as the cause of the second outbreak. The following whole-genome analyses of these virus strains provides more convincing evidence for this view. The third outbreak occurred from June to July 2016, in Kunming city, Yunnan province. According to the result of epidemiological investigation, the third outbreak was occurred independently from the previous two outbreaks.To further analyze the source of the pathogens and clarify the mutation characteristics of viral genome sequences, adenoviruses were isolated from swabs of patients from these three outbreaks. The three isolated HAdV strains were named as LS89/Tibet/2016, SF04/SC/2016 and KM03/YN/2016 respectively. The whole genome sequences were obtained and phylogenetic trees were constructed. Through sequencing, homology analysis, and evolutionary tree construction, these three strains have highly similar sequences with previous reported HAdV-55 strains in China. All the three isolated HAdV strains were in the same evolutionary branch with HAdV-55. Result of nucleotide sequences alignment showed that the mutation characteristics of LS89/Tibet/2016 and SF04/SC/2016 were basically consistent, while not the same as KM03/YN/2016. Moreover, the phylogenetic analysis of HAdV-55 strains also showed that LS89/Tibet/2016 and SF04/SC/2016 were in the same evolutionary branch, while KM03/YN/2016 was different. The etiological characteristics were consistent with the results of epidemiological survey, which provides further evidence for the direct relationship between the first two outbreaks. LS89/Tibet/2016 was the first HAdV-55 strain isolated from plateau hypoxia environment. Therefore we particularly concerned about its genetic variation. However, large fragment recombination of the full viral genome was not found, even not so many point mutations in the key proteins such as hexon and fiber. Actually, the real source of LS89/Tibet/2016 was not clear according to the results of epidemiological investigation. The whole-genome analyse results support that the viruses are changed from local epidemic strains by normal mutation.The infection ability of three isolated HAdV-55 strains to HEp-2 cells was also observed and compared with a WT HAdV-55 strain Y16/SX/2011 which is isolated in Taiyuan city, Shanxi province, China, in 2012. No significant differences were found in the levels of infection among the three HAdV-55 strains and WT HAdV-55 strain. To date, no human adenovirus vaccine is available in China. Heating is one of the ways in which viruses can be made into inactivated vaccines ,18. In tIn conclusion, our study demonstrates the characteristics of these HAdV-55 strains from the pathogenic and genetic level. No large gene fragment recombination was found, which indicates that the three strains of HAdV-55 was not belong to a new virus. Heat treatment is an effective way of inactivation of these virus, while UV irradiation could to some extent reduce the infection ability of these viruses. Our study provide more information for genetic evolution and differentiation of HAdV-55, and contribute to prevention and control of HAdV-55 infection in the future.S1 FigPhylogenetic tree based on human adenovirus hexon gene  was constructed using neighbor-joining method with 1000 bootstrap replicates. The strains in our study are labeled with the black solid circle.(PDF)Click here for additional data file.S2 FigPhylogenetic tree based on human adenovirus fiber gene  was constructed using neighbor-joining method with 1000 bootstrap replicates. The strains in our study are labeled with the black solid circle.(PDF)Click here for additional data file.S1 Table(PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file.


Nicotiana tabacum with TMV (Tobacco mosaic virus), and Arabidopsis thaliana with TVCV (Turnip vein-clearing virus)), but not in resistant host systems (Nicotiana glutinosa and Chenopodium quinoa with TMV). Virus infected plants had significantly lower stomatal indices in systemic leaves of susceptible systems; N. tabacum 9.8% reduction and A. thaliana 12.3% reduction, but not in the resistant hosts. Stomatal density in systemic leaves was also significantly reduced in virus infected A. thaliana by 19.6% but not in N. tabacum or the resistant systems. In addition, transpiration rate was significantly reduced in TMV infected N. tabacum.Stomata are important regulators of carbon dioxide uptake and transpirational water loss. They also represent points of vulnerability as bacterial and fungal pathogens utilise this natural opening as an entry portal, and thus have an increasingly complex relationship. Unlike the situation with bacterial and fungal pathogens, we know very little about the role of stomata in viral infection. Here we report findings showing that viral infection influences stomatal development in two susceptible host systems ( This latter process is the driving force responsible for the transport of water and mineral nutrients from the roots to the aerial parts. When the guard cells lose turgor the pore closes and this has significance during, for example, periods of reduced soil water availability as it allows the plant some degree of water conservation. Stomatal aperture is controlled by an array of internal cues and environmental signals that serve to “set” stomatal apertures to suit the prevailing environmental conditions12452 concentration exert systemic control of stomatal development with signals from old leaves contributing to the control of stomatal development in new developing leaves781011Stomata are microscopic pores located on the aerial parts of terrestrial plants and consist of a central pore surrounded by two guard cells. When the guard cells are fully turgid the pore gapes open and this permits the uptake of COStomata also allow pathogens, such as rust fungi, bacteria and nematodes, to gain access to the plant and in recent years it has been recognized that stomata form part of the plants’ innate immune system1314Nicotiana tabacum infected with TMV (Tobacco mosaic virus) and Arabidopsis thaliana (Col-0) infected with TVCV (Turnip vein clearing virus). The ‘resistant hosts’, Nicotiana glutinosa and Chenopodium quinoa plants infected with TMV were also investigated. Both viruses exhibit systemic infections in their respective susceptible hosts, and symptoms appear 2–3 weeks after inoculation. Infected N. tabacum plants displayed mosaic symptoms in leaves developed after infection (systemic leaves) while after the same period, infected A. thaliana plants displaying leaf stunting in a systemic manner. Infections in resistant hosts are not systemic but localised, as virus-infected cells that are confined to small areas known as ‘local lesions’, which prevent the virus from spreading to the rest of the plant.Four host-virus systems were investigated for developmental changes in stomata density and index following viral infection. These included ‘susceptible hosts’, nd or 3rd leaf to develop since inoculation; leaf impressions were taken of the abaxial surface and images were acquired from 3 areas of each leaf and SD  and SI /}*100) were calculated.Duplicate sets of plants were inoculated with either the virus or sterile water (mock inoculation). After 7–21 days, a leaf which developed after infection, ‘systemic leaf’, was selected from each individual, typically the 2A. thaliana a reduction in SD was also observed   infected sweet potato plants have fewer, smaller, stomataStrawberry crinkle virus (SCrV), Strawberry mottle virus (SMV) and Strawberry mild yellow edge virus (SMYEV)) had fewer stomataBeta vulgaris L. leaves when infected with Beet yellow virus (BYV)Stomatal development is influenced by a range of environmental cues and local signals45et al.A. thaliana the mutants showed abnormal stomatal patterning. This was apparent with increased levels of paired stomata compared with WT, and low levels of triplet and quartette stomatal clusters were also found in the mutantRuggenthaler A. thaliana and N. tabacum infected with TVCV and TMV respectively is associated with a reduction in stomatal density (Arabidopsis) and index (both systems) and that this is a systemic response. In marked contrast in the resistant hosts, N. glutinosa and C. quinoa infected with TMV, there was no significant change in stomatal density or index in virus free systemic leaves of mock and virus inoculated plants. We also showed that a reduction in transpiration was observed in virus infected N. tabacum that exhibited reduced stomatal index/density at 2 weeks post inoculation.Here we extend these studies and present insights into the physiological responses during viral infection, likely as a consequence of altered stomatal development. We show that