Chronic obstructive pulmonary disease (COPD) is a major burden for the health care system, but the exact costs are difficult to estimate and there are insufficient data available on past and future time trends of COPD-related costs.The aim of the study was to calculate COPD-related costs in Finland during the years 1996–2006 and estimate future costs for the years 2007–2030.COPD-related direct and indirect costs in the public health care sector of the whole of Finland during the years 1996–2006 were retrieved from national registers. In addition, we made a mathematical prediction model on COPD costs for the years 2007–2030 on the basis of population projection and changes in smoking habits.The total annual COPD-related costs amounted to about 100–110 million Euros in 1996–2006, with no obvious change, but there was a slight decrease in direct costs and an increase in indirect costs during these years. The estimation model predicted a 60% increase up to 166 million Euros in COPD-related annual costs by the year 2030. This is caused almost entirely by an increase in direct health care costs that reflect the predicted ageing of the Finnish population, as older age is a significant factor that increases the need for hospitalisation.The total annual COPD-related costs in Finland have been stable during the years 1996–2006, but if management strategies are not changed a significant increase in direct costs is expected by the year 2030 due to ageing of the population. These figures are roughly in line with those published internationally,5 but even higher figures have been reported in, e.g., Northern Sweden6 and Denmark.7 Although the age-adjusted prevalence of COPD has not changed during the last few decades in Finland,3 the overall incidence and prevalence of COPD may increase due to ageing of the population.Chronic obstructive pulmonary disease (COPD) is highly prevalent worldwide, and it is estimated to become the third most common cause of death globally by the year 2020.8 A large share of this is thought to be caused by hospitalisations due to acute exacerbations of COPD. Comorbidities related to COPD further increase the total costs, but their effects are difficult to estimate. As COPD-related health care costs are associated with the severity of the disease, early diagnosis and smoking cessation is advocated to hinder the disease progression1 and thereby hopefully also decrease health care costs.COPD is a major disease in economic terms as well. The direct annual health care costs of COPD in the European Union are estimated to be about 23.3 billion Euros.1 they might in turn decrease the costs caused by outpatient visits. Most importantly, the drugs are also shown to decrease the risk for acute exacerbations and hospitalisations,1 and this might be an important factor in decreasing the total COPD-related costs. Further, changes in treatment protocols of hospitalised patients towards shorter in-hospital periods are likely to affect the total costs. Unfortunately, it is not well known how these factors have changed the COPD-related health care costs on a nationwide basis. The Finnish health care system with its comprehensive statistics offers a good opportunity for estimating COPD-related costs and the effects of the above-mentioned parameters on the costs.New drugs, such as long-acting bronchodilators, have been introduced for the treatment of COPD during the last two decades. The use of these drugs increases the direct medicinal costs of COPD. However, as the drugs improve health status in subjects with COPD,The aims of the current study were to study the change in total national COPD-related costs in Finland during the years 1996–2006 and estimate the future annual COPD-related costs in Finland for the years 2007–2030. The study was conducted by the Nordic Health Care Group and it was funded by Boehringer-Ingelheim Finland.The annual costs of COPD during the years 1996–2006 were calculated using health care registers and statistics covering the whole population in Finland (5.1 million people in 1996 and 5.3 million in 2006). The direct costs of COPD were regarded as costs caused by hospitalisations due to COPD, outpatient visits due to COPD and the costs of COPD medication. The indirect costs of COPD included sickness allowances and disability pensions due to COPD. In contrast to some other studies, we did not first identify a certain population of subjects with COPD and then add up all their health care costs from registers using their personal identity codes. Instead, we utilised national registers of different health care services or social benefits and identified all such events where COPD was labelled as the cause of that service/benefit.9 The costs for private health care or occupational health care systems were excluded, as no register data of COPD patients treated in those sectors could be obtained.The core of the Finnish health care system is public health care, which consists of primary (both outpatients and inpatients) and secondary health care (both outpatients and inpatients) services. All outpatient visits, hospitalisations and medical procedures in secondary public health care and hospitalisations in primary health care are registered with the related ICD-10 diagnosis code by the National Institute for Health and Welfare.10 and are given in 10 The unit costs for other years were derived from these by using inflation corrections with price index on public expenditure provided by Statistics Finland.11We included in the analysis all outpatient visits, days in hospital and medical procedures with ICD-10 diagnostic codes J43 (emphysema) or J44 (COPD) as the primary cause. The unit costs in 2006 for a day in hospital and for an outpatient visit were obtained from the National Institute for Health and Welfare12 but this register does not include the diagnostic codes. However, patients with severe enough asthma or COPD gain a special reimbursement for their medication. If a person entitled to such a reimbursement buys medication, data on this can be retrieved from the registers. Subjects with asthma and COPD are not separated in this register but are considered a single group of patients with obstructive pulmonary diseases. To avoid including medication used to treat asthma, we defined COPD-related drugs as drugs for the treatment of respiratory diseases bought by persons who were entitled to a special reimbursement for obstructive pulmonary diseases and who also used inhaled anticholinergics (these were only seldom used to treat asthma in Finland during 1996–2006). The costs for these COPD-related drugs were gained from the Finnish Statistics on Medicines.12In Finland, all drugs bought from pharmacies are registered with the buyers' identity to the Finnish Statistics on Medicines,13 and disability pensions with the ICD-10 code of the cause are registered by the Finnish Centre for Pensions.14 The data we retrieved from the registers included the number of days on sickness allowance and the number of subjects on disability pension due to COPD (J33 or J44). These were then transformed into lost man-years, and the costs for these were calculated using the unit cost, quoted by the National Institute for Health and Welfare, of 24,600€ per year in 2006.10Indirect costs of COPD, i.e., the loss of productivity due to illness, were calculated on the basis of sickness allowances and disability pensions due to COPD. In Finland, all sickness allowances with the ICD-10 code of the cause are registered by the Social Insurance Institution,15 work outside of paid work, care-giving by family members and immaterial costs, the loss of productivity or decrease in retirement pensions due to mortality caused by COPD.Mortality caused by COPD was not included in the calculations of loss of productivity, as most COPD-related deaths occur at older age when the subject has already retired. We excluded also the following indirect costs that are difficult to define and calculate: impaired productivity at work while still present (presenteism),The estimation model was built on an assumption that smoking history, age and sex are the major factors determining (the risk of COPD and) COPD-related costs. In this model, we did not try to estimate the actual number of subjects with COPD, but our model assumes that the fractions of active smokers and recent quitters that have COPD and cause COPD-related costs remain the same in the future.16 We then calculated in each age group and sex ‘smoking standardised COPD costs’ in 2006 by dividing the actual COPD-related costs in 2006 by the number of active smokers and recent quitters with the following weighing factors: 1 for active smokers and those ex-smokers who had quit 1–3 years ago; 0.75 for ex-smokers who had quit 3–5 years ago; and 0.5 for ex-smokers who had quit 5–10 years ago.First, we assessed the total COPD-related costs in 2006, as described in the first part of the manuscript, separately in each age group  in both sexes. Second, we retrieved the percentages of active smokers and recent quitters (during the previous 10 years) separately in each corresponding age group and both sexes from the Tobacco Statistics in Finland.16 We then multiplied these percentages of smokers and recent quitters by the estimated numbers of subjects in each age group and sex retrieved from the population projection in Finland17 to get the actual numbers of smokers and recent quitters in each group and year. Finally, these numbers of active smokers and recent quitters in each age group, sex and year were multiplied by the ‘smoking standardised COPD costs’ in 2006 using the same weighing factors for smokers and quitters as mentioned above. These total annual COPD-related costs during the years 2007–2030 were then corrected by an assumed annual inflation factor of 2%.To calculate the COPD-related costs in each age group and gender in the years 2007–2030, we needed to calculate the numbers of smoking subjects and recent quitters in each age group and gender in each of the years. We began by calculating the percentages of active smokers and recent quitters in each age group