Potato viruses cause enormous economic loss in agriculture production. Potatoes can be infected by a number of different viruses that affect negatively the harvest and the tuber quality. Direct and effective drugs against plant virus diseases are still not available and control is only applied as agricultural measures and pesticides against virus vectors. Potato virus Y (PVY) is transmitted by aphids in non-persistent manner and on that account using insecticides to prevent spread of the infection is useless. Breeding of resistant plant cultivars proved to be not always a solution of the problem because of the fast evolution of the virus strains and the constantly growing group of recombinants. In this study, we have proposed a new way of controlling the virus by blocking replication and transmission through the plant by RNAi-based vaccination of potato seedlings with specific to viral HC-Pro gene siRNAs. Thus, PVY replication is decreased without altering the valuable qualities of the sensitive to the virus potato cultivars like Agria. C. The first report of PVY in Bulgaria was made by Kovachevski,[N[O group, also called ordinary strains, was widely distributed and caused severe symptoms as mottling and leaf curling, necrosis on Physalis floridana and leaf spots on tobacco.[N strains inducted vein necrosis on tobacco, leaf spots on potatoes and necrosis on P. floridana.[NTN was first reported in Hungary in 1978.[NTN strain resembled РVYN, but induced necrotic ring spots on potato tubers. Nowadays, this strain was considered as a subgroup of PVY−N group. PVY was identified as polyphagous in many plant species. Recently, the virus has been identified as a pathogen of the medicinal plant echinacea which is known as an extremely potent immunostimulant[Potato virus Y (PVY) was first reported from Smith and was considered as a complex of virus isolates. Potato vachevski, who estahevski,[N was repohevski,[N This strhevski,[N РVYO gro tobacco. PVYN strloridana. PVYNTN w in 1978. PVYNTN sstimulant and alsostimulantN/NTN, PVYN:O, PVYN, PVYNTN, PVYO, PVYEu-NTN, PVYNa-NTN and PVYC, respectively.[NTN strain.[In Bulgaria, eight strains of PVY were distinguished in potatoes, tomatoes, pepper and tobacco – PVYectively. CharacteN strain.Figure 1Control of the virus has been very difficult. In most cases, the agricultural activities and vector control has been made to limit virus spread. A relatively useful effect of blocking viral replication by thermotherapy and electrotherapy was recently established, but at tSystemic spread of potyviruses included replication in initially infected epidermal or mesophylic cells, moving from cell to cell by the plasmodesmas then in the vascular tissues of the host plant and over long distances through the phloem following the distribution of photoassimilates. The viraHC-Pro was a suppressor of intracellular gene silencing, but the signal was continuously expressed and exported outside the HC-Pro expressing cells, with the result that can reduce the level of viral spread in the healthy tissues of infected plants.The process of PTGS is initiated by double stranded RNAs (dsRNAs) that were produced during viral replication. The dsRNAs are recognized by the plant as a ‘non-own’ and subsequently cut by Dicer-like cellular enzymes to form siRNAs. These moThere are still no effective substances for reduction of the viral infection, which is imperative for developing new approaches to block the replication of PVY.plants – 18 plant pots with potatoes cv. Agria;virus: PVY strain NTN obtained from potato cv. Desire from the virus collection of Institute of Soil Sciences, Agro Technologies and Plant Protection (ISSAPP);referent compounds: dsRNA for the S segment of Phi6 and siRNAs for the S segment of Phi6.The material consisted of:Mechanical inoculation of plants with PVY: the plants were inoculated as described by Noordam . Prior t2SO3 and 0.2% ascorbic acid. Inoculations were performed by gently rubbing the leaves with this homogenate. After 3–5 minutes, the plants were washed with water.One gram of the symptomatic plant foliage was homogenized in 1 ml of cooled to 4 °C 0.1M potassium sodium phosphate buffer, pH 8.0 containing 0.2% Nap-nitrophenyl phosphate  in diethanolamine buffer (pH 9.8) at a ratio of 1 mg/ml. The reaction proceeded in the light at room temperature and was stopped with 3N NaOH. The adsorption of the colour reaction was measured in a multifunctional detector (DTX 880) at a wavelength of 405 nm.Serological diagnostic test: DAS-ELISA (Double Antibody Sandwich Еnzyme Linked Immunosorbent Assay): the analysis was conducted by the method of Clark and Adams . We usedThe positive samples had optical density (OD) over the threshold (cut-off) which was two times the value of the negative control.RNA extraction from potatoes infected with PVY: extraction of total RNA was performed with RNEasy Plant Mini Kit . Extraction was carried out according to the instructions of the manufacturer.In vitro system for the production of dsRNA: dsRNA was synthesized by a combination of in vitro transcription and replication of DNA template . DNA template for synthesis of dsRNA was obtained by PCR using Phusion High-Fidelity DNA polymerase. Primers for the PCR were designed so that the resulting PCR fragment contained a target sequence (HC-Pro of PVY), flanked by T7 promoter sequences in the 5′ end and phi6 RdRP promoter sequences in the 3′ end. PCR DNA product was purified and transcribed by T7 viral RNA polymerase to ssRNA. The ssRNA was replicated to dsRNA by virus phi6 RdRP. The sequences of our designed primers were: HC-Pro dsRNA 1 (5′-TAA TAC GAC TCA CTA TAG GG TAG GAT TCT GTC GAA TGC CGA CAA TTT T -3′) and HC-Pro dsRNA 2 (5′-GGA AAA AAA TAC TGC AGA CCA ACT CTA TAA TGT TT -3′).Giardia intestinalis. It cleaved dsRNA efficiently, producing fragments with a length of 25–27 nucleotides, yielding a pool of siRNAs.The PowerCut Dicer is a recombinant endoribonuclease from In vitro system for generating dsRNA combined T7 RNA polymerase to synthesize ssRNA templates from PCR product template of the selected fragment of the PVY genome and viral Phi6 RdRP polymerase formed de novo initiation and synthesis of dsRNA from the used template ssRNA. Viral Phi6 RdRP-based system allowed the generation of dsRNAs to attack our target genetic sequence. This system was efficient, high quality and convenient method of obtaining high-quality dsRNAs from ssRNAs, such as PVY HC-Pro gene with length 1445 bp  for presence or absence of PVY viral infection in different plant parts (old leaves and newly grown leaves). We received high OD values of samples from old leaves of potato plants treated with HC-Pro dsRNAs and siRNAs and inoculated with PVY (Fourteen days after inoculation with dsRNAs and siRNAs and seven days after inoculation with PVYwith PVY ; 4–6 butwith PVY ; 10–12. with PVY ; 13–24.NTN in newly grown leaves of potato plants cv. Agria was established. The old leaves remained infected but later defoliated leaving the plants virus free.Blocking the HC-Pro gene of PVYNTN which effectively blocked the viral replication and the systemic spread of the virus.PTGS was induced in potato plants cv. Agria by specific siRNAs for HC-Pro region of PVY


On 1 February 2016, the World Health Organization declared Zika virus-associated clusters of microcephaly and related neurological disorders a ‘Public Health Emergency of International Concern'.Macaca monkey in Zika, Uganda, in 1947;2 human transmissions were later observed. The most important vector of Zika virus is Aedes aegypti.3 The virus can also be transmitted from mother to fetus during pregnancy or birth. Other modes of transmission such as sexual contact and transfusion with contaminated blood are also possible.Zika virus was first isolated from a 4 The disease nearly disappeared over two decades until multiple outbreaks in the Pacific islands marked the eastward movement of the virus in the 2000s. In 2007, Yap in Micronesia reported the first outbreak of Zika virus.5 The second outbreak occurred in 2013 in French Polynesia, with nearly 30 000 people becoming infected.6 From there, the epidemic gradually spread to the Cook Islands, New Caledonia and Easter Island in 2014.7 Genetic analyses have shown that the strains in the French Polynesia outbreak were more closely related to the Asian strains,8 indicating that the disease may have been introduced into the Pacific islands from Asia.Between the 1950s and 1970s, only sporadic cases of Zika virus infection were reported in western and central Africa and south-east Asia.9The Zika virus epidemic in South America first surfaced in February 2015, and the Ministry of Health of Brazil first confirmed the outbreak of Zika virus in northeastern Brazil in May 2015. Since then, the epidemic has spread further in South America. By 17 January 2016, the Pan American Health Organization reported that the Zika virus epidemic had spread to 18 countries or territories in the Americas.10 Therefore, updates on the treatment strategy for Zika virus infection have been slow, and there has been a relatively small amount of research on the Zika virus compared with other mosquito-borne viral diseases.For more than half a century since its initial discovery, Zika virus seemed to pose little threat to human beings. Zika virus infection is asymptomatic in ~80% of infected individuals, and in those who develop the disease, the symptoms are usually mild and rarely result in human death.9 constituting a 20-fold increase in incidence rate compared with the period of 2010–2014.3 Circumstantial evidence continues to accumulate. First, the period of the sudden microcephaly outbreak overlaps with the period when the Zika virus epidemic was introduced into Brazil.11 In addition, almost all mothers whose babies were diagnosed with microcephaly had symptoms of Zika virus infection in their early pregnancies.11 In the amniotic fluid of two mothers whose fetuses were diagnosed with microcephaly, Zika virus RNA has also been detected by reverse transcription polymerase chain reaction.3 However, until now, the strongest evidence may yet be the report of a case with severe brain disease and microcephaly related to vertical transmission of Zika virus.12 The fetal autopsy showed pathological changes detected only in the brain, suggesting a strong neurotropism of the virus.12By the end of January 2016, nearly 4000 suspected cases of microcephaly have been reported in Brazil,3 the epidemic scale may be exaggerated. The Brazilian outbreak affected far more people than any other previous outbreaks , it would cause the Zika virus epidemic to become even more difficult to control. Currently, controlling Aedes mosquitoes and recommending that pregnant women avoid travel to Zika-virus-endemic regions and avoid being bitten by mosquitoes seems to be our best chance. However, in the future, we urgently need more research on the relationship between Zika virus and microcephaly, and doctors should establish differential a diagnosis of suspected cases of Zika virus infection.The Zika virus outbreak is currently experiencing ‘a perfect storm', because of the widespread The Zika virus was unexpected and caught us off guard, and but it has taught us an important lesson. How many other neglected pathogens are out there, lurking in the shadows and waiting for the next ‘perfect storm'? This is a scary question to ask. In a world with increasing globalization, international travel, urban crowding and global climate change, perhaps it is time for us to rethink our disease-control strategies, strengthen our research and public health infrastructures, and devise preventive measures against possible future disease outbreaks.