viral infection in two susceptible host types; The question of whether the reduction in stomatal index seen in the infection on susceptible hosts is part of a defence response by the plant or is of benefit to the virus during infection will be the subject of future investigations. In the absence of other compensatory factors a reduction in stomatal density and index would likely be associated with reduced transpiration and possible increased host water use efficiency101112A reduction in transpiration could also lead to reduced viral movement around the host. Viruses such as TMV are known to travel long distances through the phloem24In summary, here we report that during viral infection, in susceptible hosts, there is a decrease in the number of stomata that develop on the leaf surface of leaves that have developed post-infection indicating that this is a systemic response.Nicotiana and Chenopodium spp were allowed to germinate on soil after which they were transplanted into individual pots and grown under glasshouse conditions using natural lighting supplemented with artificial illumination under a natural photoperiod. Temperatures in the glasshouse were between 16–23 °C. Arabidopsis thaliana WT (Col-0) and mutant plants were germinated on soil and were transplanted 7 days after germination. These plants were grown under short day light cycles  at 22 °C and at a light intensity of 112 μmol m−2 s−1. All seeds were obtained from the University of Bristol Experimental Glasshouses.Tobacco mosaic virus) isolate used was TMV U1 (UK) from N. tabacum infected leaves. TVCV (Turnip vein-clearing virus) was kindly donated by John Carr  from infected N. tabacum material.The TMV  in sterile water. When plants were either 3–4 weeks old  or 4–6 weeks old (Nicotiana spp), a healthy leaf was then chosen for inoculation, typically 2nd or 3rd newest leaf, which was then labelled by piercing a small hole using a sterile pipette tip, and dusted with carborundum powder. The virus homogenate was applied by gently stroking it onto the leaf with a gloved finger. After several minutes the inoculated leaf was washed with sterile water and the plant was left at specified growth conditions to allow development of infection. Mock inoculations received the same treatment but sterile water was used instead of virus inoculum.Virus inoculum was prepared for manual inoculations by homogenising a small amount of infected leaf tissue  this was 21 dpi and in resistant host species (N. glutinosa and C. quinoa) this was 7 dpi; this discrepancy was due to the nature of infections established in the respective hosts. In susceptible species, as the virus causes systemic infection and symptoms take 2–3 weeks to appear post inoculation, whereas the resistant host species produce symptoms after 1–3 dpi, as the asymptomatic leaves are virus free. Once set, the leaf was removed and colourless nail polish (Revlon) was coated on the impression. After drying, colourless sticky tape (Sellotape) was used to transfer the secondary impression onto a microscope slide and stuck down.Dental resin  was applied to the underside of the 32) could be calculated. Student’s t-tests were used to asses significant differences ]) and data was tested for normality before applying the statistical test. Statistics were performed in Microsoft Excel (2010) and IBM SPSS Statistics 21.Slides were imaged using an inverted Zeiss Axiovert 200 M microscope driven by Volocity software . For each slide, 3 areas were captured . From these images, cell counts and size measurements could be made using Volocity Demo, and stomatal index (SI = {(no. of stomata)/}*100) and density  and the length of each stomata was recorded. Data in this section is from one biological replicate for each species. The data was tested for normality and a student’s t-test was used to assess differences in means.N. tabacum plants that were mock inoculated or infected with TMV, 14 dpi. Duplicate sets of plants were either inoculated with TMV or mock inoculated with sterile water, after 14 days the pots were wrapped in plastic and sealed around the stem. Mass was recorded daily on a balance for 5 consecutive days. Measurements were recorded for each individual and regression plots were formed and homogeneity of slopes was measured using STATGAPHICS-Centurion. Data was compiled from 2 biological replicates.Transpiration assays were performed on How to cite this article: Murray, R. R. et al. Plant virus infections control stomatal development. Sci. Rep.6, 34507; doi: 10.1038/srep34507 (2016).


We modeled the potential cost-effectiveness of increasing access to contraception in Puerto Rico during a Zika virus outbreak. The intervention is projected to cost an additional $33.5 million in family planning services and is likely to be cost-saving for the healthcare system overall. It could reduce Zika virus–related costs by $65.2 million . The estimates are influenced by the contraception methods used, the frequency of ZAM, and the lifetime incremental cost of ZAM. Accounting for unwanted pregnancies that are prevented, irrespective of Zika virus infection, an additional $40.4 million in medical costs would be avoided through the intervention. Increasing contraceptive access for women who want to delay or avoid pregnancy in Puerto Rico during a Zika virus outbreak can substantially reduce the number of cases of ZAM and healthcare costs. Aedes species mosquitos  and is linked to pregnancy loss and to problems in infants, including eye defects, hearing loss, and impaired growth (A primary strategy to reduce Zika virus–associated adverse pregnancy outcomes is to assist women who want to delay or avoid pregnancy. An estimated 65% of pregnancies in Puerto Rico are unintended (unwanted or mistimed), compared with 45% in the continental United States . The objective of this analysis was to estimate the potential cost-effectiveness of increasing access to contraception in Puerto Rico during the 2016 Zika virus outbreak.We constructed a decision tree cost-effectiveness model for a target population of 163,000 women who at the time of the intervention are sexually active with a male partner, fertile, not desiring pregnancy within the next 12 months, and not using permanent contraception methods  Figure 1.We assumed an intervention in place throughout a year-long Zika virus outbreak in Puerto Rico. We evaluated the costs and outcomes of increased access to contraception compared with no intervention . Output measures included numbers of ZAM cases prevented, including stillbirths, elective terminations, and live-born infants, and healthy life years (HLY) gained. Economic benefits of the intervention included avoided costs from ZAM cases prevented and costs avoided for monitoring for Zika virus–exposed pregnancies and infants born from Zika virus–infected mothers. In addition, the avoided cost of prenatal, delivery, postpartum, and neonatal care associated with avoided unwanted pregnancies was considered an economic benefit. In cost-effectiveness analyses, if total avoided cost exceeds the cost of an intervention that improves health, the intervention is considered cost-saving. For scenarios with positive net costs, we reported the incremental cost-effectiveness ratio (ICER), which is the net cost per HLY gained in comparison to the status quo.Independent of Zika virus–exposed pregnancies and ZAM, unintended pregnancy is associated with adverse maternal and child health outcomes. Because roughly 60% of unintended pregnancies are classified as mistimed, which might result in a delayed rather than avoided pregnancy, with the same costs occurring later   costs for Zika virus testing and monitoring for Zika virus–exposed pregnancies and infants, and 2) costs of ZAM cases . The cosThe lifetime cost per live-born infant with ZAM includes direct medical and nonmedical costs. ZAM is among the most severe types of microcephaly and is associated with loss of brain tissue volume, increased fluid spaces, and intracranial calcifications. All 3 cases of live-born infants with ZAM in French Polynesia demonstrated severe neurologic outcomes with delayed cognitive development . We used the MarketScan Commercial Database  with a sample of ≈100 million US residents covered by employer-sponsored insurance at any time during 2009–2014. We used average costs for 4 newborn infants with diagnoses of microcephaly and CMV who survived and were enrolled in a health plan for We determined the estimated non–Zika virus–related medical costs associated with women’s prenatal care, labor and delivery, and postpartum care for pregnancies ending in live birth and neonatal care from a study of US commercial health plan expenditures .Because