and sex for each year from 2007 to 2030 by assuming that the fractions of active smokers and recent quitters  continue to change at the same rate they have changed during the years 1996–2006 in Finland based on the Tobacco Statistics.The numbers of outpatient visits, days in hospital, days on sickness allowance and sickness pensions retrieved from the registers are given in The time trends of key COPD-related costs in the period 1996–2006 are presented in On the basis of our calculations, the annual COPD-related costs in Finland will increase by about 60% during the years 2007–2030 from 107 to 166 million Euros . The maiThe main findings of the study were that the total COPD-related costs in Finland were stable at about 100 million Euros annually during the years 1996–2006, but due to the forthcoming ageing of the population the annual costs are estimated to increase by about 60% by the year 2030.Cost-of-illness studies can be divided into two types: top-down studies, like this one, estimating total costs on the basis of the register data on a national level, and bottom-up studies that follow individual patients recording all their costs and then extrapolating these to national level by taking into account disease prevalence. Top-down studies generally include a limited number of different costs and they exclude underdiagnosing, whereas bottom-up studies include costs of comorbidities as well, and the prediction of total national costs is dependent on the accuracy of prevalence estimates. These issues must be taken into consideration when comparing different studies.18 estimated that in 1991 the total COPD-related cost in Sweden was about 3.2 million Euros per 100,000 inhabitants  in total, 1 €≈10 SEK and Sweden’s population was about 8.6 million in 1991). This estimate is somewhat higher than our estimate of total COPD-related costs in Finland in the year 1996 . This difference may be, in part, explained by the fact that the Swedish study also estimated mortality-related costs and by the fact that the mean length of hospitalisation may have decreased already between 1991 and 1996 in both the countries. A more recent study from Sweden19 gave a much higher estimate of the total COPD-related costs, totalling 15.7 million Euros per 100,000 inhabitants , but this may be explained by the bottom-up design of the study. In Denmark in the year 2002, COPD-related costs were estimated by a top-down study20 to be 4.7 million Euros per 100,000 inhabitants , which is more in the same range with our study and the top-down study from Sweden.A Swedish top-down study21 The difference from our result (about 100 million) may be explained by the broader spectrum of diagnostic labels included in the previous study  and J47 (bronchiectasis)) in retrieving health care utilisation data from the registers. Furthermore, the previous study calculated hospital treatment to be caused by COPD even if COPD was not the primary diagnosis, whereas the current study included only those treatment periods in which COPD was labelled as the primary cause of hospitalisation. Including only treatment periods with COPD as the primary diagnosis may underestimate the costs caused by acute exacerbations of COPD, as some of these periods may be labelled with a diagnosis of acute bronchitis or pneumonia. On the other hand, considering that all treatment periods are caused by COPD, where COPD is included in the list of the patient’s diagnoses, surely causes overestimation of COPD-related costs, as these subjects are treated for other diseases as well. Such differences between studies make it difficult to compare the results.A previous study in Finland reported that the annual costs of COPD were 194 million Euros.22 has estimated that COPD-related costs in Finland have decreased by 88% from 1997 to 2007. However, the costs at different time points were retrieved from different studies with considerable differences in the calculation methods used. This severely undermines the reliability of the stated huge decrease in COPD-related costs. To our knowledge, the current study is the first using the same methods consistently in assessing the annual COPD-related costs in Finland during a wide range of years. We found that there was no obvious change in total COPD-related costs during the period 1996–2006.A previous study19 in which costs of hospitalisations due to COPD decreased between the years 1999 and 2010 but there was a simultaneous increase in medication costs.Although the total COPD-related costs were stable during the years 1996–2006, there was a decrease in the direct costs and a corresponding increase in the indirect costs. The direct costs decreased mainly due to a decrease in the number of in-hospital days. This reflects changes in clinical practice regarding the treatment of acute exacerbations of COPD. The number of hospital treatment periods was about the same in 1996 and 2006, but the mean length of the treatment periods decreased, which also decreased the total number of treatment days. Our finding is in line with a recent report from Sweden23 estimated a 53% increase in national COPD costs in the United States from 32.1 billion dollars in 2010 to 49 billion dollars by the year 2020. This is in line with our estimate of a 60% increase in COPD costs by the year 2030 in Finland.There are not many prediction studies of future COPD costs available internationally, but a recent studyThis study is based on national registers covering the public health care system in the whole of Finland and gives a reliable estimate on the true costs of COPD. However, our estimate of total costs is likely a conservative one because of several reasons. First, the exact reason and primary diagnosis for hospitalisation of a subject with COPD and many comorbidities is not always straightforward, and some exacerbations of COPD are certainly treated under other diagnoses, which may lead to underestimation of hospitalisation costs. Second, outpatient visits in private health care systems or in primary public health care could not be included because of lack of reliable registers. However, this is probably not a significant issue, as the costs of outpatient visits are small when compared with hospitalisations and indirect costs, and the number of visits of elderly people in private health care is very small. Third, not all patients with mild or moderate COPD have a special reimbursement for medication, and the medication costs of subjects without this reimbursement are not included in this analysis. As medication costs are higher in subjects with severe COPD, this may not be a marked flaw but leads to underestimation in any case. Furthermore, the indirect costs are always difficult to define and calculate, and we did not include, e.g., the possibility that mortality due to COPD might decrease the expenses of retirement pensions due to earlier death of subjects with COPD.Our estimate of future costs assumes that the effectiveness of the health care system and the age-adjusted need for hospitalisation will be at the current level in the future also. However, if new treatments emerge that significantly decrease the need for hospitalisation or shorten hospitalisation periods, the increase in future costs might be smaller than estimated here. Our model on the future prevalence of COPD also assumed that the age-adjusted proportion of smokers in the population will continue to decrease at the same rate as it has in previous years. If the smoking trends deviate from the assumption either up- or downwards, this might decrease the liability of our estimate on the prevalence of COPD. However, there is a considerable latency period between changes in smoking habits and changes in COPD prevalence, and our time period for the estimate is only 24 years. Therefore, the accuracy in predicting future smoking habits is not that important for the time period of 24 years, but it would be much more pivotal for longer time periods.A weakness of the current study, as with all prediction models, is that in predicting future COPD-related costs we needed to estimate several parameters on the basis of their time trends in the past. However, while doing this we tried to be as realistic, or even conservative, as possible.In the past, the prevalence of COPD has been on the rise due to ageing of the population, and the medicine costs have also risen as new drugs have become available. However, these factors increasing the costs have been balanced out by increased efficiency of the health care system that has decreased the length of in-hospital periods. The Finnish population will be ageing much more rapidly during the next 20 years than during the years 1996–2006. The number of patients with COPD will increase especially in the older age groups, in which also the need for hospital treatment is the greatest. This is why there will be ageing-related increase in total COPD-related costs in the future, although this did not take place in the past. As the older age groups have already retired, the higher prevalence of COPD among these subjects does not cause an increase in indirect costs that are related to loss of productivity.The ageing-related increase in COPD costs should be taken into account in Finland when planning the national health care strategy. The results can be expanded to other western countries with similar smoking habits and ageing of the population. As the streamlining of the health care system in treating exacerbations has already taken place, this is likely not a significant option in reducing the costs anymore. Instead, focus should be on smoking policy to decrease age-adjusted COPD incidence and on the management of stable COPD to decrease the risk for exacerbations.In conclusion, the annual costs of COPD in Finland have been stable during the years 1996–2006, but the costs are estimated to increase by 60% until the year 2030 mainly due to ageing of the population. A strict smoking policy is needed to decrease COPD-related morbidity and the future rise in costs, and possible new treatment options need to be assessed in terms of cost effectiveness as well.