Alternanthera philoxeroides was conducted. High (five fragments) or low (one fragment) propagule pressure was established either in bare soil (open habitat) or dense native vegetation of Jussiaea repens (vegetative habitat), with the stolon connections either severed from or connected to the relatively older ramets. High propagule pressure greatly increased the establishment and growth of A. philoxeroides, especially when it grew in vegetative habitats. Surprisingly, high propagule pressure significantly reduced the growth of individual plants of A. philoxeroides in open habitats, whereas it did not affect the individual growth in vegetative habitats. A shift in the intraspecific interaction on A. philoxeroides from competition in open habitats to facilitation in vegetative habitats may be the main reason. Moreover, clonal integration significantly improved the growth of A. philoxeroides only in open habitats, especially with low propagule pressure, whereas it had no effects on the growth and competitive ability of A. philoxeroides in vegetative habitats, suggesting that clonal integration may be of most important for A. philoxeroides to explore new open space and spread. These findings suggest that propagule pressure may be crucial for the invasion success of A. philoxeroides, and such an effect also depends on habitat conditions.Many notorious invasive plants are clonal, spreading mainly by vegetative propagules. Propagule pressure (the number of propagules) may affect the establishment, growth, and thus invasion success of these clonal plants, and such effects may also depend on habitat conditions. To understand how propagule pressure, habitat conditions and clonal integration affect the establishment and growth of the invasive clonal plants, an 8-week greenhouse with an invasive clonal plant,  Plant invasion has posed a great threat to biodiversity, environment, and economy both globally and locally . PreviouPropagule pressure (propagule supply) has been considered one of the most important factors for explaining the invasion success of plants . It has According to the “ecological resistance hypothesis,” resident native communities may indirectly control invasion success by reducing the input of propagules and resource availability, thereby inhibiting the establishment and spreading process of the introduced species . TherefoEichhornia crassipes (water hyacinth), Alternanthera philoxeroides , and Myriophyllum aquaticum (parrotfeather) can grow and spread mainly by vegetative growth and clonal propagation (no seeds or seed sterility) in their introduced regions (A. philoxeroides and Carpobrotus edulis) in different habitat conditions  and J. repens (native). In an 8-week greenhouse experiment, we grew one fragment (low propagule pressure) or five fragments (high propagule pressure) of A. philoxeroides either in bare soil (open habitat) or dense native vegetation of J. repens (vegetative habitat), with the stolon connections either severed from or connected to the older ramets to test the effect of clonal integration. Specifically, we test the following hypotheses. (1) Increase in propagule supply will increase the establishment and growth of A. philoxeroides, especially when it grew in vegetative habitats. (2) Clonal integration will promote the growth and competitive ability of A. philoxeroides under high propagule pressure. (3) High propagule pressure of A. philoxeroides with clonal integration will reduce the growth of J. repens.To investigate the effects of propagule pressure, habitat conditions and clonal integration on the establishment, growth and thus invasion success of introduced invasive clonal plants, we selected these two co-occurring stoloniferous clonal plants, A. philoxeroides in China . Then plants from different locations were mixed and propagated in the greenhouse. After 2 weeks of adaptive culture, about 200 tip cuttings of A. philoxeroides and about 1000 tip cuttings of J. repens were selected and planted vertically into 20 plots (30 cm diameter × 15 cm height) with lake soil  for continued culture.Given that genetic diversity of wetland clonal plants is relatively low , especiain China , source Figure 1). The tested plants used in this experiment were 120 similar-sized clonal fragments of A. philoxeroides , each consisting of a stolon with five ramets. No differences between treatments were detected in initial size of this plants . Each clonal fragment was divided into two parts, one termed as ‘basal part’ consisting of three relatively old ramets (close to the mother ramets) and the other as ‘apical part’ consisting two relatively young ramets  and a stolon apex  and ambient temperature at the Field Station of Jiangsu University. The experiment was conducted with a factorial design involving propagule pressure , habitat conditions (open or vegetative) and clonal integration . The basal section was 20 cm long and the apical section was 30 cm long. Resources (nutrients and water) and roots in the two sections did not interfere with each other. All the containers in both sections were filled with a mixture of sand and lake mud at a volume ratio of 3:1 and with 2.0 g slow-release fertilizer . On July 5th 2014, the apical sections of 25 containers were planted vertically with cultured plant fragments  of J. repens (monoculture) in the greenhouse to mimic natural plant populations (vegetative habitats), with a density of 200 plants m−2 , each having two separated sections in this experiment  or five (high propagule pressure) fragments of A. philoxeroides were horizontally positioned in both habitat conditions of 40 containers , the remaining five containers with vegetative habitats were used as a control for plant population growth of J. repens without competition. For each clonal fragment, three ramets of basal part were placed within basal section of a container and the other two ramets and apex of the apical part were within the apical section of the same container . The stolons of basal and apical ramets were both anchored to the soil surface to facilitating rooting. Five days later, when the clonal fragments were successfully rooted, the stolon connections between the apical and basal parts were severed in 20 containers, while the other 20 ones were kept intact. Therefore, each treatment was replicated five times . The experiment was conducted for 8 weeks and ended on October 6th 2014. The experimental containers were randomly repositioned every 2 weeks to avoid the effects of possible environmental heterogeneity (such as light), and watered every other day to keep the soil in the containers wet. The mean light intensity at the top of the plant canopy was 1200–1400 μmol m−2 s−1 on the cloudless days, and the mean air temperature was 25–30°C during the experimental period.After 4 weeks adaptive growth of A. philoxeroides was measured for the apical sections of all treatments. Then the plants of A. philoxeroides in the apical part of the container were harvested and separated into leaves, stolons and roots, and their biomass was determined after drying at 70°C for 72 h. Neighboring vegetation of J. repens (entire plants including roots) in the apical sections of each container were also harvested and their dry mass was also determined in the same way.At the final harvest, the number of ramets and leaves were recorded, and total stolon length of A. philoxeroides in the apical part were calculated at both individual level and population (container) level. Prior to analysis, data were log-transformed if necessary to meet the assumptions of normality and homoscedasticity.Data were expressed as means ± SE. Growth measures  of A. philoxeroides was calculated as intraspecific RCI = (Glow –Ghigh)/Glow, where Glow is the mean growth measure of A. philoxeroides in low propagule pressure and Ghigh is that measure in high propagule pressure (open –Gvegetative)/Gopen, where Gopen is the mean growth measure of A. philoxeroides in open habitats and Gvegetative is that measure in vegetative habitats (J. repens was not considered because a significant competition effect was not observed (see Results).The intraspecific relative competition intensity (RCI) of pressure . The indhabitats . The indhabitats . The RCIA. philoxeroides in the apical section at individual level and container level. Two-way ANOVA was employed to investigate the effects of habitat conditions and clonal integration on intraspecific RCI of A. philoxeroides, and the effects of propagule pressure and clonal integration on interspecific RCI of A. philoxeroides. One-way ANOVA was used to test whether total biomass of vegetation (J. repens) in the apical section differed between the four competition treatments and the control. Post hoc pair-wise comparisons of the means were performed to examine differences between the treatments using Studentized Tukey’s HSD for multiple comparisons. Statistical significance was assigned at P < 0.05. All data analyses were performed using SPSS 17.0 .Three-way ANOVA was used to assess the effects of propagule pressure, habitat conditions and clonal integration on the growth measures of A. philoxeroides in the apical sections (Table 1). High propagule pressure greatly increased the growth of A. philoxeroides in both open and vegetative habitats , and the effect of propagule pressure on the growth of A. philoxeroides was more significant in vegetative habitats than in open habitats . Moreover, vegetation of J. repens significantly suppressed the growth of A. philoxeroides in both low and high propagule supply . In open habitats, clonal integration greatly promoted the growth of A. philoxeroides either in low propagule supply or in high propagule supply, however, such effect of clonal integration on plant growth disappeared when grown with J. repens .At the population (container) level, propagule pressure, habitat condition and clonal integration significantly affected all the growth measures , and propagule pressure × habitat conditions, habitat conditions × clonal integration had also significant effects on growth of A. philoxeroides in the apical sections (Table 1). The growth measures of A. philoxeroides were greatly higher in open habitats than in vegetative habitats . There were no differences in growth of A. philoxeroides among the treatments in vegetative habitats . However, in open habitats, the growth measures were significantly lower in high propagule pressure than in low propagule pressure, and clonal integration significantly increased the growth of A. philoxeroides only in low propagule pressure .At initial individual level, propagule pressure, habitat condition, clonal integration and their interactions had significant effects on the growth of A. philoxeroides were significantly affected by habitat conditions (vegetation), however, those values were not affected by clonal integration except for the intraspecific RCI on stolon length (Table 2). The values of the intraspecific RCI of biomass, ramet number, leaf number, and stolon length were significantly lower in vegetative habitats than in open habitats . Interestingly, the values of the intraspecific RCI of biomass and stolon length were negative in vegetative habitats , indicating that the intraspecific interactions between A. philoxeroides individuals changed from competition in open habitats to facilitation in vegetative habitats.The intraspecific RCI of all the growth measures of A. philoxeroides were significantly affected by propagule pressure, whereas those values were not affected by clonal integration (Table 2). The values of the interspecific RCI of the four growth measures were significantly lower when propagule supply was high than when it is low , suggesting that interspecific competition that vegetation of J. repens posed on A. philoxeroides became weaker when propagule supply was higher.The interspecific RCI of all the growth measures of J. repens vegetation in the apical sections had no significant differences among all the treatments . Biomass in the apical sections of J. repens in five treatments  were 82.68 ± 4.30, 87.18 ± 5.78, 84.02 ± 3.74, 78.59 ± 6.12, and 80.82 ± 4.89 g (means ± SE), respectively.Total biomass of A. philoxeroides with high propagule supply grew better and thus probably had higher chance to establish and invade into new habitats than with low propagule supply in both open and vegetative habitats  due to increase in intraspecific competition . Surprisingly, however, when grown with native vegetation, the enhanced performance of A. philoxeroides by high propagule supply did not sacrifice the growth of individual plants. These results support our first hypothesis, suggesting that the role of propagule pressure in the growth and invasion of A. philoxeroides population may be more important in vegetative habitats than in open habitats  translocation of carbohydrates, suggesting that clonal integration plays an important role in exploring new open space and rapid expansion for this invasive plant . This result does not support our second hypothesis, probably because the role of clonal integration in the invasion process of A. philoxeroides is occupying open new space and spread, but not increasing competitive ability . However, in vegetative habitats, there are both intraspecific and interspecific interactions , whereas the intraspecific interaction on A. philoxeroides was facilitation, as demonstrated by the negative values of RCI . The shift in the intraspecific interaction on A. philoxeroides from competition in open habitats to facilitation in vegetative habitats was also found by Hydrocotyle vulgaris. The shift in the intraspecific interaction occurred most likely because the native vegetation J. repens imposed a severe interspecific competition on A. philoxeroides, as shown by the relatively high positive values of RCI  was not affected by the presence of A. philoxeroides. This result did not accord with our third hypothesis, suggesting that invasion of A. philoxeroides in present study did not suppress growth of native plant populations. This is most likely because that competition between apical ramets and native vegetation was asymmetrical because of low density of A. philoxeroides in this experiment and their biomass was too small to influence J. repens (A. philoxeroides in vegetative habitats was sharply decreased to less than 35% as compared with that in open habitats. These findings suggest that the propagule pressure (propagule number) examined in this study did not reach or exceed a certain level to influence native vegetation, indicating that A. philoxeroides needed more propagules or more time to accumulate enough propagules to establish itself and then invade native plant communities (Interestingly, total biomass of native vegetation (. repens . This obmunities .A. philoxeroides, especially when it grew in vegetative habitats. This is probably due to the shift in the intraspecific interaction on A. philoxeroides from competition in open habitats to facilitation in vegetative habitats. Moreover, clonal integration did not affect the growth and competitive ability of A. philoxeroides in vegetative habitats, even with high propagule supply, suggesting that clonal integration may be of most important for A. philoxeroides to explore new open space and spread, especially when propagule supply was low, but may contribute little to its competitive ability and invasion to native vegetation. Using a control experiment, we show that even a relatively small difference in the number of propagule supply can greatly affect the invasion success of invasive clonal plants, and such an effect also depends on habitat conditions. Furthermore, our study may add support to the argument that high propagule pressure may facilitate invasion (In conclusion, increased propagule pressure greatly facilitated the growth and potential invasion of invasion , suggestConceived and designed the experiments: W-HY and D-LD. Performed the experiments: W-HY, C-MH, and L-XF. Analyzed the data: W-HY and C-MH. Contributed reagents/materials/analysis tools: W-HY and D-LD. Wrote the paper: W-HY and DLD.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.


Introduction: Arboviruses cause emerging and re-emerging infections affecting humans and animals. They are spread primarily by blood-sucking insects such as mosquitoes, ticks, midges, and sandflies. Changes in climate, ecology, demographic, land-use patterns, and increasing global travel have been linked to an upsurge in arboviral disease. Outbreaks occur periodically followed by persistent low-level circulation.Aim: This study was undertaken to determine the seroepidemiology of selected arboviruses among febrile patients in selected lake/river basins of Kenya.Methods: Using a hospital-based cross-sectional descriptive survey, febrile patients were recruited and their serum samples tested for exposure to immunoglobulin M (IgM) and IgG antibodies against Crimean–Congo hemorrhagic fever virus (CCHFV), Rift Valley fever virus (RVFV), West Nile virus (WNV), and chikungunya virus (CHIKV). Samples positive for CHIKV and WNV were further confirmed by the plaque reduction neutralization test (PRNT).Results: Of the 379 samples examined, 176 were IgG positive for at least one of these arboviruses . Virus-specific prevalence for CCHF, RVF, WN, and CHIK was 25.6%, 19.5%, 12.4%, and 2.6%, respectively. These prevalences varied significantly with geographical site (p<0.001), with Tana recording the highest overall arboviral seropositivity. PRNT results for Alphaviruses confirmed that the actual viruses circulating in Baringo were Semliki Forest virus (SFV) and CHIKV, o'nyong nyong virus (ONNV) in Naivasha, and SFV and Sindbis virus (SINDV) in Tana delta. Among the flaviviruses tested, WNV was circulating in all the three sites.Conclusion: There is a high burden of febrile illness in humans due to CCHFV, RVFV, WNV, and CHIKV infection in the river/lake basin regions of Kenya. The study was conducted at three selected health facilities: Marigat District Hospital in Baringo, Maai Mahiu Health Centre in Naivasha, and Kotile Health Centre in Tana River districts of Kenya . There aBaringo district (latitude 0.28°N and longitude 35.58°E) and Naivasha (latitude 0.72°S and longitude 36.43°E) in Rift Valley province have populations of 173,815 and 225,547, respectively. Tana basin (latitude 2°32′S and longitude 40°31′E) borders Ijara district in North Eastern Province and has a population of 164,115  aged ≥5 years attending the three health facilities (one in each area) selected on the basis of their proximity to the lake/river basin areas as well as the number of reported unexplained febrile patients being seen at the facility on a daily basis. The patients presenting with a clinical case definition of acute febrile illness characterized by fever of temperature ≥38°C and with at least one of the following clinical manifestations: Cough, joint pains, headache, chills, general body malaise, and any signs of bleeding were recruited into the study. A patient was excluded from the study if no assent or consent was provided to participate in the study.A total volume of 5 mL of blood was collected from the study participants using Vacutainer tubes with a clot activator. Serum was processed by centrifugation at 1500 rpm for 10 min. The serum was aliquoted into 1-mL volumes, placed in sterile bar-coded cryovials, and stored in liquid nitrogen at the health facilities until collection and transportation in dry ice to the KEMRI laboratories, where they were stored at −70°C, processed, and tested.www.vector-best.ru), BDSL, United Kingdom, for RVFV, Novatec Immunodiagnostica GMBH for CHIKV, and Panbio, Germany, for WNV. These tests were conducted strictly based on manufacturer's instructions.All of the serum samples brought to the laboratory were heat inactivated at 56°C for 30 min and screened for exposure to Crimean–Congo hemorrhagic fever virus (CCHFV), CHIKV, WNV, and RVFV using immunoglobulin M (IgM) and IgG capture