many parameters used in the model are uncertain, we conducted sensitivity analyses on selected parameters, including different scenarios for the baseline and postintervention contraception use distributions in Puerto Rico. We tested alternate baseline contraception use distributions in Puerto Rico for women at risk for unintended pregnancy by using the actual distribution of method use reported in 2002  when considering the benefits from preventing ZAM and avoiding Zika virus–exposed pregnancy costs in the main scenarios and in most of the scenarios we tested. In scenarios in which the intervention is not cost-saving, it is still cost-effective relative to accepted cost-effectiveness thresholds (Our study has several limitations. First, we project the effects of a hypothetical intervention in place in Puerto Rico during the 2016 Zika virus outbreak. However, the qualitative results would apply in future outbreaks. Second, the baseline contraception use distribution is based on a 2002 survey; the current distribution in Puerto Rico might be different. Third, uncertainty exists about the effect of the proposed intervention on postintervention contraceptive use distribution; however, the sensitivity analyses indicate that different distributions of LARC types among switchers does not have a substantial influence on the results. Fourth, our study assumes that women have full access to healthcare providers. In areas with limited access to providers, the effectiveness of the intervention might be lower, although Puerto Rico has a similar ratio of physicians to population as the United States as a whole (Despite its limitations, our study has several strengths. First, the study is based on the most current available information. Second, the contraception scenarios are based on real-world programs and have resulted from consultation with subject matter experts. Third, expenditure data from a large sample of US residents with commercial health insurance were used to calculate the potential medical cost of ZAM on the basis of combinations of diagnostic codes for virus-associated microcephaly, although costs might be lower for similar children with public insurance. Finally, sensitivity analyses give consistent results indicating expected net cost savings associated with an intervention that would increase access to contraception in response to the Zika virus outbreak in Puerto Rico.Zika virus can cause devastating birth defects, and infants born with ZAM and their families will require lifelong support. Avoiding unintended pregnancies is a critical intervention to mitigate the effects of ZAM. Efforts to prevent adverse Zika virus–related pregnancy outcomes in Puerto Rico are especially important because of limited resources (Process for deriving the size of the target population, decision tree structure, and probabilistic sensitivity analysis of cost-effectiveness for a proposed intervention to increase access to contraception to women during the Zika virus outbreak, Puerto Rico, 2016.


Mayaro virus has been associated with small outbreaks in northern South America. We isolated this virus from a child with acute febrile illness in rural Haiti, confirming its role as a cause of mosquitoborne illness in the Caribbean region. The clinical presentation can mimic that of chikungunya, dengue, and Zika virus infections. Alphavirus, family Togaviridae) is a single-stranded positive RNA virus that was first isolated in Trinidad in 1954  swept across the island of Hispaniola, researchers at the University of Florida  have studied alphavirus and flavivirus transmission in Haiti in collaboration with the Christianville Foundation. This foundation operates 4 schools in the Gressier/Leogane region of Haiti (≈20 miles west of Port-au-Prince) that serve ≈1,250 students from prekindergarten to grade 12  for CHIKV and dengue virus (DENV); samples that were negative for CHIKV were cultured by using cell lines and conditions as previously described , as previously described . The firAlthough MAYV was originally isolated in Trinidad in 1954, subsequent reports of illness associated with this virus have tended to be associated with small, occasional outbreaks (30–100 cases) in northern South America (Our findings suggest that MAYV is actively circulating in the Caribbean region and that there may be a link between the strain circulating in Haiti and the strains that have been circulating in Brazil since isolation of the virus in the 1950s. The patient from whom we isolated the organism had fever and abdominal pain but no rash or arthralgia. However, given that the patient was co-infected with DENV-1, it is difficult to separate out symptoms that are specific for MAYV infection. Of note, the clinic physician empirically diagnosed typhoid and treated the patient accordingly. The patient was from a rural/semi-rural area of Haiti, reflecting an ecologic setting that differs greatly from sylvan Amazon regions where many of the other reported MAYV infections have occurred. Little is known about vectors for MAYV in Haiti; potential animal reservoirs, if any, remain to be identified.Aedes and Haemagogus spp. mosquitoes, inclusion of MAYV in studies of arbovirus transmission seems to be indicated.The recent emergence of Zika virus infection in the Caribbean region, and its identification as a major cause of birth defects, has brought a great deal of attention to arboviruses. Our findings highlight the multiplicity of arbovirus species in Haiti and the evolutionary relatedness among the viruses in Haiti and those circulating in Brazil, in keeping with prior work on Zika virus (Additional methods used for virus isolation and sequencing.


The aim of the present study was to calculate non-medical costs during year 1 after diagnosis in two cohorts of patients with early rheumatoid arthritis enrolled 1996–1998 and 2006–2009. Clinical data were collected regularly in both cohorts. Besides information about healthcare utilization and days lost from work, patients reported non-medical costs for aids/devices, transportation, formal and informal care. Formal care was valued as full labour cost for official home help (€42.80/h) and informal care from relatives and friends as opportunity cost of leisure time, corresponding to 35% of labour cost (€15/h). In both cohorts, only 2% used formal care, while more than 50% used informal care. Prescription of aids/devices was more frequent in cohort 2 and more women than men needed aids/devices. Help with transportation was also more common in cohort 2. Women in both cohorts needed more informal care than men, especially with personal care and household issues. Adjusting for covariates in regression models, female sex remained associated with higher costs in both cohorts. Non-medical costs in cohort 2 were €1892, €1575 constituting informal care, corresponding to 83% of non-medical costs. Total non-medical costs constituted 25% of total direct costs and 11% of total direct and indirect costs. Informal care accounted for the largest part of non-medical costs and women had higher costs than men. Despite established social welfare system, it is obvious that family and friends, to a large extent, are involved in informal care of patients with early RA, and this may underestimate the total burden of the disease. Rheumatoid arthritis (RA) is a chronic and disabling disease, and the economic consequences of the disease are substantial for patients and for society –3. CostsWe have previously reported the development of disease activity and costs in a cohort of patients with early RA enrolled 1996–1998  . A new RDuring the first year after diagnosis, the total costs in the 1996–1998 cohort amounted to €15,868, direct costs accounting for 30% and indirect costs 70% .From a healthcare perspective, direct costs typically include costs for outpatient visits, hospitalization, surgery and drugs, while adopting a societal perspective, relevant non-medical costs such as costs for medical aids, assistive devices, transportation, formal and informal care, regardless payer, should also be included . There iThe aim of the present study was to calculate the non-medical costs during the first year after diagnosis in two similar cohorts of patients with early RA, enrolled 10 years apart, from basically the same catchment area.During 1996–1998, 320 patients with early (≤1 year) RA were recruited from 10 rheumatology units in Sweden, corresponding to a catchment area of >1 million inhabitants . A similar cohort, TIRA2 (T2), was launched 10 years later, and 463 patients with early RA were enrolled 2006–09.Clinical and laboratory data was collected regularly in both cohorts. The 28-joint-count disease activity score (DAS28) was calculated , and patwww.gulare.com, http://plus.rjl.se). Transportation, regardless mode of travel, be it somebody driving or using public transportation, was reported as the patient’s expenses for the trips or as a calculation of the travel distance, using official tariffs for costs per kilometre . Formal care was valued as full labour cost for official community home help (€42.80/h) (www.skl.se), while cost for informal care from relatives and friends was valued as the opportunity cost of leisure time, corresponding to 35% of full labour cost (€15/h). The questionnaire in T2 was slightly revised compared to the questionnaire in T1, but basically similar questions were asked.Besides a baseline questionnaire with sociodemographic data including age, sex, marital status, educational level and employment status, patients in both cohorts were provided with health economic questionnaires every 6 months. The questionnaires were kept as diaries, and patients reported continuously all health care utilization and number of days lost from work. The health related quality-of-life instruments EQ-5D and EQ-VAS (0–100) were also completed . In addiwww.riksbank.se). Costs were calculated, applying a societal perspective, including all costs, regardless payer.Healthcare in Sweden is tax-financed, and apart from minor co-payments, basically all medical and non-medical costs, except informal care, are included in the welfare system, with almost full coverage of healthcare services. Costs in the first cohort were calculated, using unit costs from 2001, inflation adjusted to 2013, using the Swedish Consumer Price Index (CPI), and in the second cohort, unit costs from 2009, inflation-adjusted to 2013 with CPI. All costs were converted to 2013 euros, using the average exchange rate in 2013, €1 = 8.6494 SEK  and categorical variables as numbers and proportions. Differences were analysed by Student’s All patients gave written informed consent to participation. The study protocol was approved by the local ethics committee in Linkoping (Dnr M 168-05).Complete health economic questionnaires were available in 276/320 (86%) patients, 68% women in T1 and in 340/463 (73.4%) patients, and 70% women in T2 at 1-year follow-up. There were no differences in age, educational level, marital status, levels of sick leave and disability pension between patients with health economic data and patients with missing data.Patients included in the present study had similar clinical characteristics at inclusion, except for women in T2, being older and reporting more pain and higher EQ-5D. Women also had slightly longer education, 11 vs 10.6 years, but there were no differences in DAS28 and HAQ between the cohorts (Table p < 0.001) and 57.3% women vs 30.7% men in T2 (p < 0.001). In T2, more women needed help with personal care, 25.5% women vs 9.9% men (p = 0.001) . Prescription of devices facilitating everyday activities of daily living (ADL) was consistently higher in women than in men. Women were also to a higher extent prescribed orthoses, splints and orthopaedic footwear. Only few patients used canes or crutches in T2. As the specific question about walking aids was not asked for in T1, comparisons cannot be made. On the other hand, workplace adjustments paid for by the employer was reported in T1, but not in T2. Such adjustments, like special chairs and height adjustable desks, were available for 4.7% of patients in T1, the vast majority women.Total prescription of aids and devices was more frequent in T2 compared to T1, 38.8 vs 18.5% (p < 0.001) . The costs for informal care in T2 amounted to €1575, which is 5 times higher than total costs for remaining non-medical costs in T2 .Non-medical costs are often excluded in cost calculations, but for RA-patients, costs for medical aids, assistive devices, transportation, formal and informal care can be considerable . In the When costs for informal care were included in total non-medical costs, the proportions grew and increased from 2 to 11% of total costs and from 5% of direct costs to 25%. Since information on number of hours was available only in T2, costs for informal care could be calculated only in T2. However, since the proportion of patients using informal care was similar in both cohorts, 51.4 and 52.4%, respectively, we believe that costs for informal care in T1 are likely to be of the similar magnitude in T1 as in T2.Direct comparisons between various studies are hazardous since healthcare systems differ between countries, depending on various social systems and availability of healthcare. Levels of remuneration differ, and availability of aids and devices as well as formal care can be limited. Bearing this in mind, some comparisons can still be made.In a Belgian study, informal care, valued at €8.70/h, made up for 51% of total direct costs . This isThere are many difficulties in estimating the value of informal care, in both quantifying the number of hours used and valuing the hours. The main methods used for quantifying the time for informal caregiving are the diary method and the recall method. Comparative studies have shown that the recall method may give a higher estimate of informal care hours . In addiWhen estimating the value of an hour of informal care, there are different theoretical approaches, as well as a variety in how these approaches are applied. The opportunity cost method calculates the costs of informal care as the value of the best alternative use of the time used for informal care. If the caregiving hours could be used for formal (paid) production, the value of informal care is equal to the value of this production. If the informal caregiver uses his or her leisure time, the cost of informal care is equal to the value of this leisure time. An alternative to the opportunity cost method is the replacement cost method or shadow price, which means that the value of the informal care is given by the cost of hiring formal care, adjusting for different efficiency between informal and formal care . In the Despite the various methods of calculating costs, costs for informal care often represent the largest part of total non-medical costs , 25. In One might assume that non-medical costs could be lower in the 2006–2009 cohort, compared to the 1996–2008 cohort, due to more active treatment strategies in the recent decade . This waThere are a number of limitations in the present study. The use of self-reported information from questionnaires may be biased by selective information. The questionnaires were however kept as diaries over the period, and the patients registered continuously all medical and non-medical healthcare utilization.The T2 questionnaire was slightly revised compared to the T1, and this lead to some cost components being grouped differently in the cohorts. For instance, in T2, costs for canes and crutches were reported separately while in T1, these costs were included in costs for aids and devices. Costs for workplace adjustments paid for by the employer was reported in T1, but not in T2. Although only 4.7% of patients in T1 used this, the adjustments were rather costly and corresponding costs are probably not included in any other cost domain in the T2 questionnaire. Since workplace adjustments for employees are available to the same extent in 2006–2009 as in 1996–2008, we believe that patients in T2 were likely to have basically similar average costs paid for by the employer. However, if costs for work adaptation, for equal comparisons, are deleted from T1 , the difference between the cohorts remains and costs in T2 increases further compared to T1.Some patients were offered arrangements by the employer such as allowing rest periods during the day and changing the time that work started in the morning. These measures were difficult to quantify and have not been taken into account.A strength of the present study is the well-characterized patient material and the longitudinal prospective design with regular follow-ups, allowing analyses of long-term outcomes in two cohorts of patients with early RA, included from basically the same catchment area 10 years apart. The bottom-up approach with questionnaires in our study is also important, since information on most non-medical costs is available only through patient-derived data.To conclude, informal care accounted for the largest part of non-medical costs and women had higher non-medical costs than men, during the first year after diagnosis. Despite an established social welfare system, it is obvious that family and friends, to a large extent, are involved in informal care of patients with early RA, and this may underestimate the total burden of the disease for patient and for society.