Bacillus thuringinsis (Bt) subsp. aegypti isolate Bt7 was engineered into the genome of the baculovirus Autographa californica multiple nuclearpolyhedrosis wild type virus, in place of the polyhedrin gene by using homologous recombination in Spodoptera frugiperda (Sf) cells between a transfer vector carrying the Bt gene and the wild type virus linearized DNA. Recombinant wild type virus containing the cry1Ab gene was detected as blue occlusion-negative plaques in monolayers of Sf cells grown in the presence of X-Gal. In Sf cells infected with plaque-purified recombinant virus, the cry1Ab gene was expressed to yield a protein of approximately 82-kDa, as determined by immunoblot analysis. The toxicity of the recombinant virus expressing the insecticidal crystal protein (ICP) was compared to that of the wild-type virus. Infected-cell extract was toxic to cotton leaf worm Spodoptera littoralis second instar larvae and the estimated LC50 was 1.7 μg/ml for the recombinant virus compared with that of wild-type virus which was 10 μg/ml.Baculoviruses have been genetically modified to express foreign genes under powerful promoters in order to accelerate their speed of killing. In this study a truncated form of cry1Ab gene derived from  Autographa californica nucleopolyhedrovirus wild type virus. These recombinant baculoviruses were evaluated under laboratory conditions for their improved pesticidal properties [Bacillus thuringiensis into a baculovirus in order to enhance its insecticidal activity.The development of synthetic pesticides since 1940 coupled with the improvement in chemical applications technology dramatically increased the potential for agricultural pest control . Moreoveoperties ,14. Becaoperties ,16 and Boperties ,18. Moreoperties  enhancedBacillus thuringiensis local Egyptian isolate Bt7 was provided by Prof. G. Osman Agricultural Genetic Engineering Research Institute (AGERI). Escherchia coli strain XL-1 blue  was used as the host for plasmid DNA preparation and propagation.Autographa californica nucleopolyhedrovirus wild type virus, strain E2 [train E2  was usedSpodoptera frugiperda cell line IPLB SF21-AE (Sf 21) was kindly provided by Dr. Hussen, A. Virology Dept. Faculty of Veterinary, Cairo University, Giza, Egypt. Sf21 cells were maintained at 27°C and grown in a TNM-FH medium that was supplemented with 10% (w/v) fetal bovine serum and antibiotics, and subcultured every 3 to 4 days.Spodoptera littoralis – Boisd – Noctuidae - Lepidoptera), was provided by Insectary of (AGERI), Giza, Egypt. The Egyptian cotton leaf worm Spodoptera littoralis (Biosd) were reared in insectary of AGERI under highly controlled conditions the larvae were fed on semisynthetic diet described by Shorey and Hale [Cotton leaf worm  based on the nucleotide sequence of the published cry1Ab gene.The cry1Ab fragment 2.2 kb was amplified by using a pair of specific primers i.e.WG1 forward (5′ATGGATAACAATCCGAACATC3′) and WG2 reverse (5′ TAGCGTAACGTAATT CTCTTT 3′). The primer was designed according to cry1Ab gene in gene bank accession number: gbKF93868.2.1. The amplified DNA fragment was cloned into pGEM-T easy vector . The new construct was named wpGM-Bt7. The wpGM-Bt7 was subcloned into pBlueBacIII transfer vector (Invitrogen) by restriction digestion using in vitro homologues recombination as described in the Invitrogen instruction manual. Linear viral DNA (500 ng) was co-transfected with (1-2 ug) wpBac-Bt7 containing the cry1Ab into S. frugiperda Sf21 cells using liposome-mediated transfection method according to Webb and Summers [-1, 10-2 and 10-3) were mixed with 1 × 106 S. frugiperda cells Sf 21 and seeded in complete TC-100 medium in 60 mm dishes  for 1 h. After incubation, the media were completely removed from all plates and the infected cells were overlaid with the supplemented TC-100 medium containing 0.5% baculovirus agarose and 150 μg ml-1 X-gal  as a chromogenic substrate. The plates were incubated at 27°C for five days and screened for the presence or absence of polyhedra by light microscopy. Plaque forming units (pfu) were detected as blue spots after five to six days post-infection (p.i.) and polyhedrin negative blue plaque were visualized using an inverted light microscope. The presence of Bt gene cry1Ab in the recombinant virus AcW-Bt7 were confirmed by PCR using WG1 and WG2 specific primers.Recombinant wild type baculovirus, containing the cry1Ab gene under transcriptional control of the polyhedrin promoter was introduced by  Summers . After 4-1 Tris-HCl pH 6.8, 2% SDS, 10% glycerol, 5% b-mercaptoethanol and bromophenol blue). Samples were denatured for five min in boiling water and separated in 10% polyacrylamide gel [Insect cells were infected with AcW-Bt7 at a multiplicity of infection (moi) of (10 moi/cell) recombinant virus. The expression of the crystalline protein of cr1Ab in the insect cells was detected by SDS-PAGE and immunoblotting assay. Sf cells infected with the recombinant virus were lysed in electrophoresis sample buffer (0.06 mol mide gel . Gels we-1 of polyhedra then assayed against second instar larvae of cotton leaf worm (CLW) as described by Ibara and Federici [50 was determined by probit analysis plot [The bioassay of infected Sf 21 cells with recombinant virus against second instar of cotton leaf worm compared to the wild type virus. Sf 21 cells were infected at an moiof 10 and collected at 72-h post infection (p.i.). For each virus, the cells were washed twice with PBS freeze-thawed several times to disrupt the cell membrane then the samples were diluted over a concentration range of 1.5-300 μg mlFederici . For eacsis plot . ControlPCR was carried out to amplify a 2.2 kb fragment by using whole Bt7 genomic DNA as a template. The PCR-amplified DNA fragment was purified and cloned in TA cloning vector . Screening for the positive clones (white colonies) was done using of both single and double restriction enzyme digestion. The new construct was named wpGM-Bt7. DNA sequencing of cry1Ab gene was carried out using the automated sequencer ABI PRISM 310. The sequence analysis of cry1Ab indicated that the amplified fragment is a typical Bt gene with low G + C content (37.63%) and high A + T content (62.37%). Moreover, the nucleotide sequence alignment of the cry1Ab gene revealed that the cry1Ab of Bt7 had an identity of 98% with the Genbank cry1Ab sequence while it was 100% identity with the Genbank at amino acid level. The sequence of cry1Ab gene of the isolate was submitted to GenBank with the accession #KC581790.NcoI and PstI endonucleases showed an 2.2 kb DNA fragment corresponding to the expected size of the cry1Ab gene. The resulting hybrid plasmid was named wpBac-Bt7.cry1Ab was subcloned into non-fused pBlueBacIII baculovirus transfer vector under the control of the polyhedrin gene promoter. Screening of the baculovirus hybrid plasmids using double digestion with Wild type baculovirus was chosen to be the parental virus for generation of recombinant virus by using the homologous recombination method. Sf cells were co-transfected with baculovirus hybrid plasmid wpBac-Bt7 and the linearized wild type virus DNA to facilitate the replacement of the cry1Ab gene (located in the hybrid plasmid) in place of the polyhedrin gene (located in the wild type baculovirus genome) via homologues recombination in the presence of Cellfectin cationic liposome reagent Figure . SDS-PAGSpodoptera littoralis 2nd instar larvae. The LC50 value for Acw-Bt7 and the wild type virus were determined by feeding of 2nd instar larvae of cotton leaf worm on a semi-artificial diet with different concentrations of the final whole culture (FWC) ranging from 1.5-300 μg ml-1 for both the recombinant virus Acw-Bt7 and virions of the wild type virus. The results of the bioassay revealed that the LC50 value for the recombinant virus was 1.7 μg ml-1, compared to the LC50 value for the wild type virus which was 10 μg ml-1. Regression analysis of the cotton leaf worm larval response to Acw-Bt7 (recombinant virus) and wild type virus are illustrated in  Cry1Ab of 82 KDa was produced in the infected Sf cells. These results proved that wild type baculovirus can be used to express and study the properties of the insecticidal Bacillus thuringiensis protein yielding information relevant to an understanding of the molecular biology of this protein as well as to improve the baculovirus insecticidal activity. These results agree with the findings of [orf) gene coding for the N-terminal 645 amino acids of the protoxin in a similar way into a wild type virus to avoid crystal formation. This protein produced in considerable amounts was biologically active, but as expected, did not precipitate into crystals confirming that the C-terminal part of the Cry1Ab ICP is required for crystal formation [orf) crystal protein expressed by AcNPV/JM3 recombinant virus from the polyhedrin promoter in Sf cells was highly toxic to P. brassicae larvae. These results indicated that the wild type baculovirus-expressed ICP is authentic and could in principle enhance the insecticidal action of a recombinant baculovirus. The results of the bioassay revealed that the LC50 value for the recombinant virus was 1.7 μg ml-1, compared to the LC50 value for the wild type which was 10 μg ml-1. These results of the bioassay revealed that the recombinant virus is 5-fold more effective than the wild type virus, and this results will encourage us to test this recombint virus on other lepidoperan pests. This is in agreement with the results of [AcOBtm and AcOBt73 had at least 1/5 of the expression of the same proteins in insects infected with the non-occluded viruses (polyhedrin-based promoter), AcBtm and AcBt73. This lower expression could be due to the simultaneous expression of the polyhedrin gene, since they might compete for the same resources or transcription factors inside the cell. The toxic action of Bt in vivo is known to be dependent on the binding of activated toxin to the external surface of midgut microvilli, where the toxins appear to bind to specific proteins receptors [Sf cells indicated that this Bt toxin may act inside the cell and is capable of inserting into the cell membrane from the cytoplasmic side [Because the C-terminal half of 135-kDa Cry1 is not toxic, it could be eliminated and use only the N-terminal half, in the same time the truncated proteins do not form inclusions. A recombinant virus containing the truncated cryIAb gene from dings of ,30, who ormation ,32. Chansults of -35, who eceptors ,37 and teceptors  that leaeceptors . Moreovemic side ,41. Thesmic side ), who remic side ,43. Charmic side . With grThe authors declare that they have no competing interest.Developing original approach (GO), improving and confirming the technique , performing experiment , analyzing data , writing the manuscript , molecular genetic studies , participated in the sequence alignment and drafted the manuscript . All authors read and approved the final manuscript.