enzyme-linked immunosorbent assay (ELISA) (for CCHFV and CHIKV) and IgM and IgG indirect ELISA (for WNV and RVFV). The commercial kits used were manufactured by Vector Best for CCHFV  to rule out cross-reactivity with other alphaviruses and flaviviruses known to be endemic in the area, specifically Semliki Forest virus (SFV), Sindbis virus (SINDV), ONNV, DENV, Uganda S virus (UGSV), and YFV. Each virus isolate was diluted to a standard concentration that produced approximately 20–50 plaques. Serum samples were heat inactivated at 56°C for 30 min. Each sample was serially diluted in a sterile 96-well plate to determine the end point titer or highest dilution that neutralizes at least 99% of the virus at 1:20 to 1:640 concentration in maintenance medium . A constant amount of diluted virus was added into each well of the 96-well plate containing serially diluted serum samples and incubated for 1 h at 37°C. The virus–antibody mixture was then transferred to a 24-well plate with a confluent Vero cell monolayer and incubated in COField data captured in the study questionnaires were stored in a password-protected database linked to the laboratory results, and the data were analyzed using Stata v. 10.1 . The main outcomes of interest were IgG antibodies against CCHFV, CHIKV, WNV, and RVFV. The IgG status was binarized as either positive or negative based on the optical density (OD) values. Data on prevalence were compared using chi-square or Fisher exact test. The 95% confidence intervals (CIs) for the proportions positive for a virus were estimated using the Agresti–Coull method . Informed consent was obtained from all enrolled patients.n=152), Baringo (n=117), and Tana (n=110). The mean/median age for the participants was 24.4 years/22 years and the age range was 5–80 years. Ten individuals did not give their age details for unknown reasons. The ages were further classified into four age categories of size 10. Because those aged below 10 years were few (n=57), they were lumped into a single age category with those aged 10–19 years. The results indicated that most of the subjects (84%) were less than 40 years old. Of the total participants, 224 (59%) were females and 155 (41%) males  males .Seventy-six percent of the patients reported to have had contact with animals and 66% with birds; 21% reported to have suffered from tick bites. They presented with the following symptoms: Fever (100%), headache (91.03%), joint aches (75.99%), muscle aches (69.93%), chills (64.64%), abdominal pain (57.52%), cough (45.91%), sore throat (36.15%), and vomiting (35.09%).None of the subjects was positive for anti-CCHFV, anti-WNV, and anti-RVF IgM. An acute case of anti-CHIKV IgM was detected in Tana (1/379). A total of 176  patients had IgG antibodies against at least one of the four arboviruses. The prevalences of each virus calculated separately and for all four viruses combined by site are presented in p<0.001), and it was the most prevalent virus in all the three sites. The prevalence was highest among those aged 5–19 and 40+ years old at 31.08% and 28.85%, respectively. The prevalence among those who had contact with animals  was 28.47% (16.48%).The anti-CCHFV IgG prevalence was 25.6% (95% CI 21.2–30.0). The prevalence of CCHFV varied by site (p=0.667). Of the 10 samples positive for alphaviruses by ELISA, one (10%) neutralized with CHIKV, five (50%) with SFV, three (30%) with ONNV, and one (10%) with SINDV. The overall prevalences of CHIKV and SINDV confirmed by PRNT were both 0.26%, whereas the prevalence of SFV was 1.3% and ONNV was 0.8% . There was no statistically significant difference in alphavirus prevalence among the sites (was 0.8% .p<0.001) and increased with age. Those aged 40 years and above were more likely to be exposed than the younger populations . The prevalence of WNV varied by site (ulations . All of ulations .p=0.053). Its prevalence increased with age . There was a borderline significant association between RVFV prevalence and site . The results also showed that the risk of infection by WNV increased significantly with age and was higher in males compared to females. Relative to 5–19 years and after adjusting for other factors, those in the age groups 20–29 years were almost twice , 30–39 years were three times , and 40+ years were four times  more likely to be infected by WNV.The odds of infection with RVFV were higher in Baringo  and Tana  as compared to Naivasha. These odds were also higher in females than males, but the difference was not statistically significant. Those aged 40+ years were more likely to be infected by RVFV  than those aged 5–19 years. Those who made contact with animals and/or birds through farming and slaughtering had 44%  and 5% , respectively, higher  odds of being infected by RVFV than those who did not make any contact with animals and/or birds through farming and slaughtering .e.g., Passeriformes) that have been implicated as a preferred avian host for SINV and WNV . This is an interesting finding because other studies have shown an increase in infection with age  in the region compared to Naivasha and hence high risk of exposure to ticks and viremic animals by the population through contact by herding, slaughtering, and tick bites. There is a big livestock market in Baringo as well as an active slaughterhouse where exposure may occur.The odds of infection with CCHFV in Baringo were higher compared to Naivasha, probably because of geographic, environmental, and cultural differences in the two sites. Baringo is more arid compared to Naivasha, with most of its population practicing nomadic pastoralism. It experiences warmer temperatures and is close to two big lakes, namely Lake Baringo and Lake Bogoria, providing humidity and arid conditions suitable for the Alphavirus infections circulate in low levels in all the three study sites. Cross-reactivity was observed in the family with 10 samples that had tested positive for anti-CHIKV IgG ELISA neutralizing with CHIKV (1/10), SFV (5/10), ONNV (1/10), and SINDV (1/10) by PRNT. This can be attributed to the antigenic similarity among alphaviruses that results in cross-reactivity, posing a challenge in diagnosis of arboviral infection  isolated previously from these sites  and Flavivirus (WNV) by PRNT. Cross-reactivity among alphaviruses and flaviviruses is a common phenomenon that creates challenges in arbovirus detection and confirmation in endemic places. Co-infection with more than one virus was observed in 19% of the patients by ELISA and in one sample by PRNT. This was also reported in a study conducted in Nigeria, where 92% of the patients were co-infected with more than one arbovirus, typhoid, and/or malaria (Baba et al. Cross-reactivity was observed in the The most common mosquito-borne arbovirus in the three sites was RVFV, followed by WNV; alphaviruses were least prevalent. These viruses have been shown to be circulating during the interepidemic periods in these areas (LaBeaud et al. This study suggests circulation and exposure of human populations to CCHFV, RVFV, WNV, and CHIKV in the river/lake basin areas of Baringo, Naivasha, and Tana. Underreporting of arboviral infection poses a serious public health concern in the study area due to misdiagnosis as malaria/typhoid and the presence of multiple co-infections. Efforts to recognize and identify cases should be made through awareness creation among clinicians in the affected areas. Additionally, infection control measures targeting the significant risk factors should be put in place to alleviate the burden of disease in these areas. There is a need for active surveillance and use of improved diagnostic tools to monitor circulation to inform public health decisions for early warning and response.


Growing unmet need in treatment of very severe and rare diseases is enhancing a development of new innovative drugs. Consequently, the inclusion of these drugs in the reimbursement system is becoming a critical issue in many countries, as most of them prove their clinical and cost effectiveness just partly at the time of submission.This study aims to describe a specific system of reimbursement of highly innovative drugs (HID) in the Czech Republic, representing a time-limited reimbursement under special conditions. The second objective is to evaluate the total number of drugs approved as HID, as well as the number of drugs entering or failing to enter the system of regular reimbursement. The third objective is to describe the development of costs and impact of HID on the overall public pharmaceutical expenditures.This is a retrospective observational study. Data for HID submissions and approvals were searched for 2008-2015. Data were analysed in order to capture the main characteristics of drugs . The length of stay in the temporary reimbursement and reasons for non-acceptance/acceptance into the regular reimbursement system were analysed for each drug individually. The overall costs of HID covered by health care payers were evaluated for 2009-2014.There were 52 drugs approved as HID during 2008-2015, with 21 already having entered the system of regular reimbursement. Most of the drugs belong to the group of antineoplastic agents. The highest number of submissions was reported in 2011 with 13 submissions, while 10 of these drugs succeeded to enter the regular system. Since 2009 expenses of HID doubled from 36 to 79 million Euro/year in 2014, while the overall out-patient expenditures on drugs remained stable .Reimbursement of drugs in the specific system of HID represents a way of early introduction to the market while giving an opportunity to deal with unmet need and the time for proving clinical and cost effectiveness. Limitations of the system are driven mainly by insufficient data from real clinical practice at the end of the limited time frame and subsequent inability to prove cost effectiveness in clinical practice.Despite some disadvantages, the system of HID is a good tool to provide treatment of very severe and rare diseases.