Vitivirus in the family Betaflexiviridae.Here, we report the genome sequence of grapevine virus K (GVK), a novel single-stranded RNA virus identified from a transcriptome of grapevine. The genome of GVK is 7,476 nucleotides in length and encodes 5 open reading frames. GVK is a putative member of the genus  Vitis vinifera) is a commercially important fruit crop worldwide that is used for wine production. Grapevines are clonally propagated by cutting and grafting. Thus, the infection rate of diverse viruses and viroids in grapevine is usually very high. To date, more than 70 different viruses infecting grape plants have been identified .In the search for viruses and viroids from grapevine transcriptomes, we identified a novel virus from a transcriptome of the grapevine cultivar Lambrusco salamino . To summrameters . The assE value (9e−59). Based on the BLASTp results, GVK could be a potential member of the genus Vitivirus in the family Betaflexiviridae along with GVA and GVD. Taken together, we identified and de novo assembled a novel virus referred to as GVK, a putative member of the genus Vitivirus from the grapevine transcriptome.We identified 143 contigs associated with viruses and viroids. Of these, three contigs were associated with a novel virus referred to as grapevine virus K (GVK) isolate Jo (GenBank accession number MF072319). GVK is a positive single-stranded RNA virus with a length of 7,476 nucleotides (nt). The obtained GVK genome sequence is a nearly full-length genome containing sequences for the 5′ and 3′ untranslated regions . GVK encodes five open reading frames (ORFs), ORF1 (RNA-dependent RNA polymerase), ORF2 (unknown protein), ORF3 (movement protein), ORF4 (coat protein), and ORF5 (nucleic acid-binding protein). A BLASTp search using five GVK ORFs against NCBI’s nonredundant protein database revealed that ORF1 of GVK was homologous to the known grapevine virus A (GVA) with 52% protein sequence identity. No protein showed sequence similarity to ORF2 of GVK. ORF3 and ORF4 for GVK showed sequence similarity to GVA (50% protein identity) and grapevine virus D (GVD) (97% protein identity). The ORF5 of GVK showed sequence similarity to GVD (96% protein identity) with the MF072319.The full-genome sequence of grapevine virus K isolate Jo has been submitted to GenBank under the accession number


Hepatitis D virus  can establish a persistent infection in people with chronic hepatitis B, leading to accelerated progression of liver disease. In sub-Saharan Africa, where HBsAg prevalence is higher than 8%, hepatitis D virus might represent an important additive cause of chronic liver disease. We aimed to establish the prevalence of hepatitis D virus among HBsAg-positive populations in sub-Saharan Africa.We systematically reviewed studies of hepatitis D virus prevalence among HBsAg-positive populations in sub-Saharan Africa. We searched PubMed, Embase, and Scopus for papers published between Jan 1, 1995, and Aug 30, 2016, in which patient selection criteria and geographical setting were described. Search strings included sub-Saharan Africa, the countries therein, and permutations of hepatitis D virus. Cohort data were also added from HIV-positive populations in Malawi and Ghana. Populations undergoing assessment in liver disease clinics and those sampled from other populations  were analysed. We did a meta-analysis with a DerSimonian-Laird random-effects model to calculate a pooled estimate of hepatitis D virus seroprevalence.Of 374 studies identified by our search, 30 were included in our study, only eight of which included detection of hepatitis D virus RNA among anti-hepatitis D virus seropositive participants. In west Africa, the pooled seroprevalence of hepatitis D virus was 7·33% (95% CI 3·55–12·20) in general populations and 9·57% (2·31–20·43) in liver-disease populations. In central Africa, seroprevalence was 25·64% (12·09–42·00) in general populations and 37·77% (12·13–67·54) in liver-disease populations. In east and southern Africa, seroprevalence was 0·05% (0·00–1·78) in general populations. The odds ratio for anti-hepatitis D virus detection among HBsAg-positive patients with liver fibrosis or hepatocellular carcinoma was 5·24  relative to asymptomatic controls.Findings suggest localised clusters of hepatitis D virus endemicity across sub-Saharan Africa. Epidemiological data are needed from southern and east Africa, and from patients with established liver disease. Further studies should aim to define the reliability of hepatitis D virus testing methods, identify risk factors for transmission, and characterise the natural history of the infection in the region.Wellcome Trust, Royal Society. Liver cirrhosis and hepatocellular carcinoma accounted for more than 3% of adult deaths in sub-Saharan Africa in 2013, and this proportion is rising.Chronic hepatitis B and D virus co-infection is associated with expedited progression to cirrhosis, and has been characterised as the most severe form of viral hepatitis.11Despite widely held assumptions of hyper-endemicity, no previous systematic analyses have been done of the epidemiology of hepatitis D virus in sub-Saharan Africa. Routine testing for hepatitis D virus is rare in clinical practice, and because of low rates of case ascertainment, the overall contribution of the virus to the burden of liver disease in the region is unknown.Evidence before this studyWe searched PubMed, Embase, and Scopus with the terms “hepatitis delta” and “sub-Saharan Africa” and the countries therein for studies published in any language between Jan 1, 1995, and Aug 30, 2016 . We idenAdded value of this studyIn our analysis, we systematically compiled available data for hepatitis D virus epidemiology in sub-Saharan Africa, added novel cohort data from Malawi and Ghana, and provided pooled estimates of seroprevalence by region and population group. Our findings suggest substantial geographical variability, with pockets of hyperendemicity in central Africa and low seroprevalence in southern Africa. Overall, hepatitis D virus seroprevalence among HBsAg-positive populations in west and central Africa exceeds the estimated global seroprevalence. Published data do not allow firm conclusions to be drawn about risk factors for hepatitis D virus infection, including any possible association with HIV status. In most studies, detection of anti-hepatitis D virus was not confirmed with a second assay, and hepatitis D virus RNA detection was reported in only a few studies.Implications of all the available evidenceHepatitis D virus infection makes a potentially important contribution to the burden of liver disease in sub-Saharan Africa, particularly in west and central Africa, but important gaps in knowledge remain and more research is needed. Epidemiological data for east and southern Africa are insufficient. The natural history of infection with genotypes 5–8, which are endemic to Africa, remains to be characterised, including ascertainment of prevalence among patients with well characterised liver disease. Methods of detection of hepatitis D virus RNA need to be standardised, and the reliability of hepatitis D virus antibody and RNA testing in African settings should be investigated. Studies are needed to identify specific risk factors for hepatitis D virus infection, and guide the formulation of prevention and management policies.We did a systematic review and meta-analysis of studies of the prevalence of hepatitis D virus in sub-Saharan Africa, including added data for anti-hepatitis D virus and hepatitis D virus RNA prevalence from two HIV-positive cohorts in Ghana and Malawi, where scarce data were previously available. AJS searched PubMed, Embase, and Scopus for studies published in any language between Jan 1, 1995, and Aug 30, 2016, in which the prevalence of anti-hepatitis D virus antibody or hepatitis D virus RNA was reported. Search strings included sub-Saharan Africa, the countries therein, and permutations of hepatitis D virus . MC and I2 statistic. Seroprevalence in patients with liver disease compared with that in those without liver disease were pooled with a DerSimonian-Laird random-effects model.AJS extracted seroprevalence data. Duplicate data from the same locations were excluded. Confidence intervals (CIs) were computed by the Wilson method and pooled seroprevalence was calculated with the DerSimonian-Laird random-effects model with Freeman-Tukey double arcsine transformation.The study funder had no role in study design; data collection, analysis, or interpretation; or writing of the report. The corresponding author had full access to all the data in the study and had final responsibility for the decision to submit for publication.Our search returned 374 records, 30 of which met the inclusion criteria . The 30 Among general populations with HBsAg, hepatitis D virus seroprevalence varied widely within geographical regions, from 0·34% to 27·27% in adults in west Africa,Among HBsAg-positive