Rodent protoparvovirus 1, TVX has a 111 nucleotide deletion in its nonstructural (NS) gene, a 24 nucleotide insertion in VP1, and a 93 nucleotide repeat initiating from the C-terminus of the capsid gene.The orphan parvovirus tumor virus X (TVX) has potent oncolytic activity. Compared to other viruses from the species  Protoparvovirus are inherently oncosuppressive and oncolytic in human tumors, while failing to kill untransformed human cells (–Rodent parvoviruses from the genus an cells . This inan cells –4.Tumor virus X (TVX) was isolated in 1965 from a permanent human amnion cell line and shown to have limited serological overlap with rodent parvoviruses Kilham rat virus (KRV) and H1  and HelREV (5′ GTGTTGGTTCAATCTGTTTGCTGCC 3′), were selected as regions of exceptional conservation identified by aligning the helicase domains of NS1 proteins encoded by rodent parvoviruses minute virus of mice (MVM) (GenBank J02275), H1 (GenBank X01457), LuIII (GenBank M81888), and KRV (GenBank AF321230). Sequences downstream of this site and extending into the stem of the viral left-end hairpin telomere were then sequenced using the the MVM-based primer MVHLP (5′ TTCACATGGTTGGTCAGTTCTAAAAATGATA 3′). Sequences upstream of the helicase and extending into the right-end viral telomere were determined using forward primers based on acquired TVX sequence and reverse primers selected from regions of high conservation identified by aligning the genomes of 17 viruses and virus strains from the species The TVX genome contains three conspicuous sequence anomalies when compared to other rodent parvoviruses: a deletion, an insertion, and a repeat. TVX has an in-frame, 111 nucleotide deletion within the coding sequence of NS1 and the second exon of NS2, deleting amino acids 587 to 624 of NS1, and 96 to 132 of NS2. This deletion bears striking resemblance to that of an H-1 variant named H-1 dr (KJ631100.The GenBank accession number of TVX is


During viral infection the first challenge that viruses have to overcome is gaining access to the intracellular compartment. The infection process starts when the virus contacts the surface of the host cell. A complex series of events ensues, including diffusion at the host cell membrane surface, binding to receptors, signaling, internalization, and delivery of the genetic information. The focus of this review is on the very initial steps of virus entry, from receptor binding to particle uptake into the host cell. We will discuss how viruses find their receptor, move to sub-membranous regions permissive for entry, and how they hijack the receptor-mediated signaling pathway to promote their internalization. Viruses are strict intracellular parasites and their life cycle is fully reliant on hijacking cellular functions to promote their replication and spread. Virus entry is the very first step of viral infection. Tremendous effort has been made to characterize the cellular receptors and the entry pathways that mediate virus internalization. With the development of high-throughput technologies and haploid genetic screens, a plethora of virus receptors and their associated endocytic pathways have been identified in the last few years ,2,3,4. WThis review will focus on the very early steps of receptor-mediated endocytosis of viruses, from receptors binding to the physical internalization of the viral particle into the host cells. We will discuss the current knowledge of the dynamic process of virus/cell surface and virus/receptor interaction. Additionally, we will detail our current understanding of the molecular signaling strategies that viruses utilize to promote their specific endocytosis and the endocytic/sorting motifs of receptors that are exploited by viruses for infection.i.e., heparin sulfate, sialic acid, gangliosides) to which virus particles can bind electrostatically. Following this primary attachment, the virus particles have to interact with the specific virus receptor(s) in order to be internalized. Emerging evidence indicates that cell signaling is strongly activated during viral infection and might facilitate viral uptake and appropriate intracellular targeting . T. T60]. TThe Kaposi’s sarcoma-associated herpesvirus (KSHV) is another excellent example of a virus that relies on signaling and actin reorganization for entry . The ectVaccinia virus likely represents the best evidence for a direct role of virus-induced signaling in uptake of virions through macropinocytosis. After reaching the plasma membrane, vaccinia virus activates a complex signaling network that involves PAK1 and leads to major alterations in the actin cortex and membrane blebbing, which result in the internalization of particles by macropinocytosis . The virIt is clear that virus binding and internalization represent a series of events that involves hundreds of cellular factors, which are highly dynamic, interconnected, and coordinated in time and space. A predominant part of the information available is for viruses that depend, directly or indirectly, on macropinocytosis for entry but, it is not always clear whether macropinocytosis is used only as an entry pathway or rather as a means to disrupt the actin cortex to facilitate virus entry through other endocytic routes. Further studies are required for viruses, which use alternative pathways for infection. For most viruses, many basic but important questions remain open C. (1) Doetc. [In the past decades, comparisons of the cytoplasmic tail of many receptors that use clathrin for endocytosis support the view of a high diversity of endocytic motifs rather the existence of common endocytic motifs . The motetc. ,89,90,91etc. . In contetc. ,93. Lastetc. ,95. HoweN-glycans [Regarding virus entry, little information is available about the endocytic and signaling motifs important for the uptake and sorting of virus particles into endocytic compartments. Experimental evidence for a direct role of such motifs in receptor-mediated internalization of virions is rare. In this respect, DC-SIGN represents one of the most interesting and documented virus receptor models. This cell surface type II transmembrane protein can bind to multiple viruses from various viral families through high-mannose -glycans . Though -glycans . Interes-glycans ,98. It iBy interacting with specific adaptor proteins, endocytic motifs determine in general the internalization pathways of cargo, and possibly of viruses. CME represents the best-documented sorting processes of cargo from the plasma membrane into the endocytic machinery. As previously mentioned, DC-SIGN contains a LL-based motif in the cytoplasmic tail, which acts as a typical docking site for adaptor proteins required for the formation of clathrin-coated pits, and therefore, receptor-induced CME ,99. The To our knowledge, there are only a few reports about endocytic motifs that are important for virus entry. The β1 integrin that serves as co-receptor for reovirus has a cytoplasmic tail with two NPXY motifs, known for their role in CME. Cells that have modified NPXY motifs lead to a reduction in reovirus infectivity . HoweverIn this review, we have summarized current knowledge of the early virus-host cell interactions from virus attachment on the cell surface, including interaction with receptor and induction of signaling, to uptake. While virus/receptor interactions and the subsequent intracellular trafficking of virus particles have been extensively studied, it is apparent that little work has addressed the transition processes between the extracellular and intracellular stages, and a substantial number of issues remains unresolved ,107.Receptor-mediated endocytosis of viruses is well established, as internalization of virus particles is fully dependent on the presence of virus-specific surface exposed cellular receptors. Viruses can only infect cells that display their specific receptors. Whether and how virus/receptor interactions induce specific signaling  that promotes internalization of the viral particle is much less understood and it remains unclear for many viruses whether the entry-associated signaling is a cause or a consequence of the entry event. Some viruses seem to not induce active signaling, however this does not necessarily imply that these viruses are randomly endocytosed in a completely passive manner. It has been suggested that the multivalent nature of the viral capsid  could constitute a “signal” that leads to particle internalization. This virus-induced receptor clustering could increase the probability of the virus/receptor complex to be up taken by stabilizing the formation of the endocytic structure . RecentlIt is frequently unknown whether viruses induce the same receptor clustering and signaling as the natural ligand of the receptors that they hijack. The reason is probably due to the absence of information about the signals produced by natural ligands themselves. EV1 is thus far the sole documented example of a virus that uses an endocytic pathway distinct from that of the natural ccligand . SimilarCell surface receptors, such as Trf receptor, GM1, and other gangliosides, often lack known signaling/endocytic motifs or a prominent cytoplasmic tail. In the latter case, this strongly suggests the involvement of additional co-receptors or microdomains in which endocytic receptors would mediate the internalization of virus/receptor complexes. The adaptor and coat proteins associated with such processes are also poorly characterized for most endocytic routes and viruses. The use of different adaptors likely generates different downstream signals, which can be useful for viruses, both in the short term, by triggering uptake, and in the mid-term, by creating a favorable and beneficial environment for the establishment of infection.While much work has focused on signaling that emanates from the plasma membrane, it is not clear whether virus-dependent signaling can occur through internalized receptors. An increasing number of viruses have been reported to remain attached to their receptors after uptake or become attached to their receptors within endosomal vesicles, at least during the early steps of the endosomal maturation . Recent Many questions about virus entry remain open (1) Whether viruses use endogenous endocytic pathways or are rather able to subvert the cell machinery to promote their own, specific entry route; (2) Whether virus uptake occurs via a constitutive endocytic process or through a ligand-induced endocytic mechanism; and (3) What are the benefits for viruses to use one of these possibilities? It clearly appears that the results gleaned from virus studies may provide valuable information, not only about viral infections, but also about hard-to-investigate, basic mechanisms that control the first stages of cargo uptake and signaling into the cells.