Human infection with avian influenza A(H7N9) virus is associated mainly with the exposure to infected poultry. The factors that allow interspecies transmission but limit human-to-human transmission are unknown. Here we show that A/Anhui/1/2013(H7N9) influenza virus infection of chickens  is asymptomatic and that it generates a high genetic diversity. In contrast, diversity is tightly restricted in infected ferrets, limiting further adaptation to a fully transmissible form. Airborne transmission in ferrets is accompanied by the mutations in PB1, NP and NA genes that reduce viral polymerase and neuraminidase activity. Therefore, while A(H7N9) virus can infect mammals, further adaptation appears to incur a fitness cost. Our results reveal that a tight genetic bottleneck during avian-to-mammalian transmission is a limiting factor in A(H7N9) influenza virus adaptation to mammals. This previously unrecognized biological mechanism limiting species jumps provides a measure of adaptive potential and may serve as a risk assessment tool for pandemic preparedness. H7N9 bird flu viruses cause mild disease in poultry but can occasionally infect humans with fatal consequences. Here, the authors show that viral genetic diversification is low in ferrets and high in chickens, suggesting that a genetic bottleneck limits H7N9 adaptation to mammals In February 2013, avian influenza AH7N9) virus crossed the species barrier in China and for the first time caused human infections34N9 virus Sporadic clusters of H7N9 virus transmission among family members have been reported8911121317202123To elucidate the transmission dynamics and transmission barriers of the emerging H7N9 influenza viruses, we evaluated the replication efficiency, genomic diversification and host adaptation of A/Anhui/1/2013 (H7N9) influenza virus after the inoculation of chickens and ferrets and during ferret-to-ferret transmission. Here we provide evidence that H7N9 virus infection of chickens, while asymptomatic, promotes genetic diversity in the virus population, increasing the likelihood of substitutions that favour mammalian infection and transmission. Conversely, the virus shows poor airborne transmissibility and no evidence of further adaptation upon replication in ferrets, likely due to a narrow bottleneck constraining genetic diversification.The H7N9 virus was isolated mainly from chickens in live-bird markets suspected to be the source of human infections. Thus, we investigated the virulence and replication of A/Anhui/1/2013 (H7N9), one of the first human H7N9 viruses isolated, in chickens. A/Anhui/1/2013 shares at least 99% identity at the nucleotide and protein levels with H7N9 influenza viruses isolated from birds and live-bird markets .10EID50 (50% egg infectious doses) per ml of virus from the upper respiratory tract at 3 days post inoculation (dpi), and shedding lasted for as long as 7 dpi . Inoculated chickens showed no signs of illness but shed on average 3.75 logas 7 dpi , suggestas 7 dpi . All theas 7 dpi , althoug10 TCID50 (50% tissue culture infectious doses) per ml of virus on 2 dpi) and protracted  virus shedding from the upper respiratory tract, as determined in nasal washes  virus except at 1 dpi, when the titre was much lower in ferrets inoculated with AC virus despite equivalent inoculum . Virus wP<0.05, two-tailed Mann–Whitney U-test) than those isolated from chickens  A/Anhui/1/2013 influenza viruses with single or multiple amino-acid substitutions in the gene/s of interest and compared their replication kinetics with the WT rg-virus in Madin Darby canine kidney (MDCK) cells. In line with the drop in NA and polymerase enzymatic activities, rg-A/Anhui/1/2013 (NAE73K+NAI300V), rg-A/Anhui/1/2013 (PB1D76N+NPI365V) and rg-A/Anhui/1/2013 (NAE73K+NAI300V +PB1D76N+NPI365V) influenza viruses replicated to significantly lower titres than the respective WT strain (0–24), was significantly lower when compared with the WT virus  and rg-A/Anhui/1/2013 (NAI300V) viruses were significantly attenuated in vitro  and rg-A/Anhui/1/2013 (NPI365V) were comparable to those of the WT virus at 37 °C  viruses to a wide array of glycans . Both thAcid stability has been described as one of the markers for regulating host range and virulence of influenza viruses212331Our study revealed a previously unknown population-level factor restricting the further adaptation of avian A(H7N9) influenza virus to mammals. Although the H7N9 virus replicates to high titres in infected ferrets, replication in this host did not select for viruses with enhanced growth, virulence or transmissibility. In contrast, our data suggest that replication in ferrets lead to an attenuated phenotype. Because adaptation depends on the extent of the influenza virus population's diversity in the host, we propose the existence of a ‘genetic bottleneck' that limits the ability of avian A(H7N9) influenza virus to adapt to a mammalian host by limiting its genetic diversity in that host. By using a population-level approach and comparing the genetic diversity of the virus population in the natural avian host and in infected mammals, we found that the genetic diversity of A/Anhui/1/2013 (H7N9) influenza virus was limited after its transmission to mammals. The loss in genetic variation imposed by the narrow genetic bottleneck appears to restrict the capacity of A(H7N9) virus to fully adapt to mammals.Genetic bottlenecks occur frequently during the natural life cycles of RNA viruses1623Several pieces of evidence, including the isolation of the H7N9 virus from chickens at live-bird markets and the exposure history of infected patients, suggest that live-poultry markets are a source of human infection4536Our data show that the H7N9 virus replicates in chicken with moderate efficiency and that the infected chickens shed virus for a protracted period. This shedding appears to be sufficient to allow contact transmission of H7N9 virus among chickens, although with poor efficiencyThe human H7N9 isolates possess, or can readily acquire, several traits that support mammalian adaptation, including the PB2-E627K mutation242743214711252329The presence of a significant subpopulation of variants with the HA mutation Q235L, which confers human receptor-binding preferencein vitro. Importantly, although a human H7N9 virus was passaged in eggs to prepare a sufficient stock for experiments, the effect of egg-selected variants was controlled by using the same parental strain to inoculate chickens and ferrets. Richard et al.et al.In this study, we found that H7N9 viruses that had been transmitted via the airborne route were not better adapted or more transmissible in mammals than the parental strain. Four residues in the three genes were uniquely enriched after airborne transmission, and in the ferrets inoculated with AC virus in round 2 experiments. Two of these substitutions (E73K and I300V) located in the NA gene resulted in a decrease in NA activity, and the PB1-D76N and NP-I365V mutations reduced polymerase activity. These mutations also attenuated virus replication In comparing the genetic diversity of the surface glycoproteins, we observed differences in genetic diversity among species in the HA gene but not in the NA gene. At the amino-acid level, none of the HA variants in the parental virus stock were detectable in the transmitted virus population. This result strongly suggests that a purifying pressure acting mainly on the HA gene (virus entry) shapes the diversity bottleneck observed in ferrets. This purifying pressure did not appear to select variants with novel traits such as enhanced transmissibility of the virus in ferrets. Similar purifying pressure on the HA gene has previously been described in association with the airborne transmission of H5N1 virus. However, in the case of H5N1 the purifying pressure appeared to favour the selection of viruses with enhanced fitness after aerosol transmissionDespite their ability to infect humans, the currently circulating strains of H7N9 viruses did not readily acquire additional adaptive mutations during their limited passage in humans or a mammalian host like ferrets. Our results suggest that viral replication in chickens may enhance the diversity of the H7N9 viruses and increase the potential of a subpopulation of variants to enter the mammalian cells and replicate. Even though these variants can replicate in mammals, the stringency of the genetic bottleneck limits their ability to acquire subsequent mutations that would allow mammal-to-mammal transmission. Whether such fitness enhancing variants get selected in nature is entirely dependent on random events, frequency of infection in the new species and time. Given the continued infection of humans with avian H7N9 viruses, the risk of adaptation and pandemic emergence remains plausible. Therefore, surveillance and risk assessment of H7N9 viruses in poultry along with monitoring infections in humans should remain a focus of pandemic preparedness efforts.L-glutamine. HEK 293T cells were maintained in Opti-MEM  supplemented with 10% heat-inactivated FBS. All the cells were maintained at 37 °C in 5% CO2.MDCK epithelial cells, African green monkey kidney epithelial (Vero) cells and human embryonic kidney (HEK 293T) epithelial cells were obtained from the American Type Culture Collection. MDCK cells were maintained in MEM containing 5% heat-inactivated fetal bovine serum (FBS) and 0.5% penicillin–streptomycin. Vero cells were maintained in DMEM (Gibco) containing 10% heat-inactivated FBS and 2 mM ad libitum to all the animals.All the experiments were performed in a biosafety level 3 enhanced (ABSL3+) containment laboratory in accordance with USDA 9 CFR 121; 7 CFR 331. All the experiments were approved by the Institutional Biosafety Committee and the Dual Use Research of Concern Subcommittee at St Jude Children's Research Hospital. The work in this study was conducted prior to the current U.S. Government gain of function stop work order. The ABSL3+ facility