patients recruited from hepatology clinics, seroprevalence ranged from 0·00% to 33·11% among nine cohorts in Ghana, Mauritania, Nigeria, and Senegal,Testing for hepatitis D virus RNA by qualitative PCR in patients seropositive for hepatitis D virus was done in 324 patients  in 16 populations.38Hepatitis D virus genotype data were included in seven studies.In our quality assessment, we identified recurrent issues with respect to incomplete description of study populations and sampling methods . TestingEpidemiological data for the prevalence of hepatitis D virus are needed to inform drug development and guide the formulation of policy on prevention, diagnosis, and management of the infection in sub-Saharan Africa, in agreement with recommendations from WHO.Enzyme immunoassay, the method of screening for hepatitis D virus that was used in the included studies, provides case ascertainment of superinfection with hepatitis D virus in carriers of hepatitis B virus, but is thought to be less reliable for the detection of simultaneous hepatitis B virus and D virus co-infection because of high rates of virus clearance and subsequent disappearance of anti-hepatitis D virus.Hepatitis D virus seroprevalence was highest among HBsAg-positive populations in central Africa, and lowest in east and southern Africa, although more data are needed from the latter two regions to confirm these findings. Even across similar populations from close geographical regions, seroprevalence was notably heterogeneous. Consistent testing methods  were used in all published studies, and thus available data suggest localised clusters of endemicity across sub-Saharan Africa. We did not identify an association between HIV status and hepatitis D virus seroprevalence, although HIV-positive and HIV-negative people in the same population were directly compared in only one study. Furthermore, HIV status was not ascertained in almost half the included studies. Thus, the absence of an association with HIV status should be interpreted with caution, and this finding contrasts with those from the USA and Taiwan, where increasing age, hepatitis C virus co-infection, and intravenous drug use were additonal risk factors for hepatitis D virus seropositivity.Hepatitis D virus infection is associated with accelerated progression to cirrhosis and increased incidence of hepatocellular carcinoma in carriers of hepatitis B virus, and cross-sectional assessments of hepatitis D virus seroprevalence can therefore be affected by survivorship bias.Our analysis has several limitations, which point to important research needs. First, we grouped various populations to facilitate summarising of the data. Published studies were few, addressed populations in various settings, and often had small sample sizes. Convenience sampling often included potentially unrepresentative populations, such as medical outpatients or health-care workers, although exclusion of these studies in sensitivity analyses did not affect estimates of hepatitis D virus seroprevalence in general populations. Substantial resources are needed to provide reliable prevalence estimates—sample sizes are large if drawn from unbiased community sampling, and have to be adjusted by the population prevalence of hepatitis B virus infection. Second, selection and method of ascertainment of the underlying hepatitis B virus infection are likely to affect hepatitis D virus estimates, and were frequently poorly described in the included studies. Third, serological results should ideally be substantiated with measurement of hepatitis D virus RNA to show active replication, but such confirmation was infrequently reported in included studies, raising the possibility of insufficient specificity of seroprevalence data even when commercial assays were used.In summary, our analysis provides a preliminary estimate of the epidemiology of hepatitis D virus infection in sub-Saharan Africa, and points to several important gaps in knowledge and directions for future research. Results suggest that co-infection with hepatitis B and D viruses could represent an important cause of liver disease in some regions and populations. Improved and systematically collected epidemiology data are needed, particularly for east and southern Africa, as is identification of risk factors for hepatitis D virus transmission. A clinical characterisation of hepatitis D virus infection is needed, particularly for the genotypes 5–8. A focus on patients with established liver disease is required to ascertain the burden of adverse hepatic outcomes attributable to hepatitis D virus. Future research initiatives should be accompanied by an assessment of the reliability of testing methods in the African setting.Hepatitis B virus infection remains endemic across sub-Saharan Africa62


NACHT, LRR, and PYD domains-containing protein 3 (NLRP3) inflammasome activation and effects during ribonucleic acid (RNA) viral infection are the focus of a wide range of research currently. Both the pathogen-associated molecule pattern derived from virions and intracellular stress molecules involved in the process of viral infection lead to activation of the NLRP3 inflammasome, which in turn triggers inflammatory responses for antiviral defense and tissue healing. However, aberrant activation of the NLRP3 inflammasome can instead support viral pathogenesis and promote disease progression. Here, we summarize and expound upon the recent literature describing the molecular mechanisms underlying the activation and effects of the NLRP3 inflammasome in RNA viral infection to highlight how it provides protection against RNA viral infection. The ribonucleic acid (RNA) virus has single-stranded (ss)RNA or double-stranded (ds)RNA as its genetic material. Infections with RNA viruses are responsible for a variety of diseases are significant threats to public health, including influenza, hepatitis, viral encephalitis, and autoimmune deficiency syndrome, to name a few of the most prominent. Host antiviral defense mechanisms are critical for clearing RNA viral infections and controlling the manifested diseases. The initiation of antiviral defense in response to RNA viruses involves interferon (IFN) response and inflammasome activation. The RNA viral infection usually leads to activation of the NACHT, LRR, and PYD domains-containing protein 3 (NLRP3) inflammasome, which is the intracellular multiprotein complex formed upon sensing pathogen-associated molecule patterns and/or damage-associated molecule patterns . The infThe NLRP3 inflammasome has been extensively investigated. Its major components include pro-caspase-1 and apoptosis-associated speck-like protein containing a CARD (ASC), in addition to the NLRP3 molecules. Upon sensing activating signals, the NLRP3 oligomerize to generate a caspase-1-activating scaffold, resulting in autocleavage, and activation of pro-caspase-1. The activated caspase-1 then shears pro-interleukin (IL)-1β and pro-IL-18 into IL-1β and IL-18, leading to production of their functional forms and release from cells to mediate protective  inflammatory responses. The activated caspase-1, however, can also promote caspase-1-dependent pyroptosis, a type of inflammatory programmed cell death; although, the consequence of pyroptosis induced by the NLRP3 inflammasome remains to be fully elucidated in RNA viral infection , human immunodeficiency virus-type 1 (HIV-1), vesicular stomatitis virus (VSV), respiratory syncytial virus (RSV), enterovirus 71 (EV71), hepatitis C virus (HCV), and SARS coronavirus (SARS-CoV), as detailed in Table NACHT, LRR, and PYD domains-containing protein 3 inflammasome activation requires two types of signals. Signal 1 primes the NLRP3 inflammasome and signal 2 initiates the sequence of its assembly, activation of the pro-caspase-1, and cleavage of pro-IL-1β and pro-IL-18.