Extreme climatic events are growing more severe and frequent, calling into question how prepared our infrastructure is to deal with these changes. Current infrastructure design is primarily based on precipitation Intensity-Duration-Frequency (IDF) curves with the so-called stationary assumption, meaning extremes will not vary significantly over time. However, climate change is expected to alter climatic extremes, a concept termed nonstationarity. Here we show that given nonstationarity, current IDF curves can substantially underestimate precipitation extremes and thus, they may not be suitable for infrastructure design in a changing climate. We show that a stationary climate assumption may lead to underestimation of extreme precipitation by as much as 60%, which increases the flood risk and failure risk in infrastructure systems. We then present a generalized framework for estimating nonstationary IDF curves and their uncertainties using Bayesian inference. The methodology can potentially be integrated in future design concepts. Human activities in the past century have caused an increase in global temperature1251791711121314Current infrastructure design concepts to deal with flooding and precipitation are based on local rainfall Intensity-Duration-Frequency (IDF) curves. These curves are widely used in municipal storm water management and other engineering design applications across the world171819202122232417262728Given the observed increase in heavy precipitation events, we argue that the IDF curves should be updated to account for a changing climate, especially for urban infrastructure design1830313233343536374041424344454647Our analyses are based on ground-based observations of precipitation extremes  from the United States National Oceanic and Atmospheric Administration Atlas 14, the basis for IDF curves in the United StatesA unique feature of this modeling framework is that it offers uncertainty bounds of IDF estimates based on a Bayesian approach , the difference between the nonstationarity (14.7 mm/hr) and stationarity (9.1 mm/hr) extreme precipitation is about 5.6 mm/hr (+61.5%); while for a 10-year 1-hr event, the difference between nonstationarity (35.0 mm/hr) and stationarity (25.9 mm/hr) extreme precipitation is over 9.1 mm/hr (+35.1%). Even for a small watershed, this extra 9.1 mm/hr (+35.1%) precipitation would lead to a significant increase in flood peak. In other words, a stationary assumption will underestimate the peak flood and as a result the actual flood risk will be higher than what the system or infrastructure is designed for.The differences between the nonstationary and stationary estimates decrease for longer durations . This implies that in this station, shorter precipitation events have been intensified more in the past decades, while longer events have not changed substantially. For the same station shown in By examining storm durations, we found that the shorter the duration the larger the differences between the nonstationary and stationary extremes. As an example, for the 100-year return period, the differences between nonstationary and stationary IDF curves of 1-hr and 2-hr events reduce from 4.7–15.6 mm/hr to 1.4–7.3 mm/hr, while for a 168-hr storm, the difference approaches zero see . SimilarInfrastructure health and safety during precipitation extremes is closely related to human health and security, particularly downstream of major structures . For this reason, methods that can account for changing precipitation extremes are essential for updating engineering standards and design codes. Potential non-uniform and climate-induced changes on heavy rainfall events calls into question the accuracy and adequacy of current infrastructure design concepts, which rely on an assumption of climate stationarity. Previous studies show that limited locations across the United States exhibit a strong nonstationary increase in precipitation extremes. The reported underestimation of extremes corresponds to the few stations investigated in this paper that exhibit a strong nonstationary increase in precipitation extremes. While still limited locations exhibit a statistically significant increasing trend in precipitation extremes, future projections indicate that extremes may become more frequent. We show that ignoring the nonstationary assumption could lead to substantial underestimation of extremes, especially at sub-daily durations .We outline a novel framework to create the next generation of IDF curves to be incorporated into infrastructure design. This paper does not argue that the proposed methodology should be used for all stations. In fact, the suggested nonstationary IDF curve estimation method would only be necessary when observations or model projections indicate a nonstationary future climate. If there is no indication of change in historical records or model simulations, current stationary IDF curve models may be sufficient for infrastructure design. The presented methodology can be used to improve our understanding of the climate-induced changes in the intensity, duration and frequency of heavy precipitation, and can potentially be integrated in design concepts. However, infrastructure design and construction require substantial investment over a long period of time and effective integration of this methodology as well as development of adaptive design frameworks will require collaborative and interdisciplinary research with engineers, policy makers, economists, climate scientists and decision makers.Finally, given that IDF curves have been widely used for design and practical applications, this study focuses on a nonstationary framework that still relies on the IDF concept. Alternative methods such as the stochastic storm transposition method offer a different way forward for flood risk assessment56x) is defined for x) is either 0 or 1μ), the scale parameter (σ) and the shape parameter (ξ) to specify the center of the distribution, the deviation around μ, and the tail behavior of the GEV distribution, respectively. For ξ→0, ξ < 0, and ξ > 0, the GEV leads to the Gumbel, Weilbull and Fréchet distributions, respectively.The GEV distribution is a combination of Gumbel, Fréchet, and Weibull distributions, and is based on the limit theorems for block maxima or annual maximaθ =  are independent of timeμ) is assumed to be a function of time  are the regression parameters.The extreme value theory of stationary random sequences assumes that statistical properties of extremes  test is used to detect trends in annual maximum rainfall intensity of the selected storm durations . The null hypothesis of no trend is rejected if the test statistic is significantly different from zero at a 0.05 significance level. Upon detection of a significant trend, the location parameter will be computed under the nonstationary assumption . This wiN annual maxima . Assuming independence between observations, the Bayes theorem for estimation of GEV parameters under the nonstationary assumption can be expressed as51y(t) denotes the set of all covariate values under the nonstationary assumption, and stationarity can be treated as a special case without the term y(t). The resulting posterior distributions μ1, μ0, σ, ξ).Providing uncertainty estimates for IDF curves is fundamental to risk assessment and decision making. In this study, a Bayesian-based Markov chain approach is integrated into the nonstationary GEV for uncertainty assessmentl maxima  and 4 inμ0 and μ1 for White Sands National Monument Station in New Mexico are presented in To estimate the parameters inferred by Bayes, the Differential Evolution Markov Chain (DE-MC) is integrated to generate a large number of realizations from the parameters' posterior distributions6666μ(t)) is derived by computing the 95th percentile of DE-MC sampled μ(t), termed as μ(t = 1),…, μ(t = 100)). The model parameters will then be used to estimate the nonstationary precipitation intensity  analogous to the standard ones as follows: p (the non-exceedance probability of occurrence) is frequency, i.e., how often a storm of specified intensity and duration is expected to occur. The n-year precipitation intensity refers to the annual maximum rainfall of specified depth/intensity and duration having a probability of exceedance of 1/n. In this approach, precipitation intensity is expressed as a function of the return period T, For a time series of annual maxima, the time-variant parameter (L.C. and A.A. conceived the study. L.C. analyzed the data and performed the experiment. A.A. and L.C. wrote the paper. All authors reviewed the manuscript.Supplementary Material