has security and containment features that eliminate risks to humans and the environmentThe human H7N9 viruses A/Anhui/1/2013, A/Shanghai/1/2013, and A/Shanghai/2/2013 were obtained through the World Health Organization network. The virus stock used in the chicken and ferret experiments was grown in 10-day-old embryonated chicken eggs at 35 °C for 48 h.50 assay in 10-day-old embryonated chicken eggs incubated at 35 °C for 48 h. Virus from chicken cloacal and oropharyngeal swabs was collected in 1-ml sterile phosphate-buffered saline (PBS) with antibiotics, titrated in 10-day-old embryonated chicken eggs at 35 °C for 48 h and titrated as EID50. Virus from ferret nasal washes and cell culture supernatants from replication kinetic experiments were titrated in MDCK cells at 37 °C for 72 h as TCID50. Virus titres (log10EID50 per ml and log10TCID50 per ml) in nasal washes, swabs, and tissues were estimated by the Reed and Muench method10 EID50 per ml and 1.5 log10 TCID50 per ml.Virus stock was titrated by plaque assay in MDCK cells at 37 °C for 72 h with a 0.4% immunodiffusion grade agarose ; and by EIDGallus gallus domesticus) were inoculated with 106 50% EID50 of A/Anhui/1/2013 (H7N9) influenza virus (0.5 ml) via natural routes . Cloacal and oropharyngeal samples were collected at 3, 5, 7 and 10 dpi with sterile cotton swabs, which were subsequently placed into 1 ml of PBS with antibiotics. Samples were stored at −80 °C for subsequent titration in 10-day-old embryonated chicken eggs as described above. The intravenous virus pathogenicity index was determined as described by Hulse et al.Three 6-week-old, specific-pathogen-free chickens  that were serologically negative by hemagglutination inhibition assay for currently circulating A(H1N1), A(H3N2) and B human influenza viruses were used in the experiments. Six ferrets per group were inoculated intranasally with 106 TCID50 of A/Anhui/1/2013 influenza virus in 0.5 ml PBS under isoflurane anaesthesia. On 3 and 5 dpi, three ferrets per group were humanely killed. Virus in the nasal cavities, tracheas, lungs, brains and the small and large intestines was titrated in MDCK cells as the TCID50. Excised tissue samples preserved in 10% neutral buffered formalin (Thermo Scientific) were paraffin-embedded, sliced into 5-μm-thick sections and stained with hematoxylin and eosin or with immunohistochemical stains for influenza A virus nucleoprotein, pneumocyte type II cells (surfactant protein c), macrophages  and neutrophils (myeloperoxidase). To collect nasal washes, ferrets were anaesthetised by intramuscular administration of ketamine HCl (25 mg kg−1) and sterile PBS with antibiotics (0.5 ml) was instilled into each nostril. The resultant washes were collected into a sterile cup. Samples were titrated by using the TCID50 assay in MDCK cells as described above.Young adult (3–4- month-old) male ferrets  and another four ferrets were placed in AC in an adjacent cage separated from the inoculated ferrets' cage by a perforated divider with controlled air flow.Two rounds of transmission experiments were performed. During round 1, four ferrets were inoculated intranasally with 106 TCID50. At 1 dpi, three naïve ferrets were placed in AC in an adjacent cage as described above. Body weight and clinical signs were monitored for 21 dpi. Virus was titrated in nasal washes collected from inoculated and AC ferrets on 1 dpi and then every other day for 14 dpi or until no virus shedding can be detected for two consecutive sneezes as described above.For the second round of experiments (secondary respiratory droplet transmission), six naïve ferrets were inoculated with a nasal wash sample containing ‘AC' virus obtained at 10 dpi (day 9 post contact) from the ferret infected via AC in the previous round of experiments. The nasal wash was diluted in PBS to a titre of ∼10Seroconversion was tested at 20 days post contact or at 21 dpi. Serum was treated with receptor-destroying enzyme (Seiken) overnight at 37 °C to destroy non-specific inhibitors, heat-inactivated at 56 °C for 30 min, and tested by hemagglutination inhibition assay with A/Anhui/1/2013 (H7N9) virus and 0.5% chicken red blood cells as described previouslyViral RNA was extracted by using an RNeasy kit (Qiagen); reverse-transcriptase PCR was then performed by using a SuperScript III first-strand synthesis kit (Invitrogen) with the Uni12 influenza primer. Multiplex PCR of all the eight gene segments was conducted by using PCR Supermix HiFi (Invitrogen) with the Uni12/13 and influenza A polymerase segment primers under previously described thermal cycler conditionsGenomic sequences were analysed by using a custom work-flow protocol. In brief, sequence reads were filtered for quality scores by using the Nextera adapter trimming protocol, discarding low-quality terminal bases with PHRED scores >20. A sample of 100,000 high-quality read pairs were aligned to the full set of complete NCBI influenza genomes in an exhaustive, competitive fashion to identify potential contaminating samples. Two rounds of alignment were performed: one pairwise alignment and one single-end alignment of unmapped reads from the first round. Genomes with at least one segment with a coverage >0.7 were selected and used to populate a new reference set. The full set of high-quality read pairs was aligned to H7N9 and this reduced reference set, by using the same approach. Genomic variations in H7N9 were detected by using an in-house single nucleotide variants (SNV) detection pipeline that considers base quality of a putative SNV, neighbourhood base quality, strand bias of SNV and several other factors. Heat maps were generated by using the R programming software package . Mean variant frequency for each sample was calculated by using positions that are variable in at least one of the examined samples/reads. If a site had a minor variant frequency >0 in any of the samples, it was included in the numerator; the denominator of the proportion was the total number of variable sites that was variable in all samples.Viral ribonucleoprotein complex genes, including NP, PA, PB1 and PB2 of A/Anhui/1/2013 (H7N9) virus, were cloned into the pHW2000 plasmid to test the viral polymerase activity50 per cell and incubated at 37 °C for 18 h to allow cell surface expression of the HA protein. Cells were then washed with PBS and treated with 5 mg ml−1 of TPCK (L-tosylamido 2-phenylethyl chloromethyl ketone)-treated trypsin (Worthington) for 5 min, followed by neutralization with FBS. Cells were then washed twice with PBS and pulsed with PBS buffers adjusted to 0.1 pH increments by using 0.1 M citric acid. The highest pH at which syncytia were observed was recorded as the pH of HA activation.The pH of HA protein activation was measured by syncytium assay, as previously describedClaI site and subcloning of the product into a pCAGGS expression plasmid, using primers with overhangs that include ClaI and NheI restriction sites  substrate  for 30 min at 37 °C. The reaction was quenched with 100 μl stop solution (150 mM sodium hydroxide in 83% ethanol), and the fluorescence intensity of the released 4-methyl-umbelliferone was measured on a Synergy 2 multi-mode microplate reader (BioTek Instruments) with excitation and emission wavelengths of 360 and 460 nm, respectively. NA activity was quantified as fluorescence above the background fluorescence of mock-transfected cells, expressed in picomoles of 4-methyl-umbelliferone product.To assess the NA activity, Vero cells were transfected with the HA-tagged NA plasmids carrying the mutations of interest (NAI300V and NAE73K), WT plasmid and an empty pCAGGS expression plasmid (mock) by using TransIT-LTI (Mirus Bio) transfection reagents. After incubation of the transfected cells for 20 h at 37 °C, cells were collected and resuspended in assay buffer . Fifty microlitres of the suspension was incubated with an equal volume of 2′-(4-methylumbelliferyl)-α-To confirm the cell surface expression of NA protein, the Vero cells transfected as described above were collected, resuspended in 1X bovine serum albumin blocking buffer in Tris-buffered saline (Thermo Scientific) and stained with monoclonal anti-HA-FITC antibody  for 1 h at 4 °C. HA-tagged NAs was then analysed by flow cytometry on a MACS Quant analyzer (Miltenyi Biotec). The mean fluorescence intensity above that of mock-transfected cells was quantified by FlowJo software.N-linked and O-linked glycans found on mammalian and avian glycoproteins and glycolipids−1. Slides were incubated at room temperature for 1 h, washed with PBS and overlaid with rabbit or goat anti-H7 antibody (1:200) for 1 h, followed by the addition of 10 μg ml−1 anti-rabbit IgG (Alexa Fluor 488) or anti-goat IgG . Slides were washed and scanned on a ProScanArray Express HT (PerkinElmer) confocal slide scanner to quantify the bound virus.Glycan array analysis was performed as previously describedE73K), rg-A/Anhui/1/2013 (NAI300V), rg-A/Anhui/1/2013 (NAE73K+NAI300V), rg-A/Anhui/1/2013 (PB1D76N), rg-A/Anhui/1/2013 (NPI365V), rg-A/Anhui/1/2013 (PB1D76N+NPI365V) and rg-A/Anhui/1/2013 (NAE73K+NAI300V +PB1D76N+NPI365V) were generated by co-transfecting 293T cells  with the appropriate eight plasmids and the virus stocks were prepared in MDCK cells. The presence of the amino-acid substitutions of interest in the recombinant viruses were verified by Sanger sequencing.The plasmids encoding the eight genes of A/Anhui/1/2013 (H7N9) virus cloned into plasmid vector pHW2000 have been described in ref. 50 per cell at 37 °C. After 1 h of incubation, the cells were washed with 0.9% aqueous NaCl solution (pH 2.2) to remove unbound virus particles, washed for three times with PBS to adjust the pH and overlaid with infection medium . Supernatants were collected at 2, 4, 6, 8, 10, 12 and 24 h post inoculation and stored at −80 °C for titration in MDCK cells as described above. To quantify the total amount of virus produced over the course of the experiment, we computed the area under virus titre hour curve from 0 to 24 h (AUC0–24), using peak virus titres at 0, 2, 4, 6, 8, 10, 12 and 24 h post inoculation (log10TCID50 per ml) by GraphPad software.To determine single-step growth curves, confluent monolayers of MDCK cells were infected with reverse-genetic viruses at an identical multiplicity of infection of 1 TCIDU-test was used to compare the genetic diversity. P values <0.05 were considered to indicate a statistically significant difference.GraphPad Prism 5 software (GraphPad Software Inc.) was used for statistical analysis. Two-way analysis of variance was used to compare the groups. Two-tailed Mann–Whitney H.Z. and T.B. designed and performed experiments, and drafted the manuscript; R.C., M.E., G.W. and J.Z. performed computational analysis; B.S.K., J.C.P., R.McB., M.