−/− mice in response to 24 h of IAV infection   and retinfection . Anothersignal 1 . Investisignal 1 , suggestPost-translation-dependent priming occurs upon the simultaneous engagement of TLRs and Nod-like receptors with their ligands, even causing rapid activation of the NLRP3 inflammasome (within 15 min) , 44. VirSignal 2 is responsive to a wide range of stimuli . However, the spectrum of different structures represented by these varied and distinctive agonists indicates that NLRP3 does not simply bind directly to each but instead relies on a common intracellular signal transmission pathway, triggered by the agonists themselves, for activation of the NLRP3 inflammasome. To date, five major signal mechanisms have been proposed as those responsible for or contributing to the NLRP3 inflammasome activation , 49. In via formation of the NLRP3-NEK7 complex (Viroporins are a group of virus-encoded proteins that enhance permeability of host cell membrane (through interactions with the lipid bilayer) to promote release of viral particles from cells , making  complex .Among the other viral proteins involved in NLRP3 inflammasome activation are IAV PB1-F2   and EV71Plasma vRNA, another common agonist for NLRP3, may bind with NLRP3 and act to subsequently activate it. Although NLRP3 contains a nucleotide-binding domain , RNA-senThe DHX family members contain some domains required for ATP binding, hydrolysis, and nucleic acid binding and unwinding, which usually acts as a cytosolic RNA sensor and triggers type I IFN response. DHX33, DDX19A, and DDX58 have been reported to regulate NLRP3 inflammasome activation. DHX33 was shown to combine directly with vRNA to bind to NLRP3 through the DEAD domain of DHX33 and the NACHT domain of NLRP3, forming a DHX33/NLRP3/ASC inflammasome complex to promote NLRP3 inflammasome activation . In thatAnother study demonstrated that DDX19A binds porcine reproductive and respiration syndrome virus (PRRSV) genomic RNA through its ATP-binding domain and C-terminal domain and directly interacts with the NLRP3 inflammasome in highly pathogenic-PRRSV-infected primary porcine alveolar macrophages, dependent on the DDX19A ATP-binding domain, and the NLRP3 NACHT and LRR domains. Knockdown of DDX19A expression led to reductions in both IL-1β secretion and virus titers in the PRRSV-infected porcine alveolar macrophages . Thus, D−/−, caspase-1−/−  , which occurs within 3–7 days of the IAV infection, and the subsequent promotion of secretion of various cytokines, such as tumor necrosis factor (TNF)-α, IFN-α, and MIP-2α , 5. Furtge fluid , and impturation . In a renfection . In WNV nfection , 17. In nfection ; the finnfection . Thus, tAberrant NLRP3 inflammasome activation usually results in progression of viral pathogenesis and the manifested disease in host, such as the robust NLRP3 activation induced by PB1-F2 during the IAV infection . The majUnlike RNA viruses, the DNA viruses, which have a large DNA genome, can encode viral homologs of some complex intracellular inflammasome-regulating molecules to regulate inflammasome activity; an example of this is the pyrin-only proteins and BCL-2 encoded by poxvirus , 71. TheNACHT, LRR, and PYD domains-containing protein 3 inflammasome activation in response to RNA virus is a fundamental innate immune response as well as an IFN response, which usually contributes to antivirus defense, although aberrant and inappropriate NLRP3 inflammasome response still results in the immunopathology and exaggeration of infectious disease, especially in some persistent virus infections. The activation itself is induced by different signals and probably occurs in different phases of the viral infection, triggering inflammatory response , and affecting outcome of the viral infection substantially. Thus, the proper regulation of NLRP3 inflammasome activation with specific NLRP3 inhibitors may have therapeutic benefit in controlling virus-related diseases and in clearance of the infecting virus.JY collecting literatures and drafting manuscript. JW design of the work, collecting literatures, drafting, and revising manuscript. YW discussing literatures, design of the work, and revising 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.


Cardiac catheterization laboratories (CLL) have continued to function as profit centers for hospitals. Due to a high percentage of material and labor costs, they are natural targets for process improvement. Our study applied a contribution margin (CBM) concept to evaluate costs and cost dynamics over a 5-year period.We retrospectively analyzed all procedures performed at a tertiary heart center between 2007 and 2011. Total variable costs, including labor time, material, and maintenance-expenses, were allocated at a global as well as a procedural level. CBM and CBM ratios were calculated by integration of individual DRG revenues.p < 0.001). Coronary angiography generated higher average CBMs per hour than coronary or electrophysiological interventions . The latter are characterized by relatively high per case material expenditures. On a procedural level, DRG-specific trends as a steady improvement of examination time or an increase in material costs were detectable.Annual case volume increased from 1288 to 1545. In parallel, overall profitability improved as indicated by a 2% increase in CBM ratio and a higher CBM generated per hour of CLL working time  contains supplementary material, which is available to authorized users. Cardiac catheterization laboratories (CCLs) are modern and technologically advanced facilities offering specialized care to patients with a wide range of cardiac and vascular disease. They are frequently considered as profit centers within a hospital system . AlthougContribution margin analysis is the preferred financial analysis tool in situations where the profitability of different treatment types has to be compared within an established service line , 9. KnowThe traditional costing systems do not separate between variable and fixed costs. As a consequence, time and material expenses are not appropriately allocated to specific procedures providing an unsafe base for strategic decision making. With the ongoing technical progress and the further development of hospital information systems, resource allocation at patient level will become widely available even paving the way for ad hoc analyses of individual procedure-related variable costs and CBMs .With the beginning of the financial crisis in 2007, many health systems worldwide had to face with an increase in cost and competitive pressure . Since cOur study was performed in a hospital setting being reimbursed by diagnosis-related groups (DRG). The DRG system is the most widely used in-patient billing standard in Europe and follows a flat-rate per case approach. The German DRG system was adapted from the Australian DRG system and today classifies patients into about 1200 DRG groups according to their diagnoses and resource consumption , 17.In total, 11,768 consecutive catheterization procedures from January 1, 2007 to December 31, 2011 of a University Medical Center in central Europe were analyzed. In a first step, patients’ baseline and procedural data were derived from the CCL database Metek  and the ongoing CCL patients’ registry. In particular, all materials utilized during the procedure were collected from the Metek database. The software provides a list of supply costs, including catheters and other disposable equipment, radiographic contrast medium, and medication. Each material position has been substituted in a synthesis step by a corresponding Euro amount, which has been generated from the purchase list of the material storage data base. In further step, the resulting database was combined with the hospitals’ information system . The corresponding DRG data were assigned to each case data set. As in most European countries, patients who had multiple CCL visits during hospital stay and such cases who received a combination of coronary and electrophysiological procedures during one visit were excluded. Furthermore, patients undergoing artificial respiration were excluded and those who received pacemaker therapy or any relevant procedure from other specialties . Patients with incomplete data sets were not included. All procedures were done by one of five interventional cardiologists with a high experience levels.n = 4632) which comprises all diagnostic left heart catheterization procedures; (b) percutaneous coronary intervention  representing all cases of coronary intervention; and (c) electrophysiological ablation  which defines all interventional cases in this sub-specialty. Furthermore, a selected cost analysis was performed on DRG level, including DRG F49E  and DRG F58B .Subsequently, the three most represented service lines were defined: (a) coronary angiography  are considered as organizational prerequisite for the value creating process within the catheterization laboratory. These costs are accordingly added to structure or fixed costs. Costs, which are procedure-dependent generated and related to material, staff, cleaning, and maintenance are considered as variable costs and compared with the DRG proceeds. The CBM amounts were analyzed on a case-by-case basis. To take an account of time constraints, a CBM per hour of CCL activity was introduced. Furthermore, the CBM ratio was calculated which is the percentage of revenues which remain after all variable costs have been covered (CBM/DRG-revenue). Further details about the single methodic steps can be found in the specifications of the accounting model . The stuAll data are given in terms of the mean ± SD. To compare group means with respect to systematic differences, an analysis of variance (ANOVA) has been performed. If variations between the means can be found, post hoc tests can be run to verify, which of both groups specifically differ. Before running post hoc analysis, the relevant factors were examined with respect to homogeneity (Levene-test). Depending on the results, either the Bonferroni test (variance homogeneity) or