The Continuing to Confront COPD International Patient Survey estimated the prevalence and burden of COPD across 12 countries. Using data from this survey we evaluated the economic impact of COPD.This cross-sectional, population-based survey questioned 4,343 subjects aged 40 years and older, fulfilling a case definition of COPD based on self-reported physician diagnosis or symptomatology. Direct cost measures were based on exacerbations of COPD , contacts with healthcare professionals, and COPD medications. Indirect costs were calculated from work loss values using the Work Productivity and Activity Impairment scale. Combined direct and indirect costs estimated the total societal costs per patient.The annual direct costs of COPD ranged from $504 (South Korea) to $9,981 (USA), with inpatient hospitalisations (5 countries) and home oxygen therapy (3 countries) being the key drivers of direct costs. The proportion of patients completely prevented from working due to their COPD ranged from 6%  to 52% (USA and UK) with 8 countries reporting this to be ≥20%. Total societal costs per patient varied widely from $1,721 (Russia) to $30,826 (USA) but a consistent pattern across countries showed greater costs among those with increased burden of COPD  and a greater number of comorbidities.The economic burden of COPD is considerable across countries, and requires targeted resources to optimise COPD management encompassing the control of symptoms, prevention of exacerbations and effective treatment of comorbidities. Strategies to allow COPD patients to remain in work are important for addressing the substantial wider societal costs. Chronic obstructive pulmonary disease (COPD) is a multifactorial and heterogeneous disease ,2, and aThe substantial burden of COPD is associated with a significant economic impact both in terms of direct  and indirect (impact on home and work productivity) costs . PreviouThe Continuing to Confront COPD International Patient Survey was conducted globally across 12 countries as a follow-up of the original Confronting COPD International Survey , and estThe study design and methodology of the Continuing to Confront COPD International Patient Survey have been reported previously . This waPatients from 12 countries  provided responses to a structured questionnaire either by telephone or in face-to-face interviews. Patients were asked about their disease severity, symptoms, medications and COPD-related healthcare resource use  visits, and healthcare professional consultations). Several patient-reported instruments were completed including modified Medical Research Council (mMRC) Dyspnoea Scale . PatientA societal perspective was used which included COPD-related healthcare resource use and lost productivity. Costs were calculated using local country specific costs and converted to US dollars (2013) using the average historical exchange rates over the survey period (Table A in 1) and severe  which were costed for the ED and/or inpatient stay as reported. Of note, the requirement of treatment in addition to self reported worsening of breathing problems to define exacerbations was implemented in this study to improve the specificity of the definition for an exacerbation. Due to this additional requirement, the reported percentage of patients with an exacerbation episode reported herein is slightly lower than previously reported for this survey.[Direct cost measures were estimated for moderate and severe COPD exacerbations, contact with healthcare professionals, COPD medications, home oxygen use and influenza vaccination during the past 12 months. For this analysis, exacerbations of COPD were defined as episodes of worsening breathing problems that required treatment with antibiotics and/or systemic corticosteroids. These were further categorized as moderate (community treated) which were costed as a general practice visit plus a course of prednisolone 40mg once daily for 5 days and co-amoxiclav 875mg/125mg twice a day for 7 days —mMRC 0–1, mMRC≥2 (moderate-to-severe dyspnoea)Health status (CAT)—CAT≤20, CAT>20 (high-to-very high impact)Self- perceived severity of COPD–mild, moderate, severe/very severeAdherence (Morisky MMAS-8)—Low adherence <6, medium adherence 6 to <8, high adherence ≥8a priori comorbidities of clinical interest Comorbidity—<2, ≥2 self reported co-morbidities from a list of 6 To minimize the effect of sex and age imbalances by country in the sampling and screening process, the reported percentages and costs were weighted by age and sex according to the latest census data available in each country.A total of 4,343 respondents fulfilled the case definition of COPD and consented to complete the full survey. A total of 45% qualified with a physician diagnosis of COPD, chronic obstructive airway disease, or emphysema; 45% had a physician diagnosis of chronic bronchitis; and 10% had symptom-based chronic bronchitis. The mean age of the population ranged from 57.2 years (Russia) to 66.8 years (France), with Brazil reporting the highest proportion of patients of working age (75%) and France reporting the lowest (31%) . The majWith respect to COPD-related healthcare resource use, patients across all countries except Japan and Brazil reported more frequent contact with general practitioners (GPs) than with specialists . The meaThe proportion of patients reporting at least one moderate exacerbation was highest in Mexico (75%), the UK (63%) and Italy (55%), and lowest in Brazil (31%) . The meaThe majority of patients (range 81% in Japan to 97% in Mexico) reported taking a prescription medication for COPD during the past year . With reThe annual direct cost of COPD per patient ranged from $504 in South Korea to $9,981 in the USA . The breThe proportion of working age patients completely prevented from working due to their COPD ranged from 6% to 52% across countries with the highest proportions reported in the UK and the USA (52%) and the lowest (≤10%) reported in Italy, Russia and Japan . PatientThe calculated annual societal per patient cost of COPD varied widely across countries, ranging from $1,721 in Russia to $30,826 in the USA . The indThe total societal costs per patient were greater in patients who reported increased levels of breathlessness (mMRC ≥2), greater impact on health status (CAT score >20), and perceived themselves to have more severe COPD, than in those reporting less symptoms and less severe disease . These fThis analysis of data from the Continuing to Confront COPD International Patient Survey evaluated the economic impact of COPD in a global population of patients across 12 countries. This dataset provides one of the largest global cost of illness evaluations to date and, importantly, allows for a comparison with the original Confronting COPD Survey, enabling a study of the changing economic impact of COPD over the last decade.The current survey showed that the economic burden of COPD is considerable in all countries studied. The annual societal costs per patient covered a wide span ranging from $1,721 (Russia) to $30,826 (USA); however costs for the majority of countries fell within the approximate range of $4,000-$11,000. The largest direct cost associated with COPD in 5 of the 12 countries was attributed to hospitalisations. Other healthcare resources that made the largest contribution to COPD direct costs were: home oxygen therapy (3 countries), GP or specialist visits , GP visits (1 country), and medication costs (1 country). Indirect costs including productivity loss were several times that of direct costs in many countries including the USA, Germany, the UK, the Netherlands and South Korea. This highlights the “hidden” burden of COPD often ignored by decision makers whilst making policy decisions.The differing local unit costs of healthcare services in each country had limited impact on the variability across different countries as shown by consistency in costs estimated using exchange rates and purchasing power parities. Variability in the COPD patients surveyed across multiple countries also had limited impact on societal costs with no consistent pattern observed. For example, the USA had high proportion of severe/very severe COPD patients and patients with two or more comorbidities consistent with high direct COPD costs. In contrast, Japan had high proportion of patients with mild disease and yet had the second highest direct costs. Direct costs were likely to be driven by healthcare systems and patients’ access to healthcare. Developed countries where access to COPD care was often free at the point of delivery tended to have higher direct costs. Patients in these countries also tended to have high indirect costs, mainly driven by higher national per capita incomes. Other factors influencing variations in cost data could be those related to cultural and healthcare practices differences. Patients in Japan reported the lowest levels of symptoms but had one of the highest proportions treated under specialist care.A consistent finding across countries was the association between increased reported burden of COPD  and greater total societal costs per patient. These findings are consistent with those reported in previous international surveys of COPD patients in developed and developing countries –8, and iet al used an economic model to assess the economic impact of COPD in Japan using data published between 1990 and 2002, and estimated the average annual total cost per patient for moderate/severe COPD to be US$3,694, compared with $9,893 in the Continuing to Confront COPD Survey, although 80% of costs were attributed to direct costs and 20% to indirect costs while the cost contribution was relatively equal in the current analysis [et al study population were receiving at least two COPD medications and also included intensive care and tertiary medical care costs [Comparing the cost