-S.S., J.C.C., J.S., E.K., M.H. and T.F. performed the experiments. J.E.R. analysed the pathology data. J.B., Hu.Z., Y.G., S.S.-C., S.K., R.G.W., E.A.G. and C.J.R. designed the influenza virus infection experiments and contributed to writing the manuscript. R.J.W. designed and supervised the study, critically reviewed and revised the manuscript.How to cite this article: Zaraket, H. et al. Mammalian adaptation of influenza A(H7N9) virus is limited by a narrow genetic bottleneck. Nat. Commun. 6:6553 doi: 10.1038/ncomms7553 (2015).Supplementary Figures 1-5 and Supplementary Tables 1-7Synonymous and non-synonymous mutations detected in chicken isolatesSynonymous and non-synonymous mutations detected in ferret isolates


As our healthcare system moves towards bundling payments, it is vital to understand the potential financial implications associated with treatment of surgical complications. Considering that surgical treatment of ankle fractures is common, there remains minimal data relating costs to postsurgical intervention. We aimed to identify costs associated with ankle fracture complications through case-control analysis. Using retrospective analysis at a level I trauma center, 28 patients with isolated ankle fractures who developed complications (cases) were matched with 28 isolated ankle fracture patients without complications (controls) based on ASA score, age, surgery type, and fracture type. Patient charts were reviewed for demographics and complications leading to readmission/reoperation and costs were obtained from the financial department. Wilcoxon tests measured differences in the costs between the cases and controls. 28 out of 439 patients (6.4%) developed complications. Length of stay and median costs were significantly higher for cases than controls. Specifically, differences in total costs existed for infection and hardware-related pain. This is the first study to highlight the considerable costs associated with the treatment of complications due to isolated ankle fractures. Physicians must therefore emphasize methods to control surgical and nonsurgical factors that may impact postoperative complications, especially under a global payment system. Healthcare costs in the United States were approximately $2.8 trillion in 2012, representing 17.9% of the national gross domestic product [Unfortunately, even with the high frequency of these injuries, there is minimal literature regarding the financial costs of ankle fracture complications. For example, in an economic analysis by Belatti and Phisitkul, foot and ankle diseases in the Medicare population were estimated to cost $11 billion in 2011, with treatment secondary to fractures contributing to 31% of these costs . AdditioDespite studies focusing on various aspects of costs related to ankle fractures, to date, no study has investigated the costs related to the types of complications that these patients with ankle fractures may develop. In a bundled-payment system under which these complications may not be reimbursed, it is vital for the practicing orthopaedic surgeon to understand the financial implications associated with post-surgical complications.To evaluate the costs associated with ankle fracture complications, we designed a case control analysis where we matched patients who developed complications after ankle fracture surgery (“cases”) to those who did not develop complications (controls). The analysis of these cohorts enables us to determine the costs associated with each type of complication. Furthermore, by categorizing the costs associated with different hospital services, we found which factors have the greatest impact on overall expenditures. Therefore, understanding these variations in costs may help drive policymaking to better prepare orthopaedic surgeons for a bundled-payment system.After institutional review board approval, all patients treated at a level I trauma center from January 1, 2000, to January 1, 2010, for ankle fractures were identified through a CPT code search of the university's orthopaedic database. Patient charts were reviewed to include only those who had isolated ankle fractures without any concomitant injuries, including additional fractures, organ injuries, or neurovascular injuries. The charts of these remaining patients were reviewed for basic demographic information including age and gender, as well as type of fracture, fracture location, length of stay for index hospitalization and readmissions, American Society of Anesthesiologist (ASA) score, and complications. Complications included those that resulted in reoperation or readmission and included nonunion, infection, hardware pain, hardware failure, and malunion.Patients with complications (cases) were matched to those without complications (controls) following principles in orthopaedic literature, which are based on correlating specific factors that may be associated with the outcome of interest. In our study, we matched by ASA score (within one score), age (within 10 years), type of surgery (based on CPT code), and type of fracture (open versus closed) as several studies found correlations between these variables and development of complications –8. CostsThrough a CPT code search, 439 patients presented with isolated ankle fractures, of which 28 developed a post-surgical complication. Basic demographic data, including age, gender, fracture type, and ASA score, for the 28 cases and their corresponding 28 controls is provided in P = 0.028).Figures P ≤ 0.001). Similarly for hardware-related pain, the cases presented with both higher professional and technical charges; however, these costs were mainly due surgical and pharmacy charges, respectively. There were no significant differences in all categories of charges between cases and controls for hardware failure, although only 3 patients had this type of complication. Finally, only 1 patient had a nonunion out of our series of 28 patients; therefore a Wilcoxon ranked test was not run. However, this 1 patient did have greater costs in all categories except radiology  and diagnostics  compared to a similarly matched patient.Tables Our study is the first to report on the specific costs associated with complications related to surgical treatment of patient with an isolated ankle fracture. Based on the results of our study, patients with ankle fractures who develop complications incur significantly greater professional , technical , and total charges  than patients who do not develop complications. Among the specific types of complications, patients who developed infections or had hardware-related pain experienced significantly higher costs compared to their corresponding control patients. Specific subcategories of professional and technical charges, like surgical charges, were higher or lower depending on the type of complication, thereby highlighting areas that may cause the greatest financial risk under a bundled-payment system.To our knowledge, no other study has compared the costs of all common ankle fracture complications. However, a few studies have investigated costs with certain aspects of ankle fractures and therefore serve as a basis for comparison. In Sanders et al.'s study of 11 open grade IIIB ankle fractures, average inpatient charges were just over $62,000 per patient . This paGiven that the annual rate of ankle fractures is 187 per 100,000 Americans, then, based on the current US population of approximately 318 million people, nearly 594,650 Americans sustain ankle fractures each year . Based oThe results of our study identify specific complications that are more likely to increase costs for postsurgical ankle fracture patients. Orthopaedic surgeons should be aware of the high costs associated with treatment of complications including infection. Such a finding emphasizes the importance of preoperative antibiotics and thorough debridement in open fractures, minimizing soft-tissue stripping intraoperatively, and employing 24 hours of postoperative antibiotics following surgical intervention , 14.Although not specifically assessed in this study, the mode of fixation utilized is known to have variable costs. Murray et al. reported that external fixation of ankle fractures results in greater financial expenditures compared to open reduction internal fixation . MoreoveOur study had some limitations. For one, our cost calculations were based only on the type of complication. However, other factors impact ankle surgery costs and may confound the relationship between costs and type of complication. Furthermore, while we matched patients on multiple comorbidities, additional factors such as open fracture grade may have been important to control for given the clear link between open fracture grade and infection risk . It may Our study determined that complications due to isolated ankle fractures greatly increase the cost of treatment. Even though complications cannot be completely prevented in surgery, practicing orthopaedic surgeons should remain vigilant in minimizing factors that potentially lead to complications. Overall, by finding avenues to reduce complications, orthopaedic surgeons can also minimize charges under a bundled-payment system.