the Tamhane-T2 test (variance inhomogeneity) was performed. By means of a correlation analysis, it has been examined whether there is significance between two factors. Statistical analyses were performed using SPSS 16.0 for Windows .p < 0.001) and a 2% increase in CBM ratio  and CBM ratio  was obvious. Examination time significantly decreased (0.53 ± 0.34 vs. 0.46 ± 0.41 h) and material expenditures remained unchanged. In contrast, an increase in CBM per hour values but not in CBM ratio was noted in coronary intervention procedures between 2007 and 2011 (Table p < 0.001), this increase in productivity did not translate into a favorable development of profitability in terms of CB ratio, since material costs significantly increased .Coronary angiography was the most frequently performed procedure and generated the highest average CBMs per hour during the entire observation period [5831 ± 52905 € vs. 3458 ± 3631 € (PCI) vs. 1495 ± 2911 € (EA); Electrophysiological ablation was characterized by the lowest profitability from a CCLs perspective. Although CBMs per case were in a similar range as in the PCI and in CA group, CCL labor time was much more ineffective as indicated by low CBM per hour values. The reason behind the low profitability of EA procedures was high material expenditures Table .Contribution margin analysis was applied in an exemplarily way to evaluate the development of DRG F49E (CA) and DRG F58B (PCI). Revenues of both DRGs increased within the observed time period. Simultaneously, the length of hospital stay and procedural examination time decreased. Shorter procedural examination times translated into higher CBM per hour amounts which indicated a higher profitability of CCL labor time. However, it is important to realize that these positive changes were paralleled by falling material costs in DRG F49E but steadily rising material costs in F58B. As a consequence of an increase in overall variables costs, CBM ratio of DRG F58B declined which indicates a gradual loss of profitability.Our study, for the first time, reports on the application of a CBM approach to evaluate overall economic performance and to analyze the procedural portfolio of a CCL. Within the reported time period, a number of measures were implemented with a view to increase CCL performance. Examples include an improved scheduling of cases, a standardization of workflows, the introduction of flexible staffing, and a reduction in changeover times. Although total case number rose by 17% from 2007 to 2011, it was less clear if this increase resulted in a revenue surplus and thus a positive contribution to the economic performance of the hospital. In addition, there was uncertainty about the relative contribution of individual procedure groups to overall CCL performance. In the worst case scenario, the increase in case volume may have been attributed to a rapid accumulation of low or negative margin procedures and thus an unfavorable economic development.To meet these uncertainties, we have in a first step applied the CBM analysis to evaluate overall CCL profitability on a year-by-year basis. It is important to recognize that an increase in CBMs per case may not necessarily indicate a better CCL performance, since there is a steady annual increase in DRG revenues which may per se contribute to higher per case margins. In our setting, however, a 2% increase in CBM ratio indicates that savings in CCL resource consumption and thus direct procedure-related costs were realized. Profitability per case rises as a larger share of DRG revenues contributes to the payment of hospitals’ fixed costs. Interestingly, these savings were solely realized by savings in examination time, since material expenses hardly changed over time. Time management has previously been identified as an important target for CCL process improvement , 18. CohFrom a hospital’s economic point of view, it is not only important to determine overall profitability but also to figure out how profitable different service lines are. The CBM concept was, therefore, applied in a second step to compare typical CCL service lines and to identify those that give the highest returns. Among the three procedure groups analyzed, CA proved to generate the highest CBMs per hour and was performed with the highest case frequency per year Fig. . In addiAlthough portfolio analysis is a proven strategy in business and industry, such concepts have only recently entered healthcare industry. On the one hand, it is important to recognize that the majority of hospitals have to fulfill public service obligation and cannot arbitrarily cease unprofitable service lines. On the other hand, information on low- and high-margin services can well be used to further develop or rebalance the case mix. If low or negative CBMs are generated, the variable costs structure of these service lines has to be questioned critically to reduce these expenditures. In addition, other options as hospital co-operations or out-patient service models may be considered to improve process efficiency. Identifying high-margin service lines may help to focus development strategies, marketing efforts, and investments into areas of future growth with the aim to increase the volume of these cases. A study on the implementation of portfolio planning in plastic surgery confirmed that such efforts are effective in improving hospital’s financial position and patients’ satisfaction , 22 Add.Third, we demonstrated that CBM analysis when converted to a case-based measure provides valuable insights into the mechanisms underlying positive or negative economic developments. The cost structure of a CCL is characterized by a high percentage of manual activity and high material expenses. Given the high proportion of variable costs, even small reductions in these expenditures could have a direct positive impact on revenue surplus as CBM will increase. In the case of DRG F49E, savings in material costs and in labor time had a synergistic effect on CBM development. A divergent behavior was obvious in DRG F58B cases. The efforts to improve time management were almost used up by a steady increase in material costs. This example makes us aware how important it is to identify relevant cost drivers and their dynamics to develop appropriate strategies to support positive and to counteract negative trends Fig. .Fig. 2PeWithin a DRG-based payment system variations in service line profitability may create incentives to invest in profitable lines over less profitable ones which, in turn, may lead to the provision of inappropriate services. Such incentives in hospital activities are inherent to a per case reimbursement mechanism and are, therefore, not completely avoidable. To overcome such unintended consequences, basic models of DRG-payment are typically modified by regulatory instruments. In Germany and other European countries, provider-level budgets or ceiling was introduced to counterbalance incentives to treat more cases. More important, data on coronary angiographies and interventions are integrated into federal or national quality assurance programs. Recent reports of these institutions ensured that the indication of these procedures were in line with the current guidelines and not driven by economic aspects .With respect to the ongoing development of the German DRG system, the observed imbalance in the profitability of different service lines is not expected to disappear in the near future. When typical interventional or EA procedures from 2011 were virtually compared with their 2015 equivalents, little increases or even decreases in total DRG revenues can be noted . Double The study represents a single center experience and generalizability may thus be limited. Processes show a considerable variation across cardiac catheterization laboratories and material costs may differ depending on hospital contracts. However, institutional characteristics do not interfere with the transferability of our CBM concept but may indeed encourage its application. With the ongoing evolution of hospital information systems, even ad hoc analyses are conceivable.The CBM concept does not include fixed costs, such as room nursing costs, laboratory costs, and overhead costs. However, those costs (variable costs) which can be directly influenced by the catheterization team are well reflected.The CBM concept allows a comprehensive analysis of costs and cost dynamics in the setting of a catheterization laboratory. Its range of application includes global profitability analysis, portfolio evaluation, and a detailed cost analysis of specific service lines. The calculation principle is simple and the concept solely includes cost components which can be influenced by the catheterization team. This fact may increase user acceptance and thus the motivation of team members. Contribution margin analysis can be viewed as an integrative tool which can manage costs and the value creating process together.