of COPD across individual studies, a USA medical expenditure survey in 2010 reported an average per person medical expenditure on COPD to be $9,800  which included direct medical expenses and costs due to work loss (absenteeism), but not costs due to lost work productivity (presenteeism) which were included in the Continuing to Confront COPD Survey . A prospanalysis . Interesre costs . A crossA main strength of the Continuing to Confront COPD Survey is that it allows for the assessment of change in the economic burden of COPD over a decade since the original Confronting COPD Survey was undertaken. In countries that participated in both surveys , the annual societal costs per patient has increased above inflation in all countries . The oriet al ranked the costs associated with COPD to be lower than those for cancer and cardiovascular disease and higher than those associated with diabetes [In addition to historical comparison, it can also be of interest to understand COPD costs in the context of other common chronic diseases. A review by Muka diabetes . For exadiabetes . We foundiabetes .The case definition used in this study included a variety of conditions reflecting how COPD may be diagnosed in different countries, including both physician-diagnosed disease and chronic bronchitis based on symptoms. Diagnosis of COPD based on symptoms overcame some of the problems associated COPD under-diagnosis and with physicians using alternative diagnosis terms when speaking to their patients. We accept that it is possible that some patients with lung diseases other than COPD may have been included under this definition, but symptom based COPD represented only 10% of the final sample. Another limitation of the Continuing to Confront COPD International Patient Survey is that outcomes were self-reported and could not be clinically verified, although this was partly mitigated by the use of validated patient reported outcome instruments. Furthermore, resource use was derived indirectly from survey responses and assumptions inevitably needed to be made in order to bridge survey responses to resource counts. A fixed cost was assigned to each type of resource use based on information from local health economics experts in each participating country, global datasets or local prescribing data on medication sales. The accuracy of these cost estimates was fundamental to the resultant estimated costs in each country.et al in the COPD Uncovered International Survey in a working age COPD population [The survey attempted to collect a variety of costs attributable to COPD, however, it was not possible to capture every single resource use parameter  or monetise every parameter recorded . Costs of out-of- pocket expenses such as non-prescription medication were also excluded. This survey did not include indirect costs due to caregiver work loss, suggesting that indirect costs of COPD may be even higher than that reported. In addition, if retirement ages increase in the future, the burden due to early retirement could also be higher as was suggested by Fletcher pulation . OverallThis economic analysis of the Continuing to Confront COPD International Patient Survey showed that COPD continues to place a high economic burden on the healthcare system and society in all countries studied. Indirect costs were higher in most countries and, patients with breathlessness, symptoms and comorbidities experienced higher costs. Attempts to reduce the economic burden will require interventions aimed at delaying the progression of disease, preventing exacerbations, optimising medication usage and reducing the risk of comorbidities in patients with COPD. In addition, strategies to allow COPD patients to remain in work are important for addressing the substantial wider societal costs.S1 FigAbbreviations: USA, United States of America, UK, United Kingdom, NL, Netherlands; SK, South Korea(TIF)Click here for additional data file.S1 Supporting InformationTable A: Historical average exchange rates (Nov 2012 to May 2013) and Purchasing Power Parities (2013) Table B: Country specific unit costs  Table C: Effective retirement age and annual average income Table D: Indirect costs using the friction cost method(DOCX)Click here for additional data file.


The strains of the genotype VIId Newcastle disease virus (NDV) induce more severe tissue damage in lymphoid organs than other virulent strains. The underlying molecular mechanisms are poorly understood.in vitro characteristics of the recombinant viruses were assessed. In addition, pathological changes, virus load, and transcriptional cytokine response in the spleen of chickens infected with these recombinant viruses were also analyzed.Genotype IV NDV Herts/33 and genotype VIId NDV JS5/05 have a distinctive pathological profile in the spleen. These two strains of viruses were selected as parental viruses to generate a panel of chimeric viruses by replacing the M, F and HN genes of Herts/33 individually or in combination with the corresponding genes of JS5/05 using reverse genetic. Virulence and In vitro characterization revealed that gene replacement did not change growth kinetics and HN expression on cell surface of the recombinant viruses. However, replacement of the M, F and HN genes resulted in apparent changes in the fusion activity. Moreover, pathological studies revealed that only inclusion of the homologous M, F and HN genes of JS5/05 in Herts/33 backbone resulted in severe pathological changes characterized by extensive necrosis in the spleen, similar to that induced by JS5/05. In addition, this gene replacement significantly increased virus replication and the levels of transcriptional cytokine response, compared to Herts/33. Conversely, inclusion of the M, F and HN genes of Herts/33 into JS5/05 backbone resulted in Herts/33-specific pathological changes and significantly decreased virus load and the expression levels of cytokine genes, compared to JS5/05.Pathogenicity test showed that all chimeric viruses are virulent. The M, F and HN genes are related to the severe pathological changes in the spleen of chickens infected with genotype VIId NDV. Based on the disease severity in chickens, NDV strains are divided into three pathotypes: lentogenic, mesogenic and velogenic . It is gd of NDV , 9.Since NDV is an evolving pathogen and has a high genetic diversity, it is necessary to determine the molecular basis for the severe pathological changes in the lymphoid organs caused by genotype VIId of NDV. Our previous studies demonstrate that genotype VIId NDV strain JS5/05 and genotype IV NDV strain Herts/33 induce contrasting pathological manifestation in lymphoid tissues in chickens , 8. JS5/The infectious clone of Herts/33 was constructed following the cloning strategy illustrated in Fig. Intracerebral pathogenicity index (ICPI) and mean death time (MDT) in eggs were measured to evaluate the virulence of the recombinant viruses. We found that ICPI values of all chimeric viruses were above 1.80, and MDT values were lower than 60 h Fig. , which aBased on the virus titers of the collected samples, the growth curves of the viruses were obtained. As shown in the Fig. Regarding the fusion activity of the viruses, JS5/05 and two Herts/33-based recombinant viruses showed stronger fusion ability than rHerts/33 Fig. . InclusiThe cell surface expression of the glycoprotein HN was determined by the FACS analysis Fig. . The datSince JS5/05 and Herts/33 induce remarkably contrasting pathological changes in the spleen, the splenic lesion was used as the primary parameter to evaluate the pathogenic role of viral genes. We found that all tested viruses killed all chickens within 5 days p.i., indicating that they are highly pathogenic to chickens, and that gene replacement does not decrease the pathogenicity of the parental virus in chickens.JS, HertFJS and HertMJS) resulted in the Herts/33-specific pathological changes characterized by splenic atrophy and lack of necrosis. Inclusion of both the F and HN genes of JS5/05 into the Herts/33 backbone was also unable to induce JS5/05-specific necrotic lesions. More importantly, chickens infected with HertMFHNJS displayed severe pathological changes in the spleen characterized by remarkable necrotic lesions, resembling that produced by rJS5/05. Conversely, simultaneous inclusion of the M, F and HN genes of Herts/33 into JS5/05 backbone led to the elimination of the necrotic foci. Scoring of gross pathological changes showed that only inclusion of the M, F and HN genes of JS5/05 significantly increased the severity of gross lesions in the spleen compared to rHerts/33, and simultaneous inclusion of the M, F and HN genes of Herts/33 in to JS5/05 backbone significantly decreased the severity of splenic damage  of the F and HN proteins of paramyxovirus are critical for their interaction. The homotypic F and HN interaction is important for paramyxiviruses for proper function of these proteins –13. In oUsing a backbone virus to replace various genes from other virus strains, several studies have identified the homotypic F and HN genes as key players in NDV virulence –15. We aRecently, Paldurai et al.  reportedOur study showed that the M, F and HN genes are associated with the high level of virus replication and intense innate immune response of genotype VIId NDV strain JS5/05. Increased virus load and strong innate immunity of JS5/05 strain may contribute to the severe pathologic changes in the spleen. Our study narrows down the pathogenicity of genotype VIId NDV virus into these three genes, laying a foundation for further fine-mapping the epitopes responsible for NDV pathogenicity.Animal experiments were conducted according to the guidelines of Jiangsu laboratory animal welfare and ethics of Jiangsu Administrative Committee of Laboratory Animals and approved by the Jiangsu Administrative Committee for Laboratory Animals (Permission number: SYXK-SU-2007–0005).Genotype IV NDV strain Herts/33  was plaque-purified for three rounds in chicken embryo fibroblasts (CEF). Reverse genetic-derived JS5/05 strain (rJS5/05) has been generated by Hu et al. . VirusesVirus RNA was extracted from the infective allantoic fluids using TRIzol Reagent . Briefly, ten overlapped cDNA fragments encompassing the complete anti-genome of the virus were amplified by reverse transcriptase-polymerase chain reaction (RT-PCR). These cDNA fragments were sequentially cloned into the transcription vector TVT (0.0) using the shared restriction sites naturally present in the virus genome, resulting in the full-length cDNA clone of Herts/33 Fig. .Virus rescue was performed as previously described . BSR-T7/As illustrated in Fig. 50) using the Reed and Muench method [The growth kinetics of the parental and recombinant viruses was assessed by a multistep growth assay in DF1 cells. Briefly, each virus was inoculated at a multiplicity of infection (MOI) of 0.001 into DF1 cells that were cultured at 37 °C in DMEM containing 5 % FBS. The supernatant was collected at every 12 h until 60 h p.i. The virus content in the collected samples was titrated as 50 % tissue culture infectious dose  against the NDV HN protein at 37 °C for 1 h. Then the cells were washed with PBS and stained with the goat anti-mouse IgG-FITC at 37 °C for 1 h. Finally, the cells were pelleted by centrifugation, washed with PBS and resuspended in a suitable volume of PBS for FACS analysis.HN is a multifunctional surface glycoprotein of NDV. It possesses the receptor recognition, neuraminidase and fusion promotion activities. Therefore, the cell surface expression of HN of the viruses was quantified using fluorescence activated cell sorter (FACS) method in this study. Briefly, monolayers of DF1 cells grown in flasks were infected with the viruses at MOI of 0.001 and then incubated at 37 °C in DMEM containing 5 % FBS. Twenty-four hours p.i., the supernatant was decanted and the cells were scraped and pelleted by centrifugation. The pelleted cells were fixed with 1 % polyformaldehyde for 1 h. After fixation, the cell pellet was resuspended with phosphate-buffered saline (PBS) and centrifuged for 2 rounds again. Following the 2The pathogenicity of the viruses was determined by measuring ICPI and MDT as described elsewhere . For ICP6 50 % egg infectious dose (EID50) of each recombinant virus and two parental viruses. Four birds were mock-inoculated with PBS as the negative control. Three birds per virus-infection group were sacrificed at day 3 and 4 p.i. for gross pathology observation, and the remaining chickens were monitored for mortality. Spleens were collected for tissue section preparation for histopathologic observation. Gross lesion in the spleen was scored based on the following standards: 0 for normal; 2.5 for atrophy without necrosis; 5 for enlarged and mottled; 7.5 for mild to moderate necrotic foci; 10 for extensive and severe necrosis and average scores for three birds were shown. Histologic changes in the spleen were scored as follows: 0 for normal; 2.5 for mild to moderate hyperplasia of lymphocytes; 5 for mild lymphocyte depletion; 7.5 for moderate (<50 %) lymphocyte depletion, histiocytic accumulation and necrosis; 10 for severe (>50 %) lymphocyte depletion, and extensive necrosis, and average values for three chickens were presented.Groups of ten 4-week-old SPF chickens were intranasally and intraconjunctivally inoculated with 1050 per gram using the Reed and Muench method [Groups of fifteen 4-week-old SPF chickens were intranasally and intraconjunctivally inoculated with the chimeric viruses as well as the parental viruses. At 12, 24, 48 and 72 h p.i., spleens from three birds were collected for virus load measurement. Tissue samples were homogenized in PBS containing the antibiotics, and virus titers were measured in CEF. The cleared tissue homogenates were serially 10-fold diluted and inoculated in CEF. At 96 h p.i., cells were fixed with the cold methanol and incubated with the first antibody mAb 6B1 at 37 °C for 1 h to determine the presence of virus. Then the cells were washed with PBS for 3 times and incubated with the secondary antibody goat anti-mouse IgG-FITC at 37 °C for 1 h. Virus titer was determined as TCIDh method .-△△CT method was used to determine the fold change of gene expression levels.Tissue samples taken at 12, 24 and 48 h p.i. for virus load assay were also processed for cytokine response profiling. Samples collected at 72 h p.i. were excluded because severe pathological changes may lead to indirect effects on cytokine response. Relative expressions of three representative cytokine genes, IFN-β, IL-1β and IL-18, were determined using quantitative real-time PCR (RT-qPCR) and normalized to the housekeeping gene β-actin. Briefly, 1 μg of DNase I-treated total RNA per sample was reverse transcribed into cDNA using RevertAid Premium Reverse Transcriptase  at 50 °C for 60 min. The RT-qPCR reaction mixture was consisted of 2 μl of cDNA, 200 nM  of each primer and 10 μl of 2 × SYBR Green PCR Master Mix . PCR reactions were performed in triplicate using the ABI Prism 7300 system . The 2p-value of < 0.05 was considered significant.The differences of the pathological changes score, cytokine expression levels and virus load among chickens were analyzed using one-way analysis of variance (ANOVA) followed by the least significant difference (LSD) multiple-comparison test. A


A horse in Finland exhibited generalized granulomatous inflammation and severe proliferative dermatitis. After euthanization, we detected poxvirus DNA from a skin lesion sample. The virus sequence grouped with parapoxviruses, closely resembling a novel poxvirus detected in humans in the United States after horse contact. Our findings indicate horses may be a reservoir for zoonotic parapoxvirus. Parapoxvirus, 4 species are currently recognized: Orf virus (ORFV), bovine papular stomatitis virus (BPSV), pseudocowpox virus (PCPV), and parapoxvirus of red deer in New Zealand (PVNZ) (Parapoxviruses (PPVs) are zoonotic viruses that have been known for centuries to cause contagious pustular skin infections in sheep, goats, and cattle worldwide. These viruses also infect other animals, such as red deer, seals, camels, reindeer, and domestic cats (All recognized PPV species except PVNZ have been identified in humans. Manifestations of human PPV infections (“farmyard pox”) are typically seen on the hands of persons who had contact with infected ruminants. Recently, Osadebe et al. , but such infections appear to be subclinical , but no OPV DNA was detectable by real-time PCR  Table. HSequencing of the PCR products showed that the ENV (GenBank accession no. KR863114) and RPO147 (GenBank accession no. KR827441) sequences shared 80%–89% nt and aa identity with other PPVs, depending on the virus species. The RPO147 sequence was 99%–100% identical at nt level and 100% identical at aa level to the sequences of the 2 recent poxvirus isolates (2012_37 and 2013_013 RPO147) from humans in the United States . The equAlthough the skin lesions showed poxvirus infection, formalin-fixed samples from internal organs contained no viral inclusion bodies and were negative for PPV by PCR. This finding is in accordance with the fact that PPVs are specialized to replicate in the highly specific immune environment of skin . The horse as an origin for zoonoses is not uncommon: as many as 58% of emerging zoonotic pathogens infect ungulates (We report a clinical equine infection with a novel poxvirus in Finland. The infection is at least of dermatitic relevance for horses, and veterinary awareness is needed. The sequence analysis based on conserved genes revealed a close relationship between this isolate and recent poxvirus isolates from humans with horse contact in the United States (Parapoxvirus species. However, many of the established PPV primer pairs did not produce PCR product, which suggests that the virus is different from the established PPV species and may represent a new poxvirus genus. More sequence data are needed to validate the taxonomic classification of the equine poxvirus. In conclusion, our results provide further evidence that horses are a possible source of the new poxvirus infection recently observed in humans.As our limited sequence analysis suggests, the virus we detected is most closely related to PPVs and may merit being classified as a new Technical Appendix. Summary of PCR results on a sample from a horse infected with possible novel parapoxvirus in Finland, 2013, and image of the horse.