Genome-wide RNAi screening has been widely used to identify host proteins involved in replication and infection of different viruses, and numerous host factors are implicated in the replication cycles of these viruses, demonstrating the power of this approach. However, discrepancies on target identification of the same viruses by different groups suggest that high throughput RNAi screening strategies need to be carefully designed, developed and optimized prior to the large scale screening.Two genome-wide RNAi screens were performed in parallel against the entry of pseudotyped Marburg viruses and avian influenza virus H5N1 utilizing an HIV-1 based surrogate system, to identify host factors which are important for virus entry. A comparative analysis approach was employed in data analysis, which alleviated systematic positional effects and reduced the false positive number of virus-specific hits.The parallel nature of the strategy allows us to easily identify the host factors for a specific virus with a greatly reduced number of false positives in the initial screen, which is one of the major problems with high throughput screening. The power of this strategy is illustrated by a genome-wide RNAi screen for identifying the host factors important for Marburg virus and/or avian influenza virus H5N1 as described in this study.This strategy is particularly useful for highly pathogenic viruses since pseudotyping allows us to perform high throughput screens in the biosafety level 2 (BSL-2) containment instead of the BSL-3 or BSL-4 for the infectious viruses, with alleviated safety concerns. The screening strategy together with the unique comparative analysis approach makes the data more suitable for hit selection and enables us to identify virus-specific hits with a much lower false positive rate. Emerging and re-emerging human viral pathogens pose one of the major public health concerns since effective countermeasures are not available to detect, prevent, and treat these viral diseases . The 201Another obstacle in working with highly pathogenic viruses is that they require high containment facilities , which are not readily available for many researchers. However this problem can be often circumvented by an HIV-1 based surrogate system in the entry studies and in the drug discovery efforts to identify and develop antivirals targeting the entry process –17. ThisTo perform a high throughput RNAi screen to identify the host factors involved in viral entry of highly pathogenic Marburg virus and avian influenza virus H5N1, which require BSL-3 and BSL-4 facilities, respectively, we adopted a surrogate system which allows us to perform the initial screening in a BSL-2 facility . This huIn this study, avian influenza virus H5N1 pseudovirions (AIV ) and MarThe overall RNAi screening protocol is outlined in Fig. Screen data quality is crucial for subsequent data analysis and hit selection. Quality control of the screen is commonly indicated by the Z’ factor  which asIn our screens, we included three controls: non-targeting siRNA as the negative control and siRNAs targeting luciferase or ATP6V0C as the positive controls. The pseudovirion carries a luciferase reporter gene, and AIV and MARV virus entry is dependent on the low-pH environment in the endosome/lysosome which is regulated by vacuolar ATPase. So siRNA targeting luciferase or ATP6V0C (a component of vacuolar ATPase) is expected to significantly reduce the final luciferase activity level. In addition, these two positive controls can serve as transfection efficiency controls, monitoring siRNA transfection. Eight control siRNAs of each type were arranged on the column 23 or 24 of a 384-well assay plate, as shown in Fig. Figure Despite of the adverse impact of the positional effects, the Z’s calculated from two control combinations (luciferase versus non-targeting and ATP6V0C versus non-targeting), are very close to 0.5, an indication of excellent assay quality Fig. . In comp2(ratioMARV/AIV), was then calculated for each siRNA to indicate specificity. Theoretically, an siRNA with relative infection index equaling to zero means no bias of the siRNA on the infections of both viruses; an siRNA inhibiting MARV infection or enhancing AIV infection results in a minus value, and an siRNA with an the opposite effect results in a positive value.In the aforementioned screen strategy, we were more interested in identifying virus-specific host proteins which could be revealed by a comparative analysis approach, due to the parallel nature of the screen. In this approach, the infection rates for AIV and MARV for each siRNA were obtained respectively by normalizing the measured luciferase signals with corresponding plate signal medians; the relative infection index, namely the logarithm of the ratio of their infection rates to base 2 - logWe used this comparative analysis as a means to correct the observed positional effects. The robust z-score, a robust version of z-score which indicates the number of standard deviations from the mean, was employed to represent data variance. The robust z-score distributions of the normalized signals for the columns and rows from both AIV and MARV screen plates are shown in Fig. Since the relative infection index approach can reduce the systematic errors, we combined it with the individual virus infection rates to identify virus-specific host factors. As presented in Fig. The use of relative infection index together with individual virus infection rate and the 2 siRNAs per gene rule is a powerful means to identify virus-specific host factors and to reduce the false positive rate. The parallel nature of the screen strategy makes the results from the two viruses highly comparable and the relative infection index obtained by comparison easily establishes virus-specificity. As shown in Fig. A selected short list of EBOV or AIV entry-related genes is shown in Table It is interesting to note that two host proteins, FOLR1  and TIM1This report describes a new strategy, referred to as a parallel genome-wide RNAi screen, to identify host factors which are important for entry of enveloped viruses. This strategy is particularly useful for highly pathogenic viruses such as Ebola/Marburg viruses and avian influenza virus H5N1 (or bird flu) since the HIV-1 based surrogate system allows us to perform high throughput screens in the biosafety level 2 (BSL-2) containment instead of the BSL-4 or BSL-3 for the infectious viruses with alleviated safety concerns. More importantly, the parallel nature of the strategy allows us to easily identify the host factors for a specific virus with a greatly reduced number of false positives in the initial screen, which is one of the major problems with high throughput screening. The power of this strategy is well illustrated by a genome-wide RNAi screen for identifying the host factors important for MARV and/or AIV as described in this study.Genome-wide RNAi screening has been widely used to identify host proteins involved in replication and infection of different viruses, and numerous host factors are implicated in the replication cycles of these viruses, demonstrating the power of this approach. However, it is clear that these RNAi screens, even performed on the same virus by different groups, do not always identify the same set of host proteins, suggesting that high throughput RNAi screening strategies need to be carefully developed and optimized prior to the actual screening. Thus several features of the screening strategy described here are attractive to identify host proteins involved in viral entry of highly pathogenic enveloped viruses. One obvious advantage of this strategy is that it can distinguish virus-specific hits from the “shared” hits. Because each virus in the screen also serves as a control for the other virus, an siRNA showing effects on one virus but not on the other one will be tentatively classified as a specific hit, as illustrated in Fig. Another merit of the current strategy is that it can reduce the false positive rate. The reasons for high false positive rates in a genome-wide RNAi screen are due to a lack of replicates and siRNA off-target effects. In this study, each siRNA was actually tested twice, but with two different viruses. For an siRNA specific to one virus entry, the possibility of obtaining similar results for two different viruses is pretty low, greatly decreasing the false positive rate. For shared hits, the false positive rate also decreases owing to the duplicate nature of this strategy. Further, an siRNA hit due to an off-target effect is likely to affect both viruses' infection and can thus be classified as a “shared” hit, resulting in a lower false positive rate for the virus-specific hit. In addition, it has been recently shown that virus infection rate is largely determined by the population context  of the target cell which can be affected by gene perturbations through RNAi . BecauseWe have observed the position effects in this study, which are a commonly observed phenomenon in RNAi screens, as a result of the long incubation period needed for RNAi assays. Thus it can bring more noise to the screen and results in distortions of the true effects of siRNAs. A number of pre and post-screening correction methods have been developed to battle these effects. It has been suggested that the controls evenly scattered over an assay plate by careful plate design can be used to adjust the systematic errors . HoweverIn our comparative analysis approach, however, we are able to alleviate the positional effects without introducing a correction factor. The positional effects are largely due to the different evaporation rates across the plate which leads to a multiplicative bias to the measured signal. Because the screens were performed in parallel, the duplicate wells with a same relative position in two plates were screened simultaneously, making them subject to a similar positional effect. This multiplicative bias can thus be corrected by simply dividing virus infection rate by the rate of the other one. In our case, the relative infection index was used to reduce the bias and the results we presented in Fig. It should be pointed out that this report describes a powerful high throughput screening strategy which allows the initial identification of host proteins which may play a role in AIV- or MARV-specific entry. However, the role of each putative host factor has to be individually confirmed and validated, and one of the challenges is to prioritize and select the host factors to be carefully characterized.We have developed a parallel genome-wide RNAi screen strategy to identify specific host factors to either AIV or MARV entry. Implementation of this strategy generated two sets of data and a comparative analysis approach is proposed. Our screening strategy together with the unique comparative analysis approach alleviates the systematic positional effects, makes the data more suitable for hit selection, and enables us to pick virus-specific hits with a much lower false positive rate. This strategy, we believe, can be easily adapted to other screens with the aim of increasing screen specificity and reducing false positive rates.−E− [Human 293 T embryonic kidney cells and human lung epithelial A549cells were cultured in Dulbecco's modified Eagle's medium  supplemented with 10 % fetal bovine serum , 100 μg/mL of streptomycin and 100 units of penicillin (Invitrogen). The avian influenza virus H5N1/HIV pseudovirions (AIV) and Marburg virus/HIV pseudovirions (MARV) were generated from the following plasmids: hemagglutinin (HA), isolated from a highly pathogenic avian influenza virus, A/Goose/Qinghai/59/05 (H5N1) ; neurami−E− , 33 was −E−) together with plasmids encoding MGP or HA/NA were used for transient co-transfection into 293 T producer cells. Six hours after transfection, cells were washed with phosphate-buffered saline (PBS), and 40 mL of fresh medium was added to each plate (150 mm). Forty-eight hours post transfection, the supernatants were collected and filtered through 0.45 μm pore size filter . The pseudovirion stocks were stored at 4 °C prior to use.AIV and MARV pseudovirions were produced by transient co-transfection of human 293 T cells using a polyethylenimine (PEI)-based transfection protocol . ReplicaThree siRNA libraries  were purchased from Ambion . These libraries contain 64,755 siRNAs targeting 21,585 human genes (each gene has three distinct siRNAs). Daughter plates were prepared in 384-well format at a 250nM concentration in water. Controls include siRNA targeting ATP6V0C or luciferase and non-targeting siRNA. All control siRNAs were purchased from Ambion.2 for 48 h. Then the medium was removed and the cells were challenged with 30 μl MARV or AIV virions in parallel. Virions were removed 24 h later and 30 μl fresh DMEM supplemented with FBS and antibiotics was added to each well. After another 24 h incubation, 15 μl of neolite luciferase substrate  was mixed in, incubated for 5 min, and luciferase activity was measured with an Envision plate reader (PerkinElmer). All multi-well pipetting procedures were conducted by the JANUS automated liquid handling system (PerkinElmer).All siRNAs were arrayed in 384-well plates. For each assay plate well, 2 μl siRNA was mixed with 0.1 μl Lipofectamine RNAiMAX (Invitrogen) and 9.9 μl Opti-MEM (Invitrogen). After 20 min incubation at room temperature, 38 μl cell suspension of 1000 A549 cells was added, resulting in a final siRNA concentration of 10nM. Cells were incubated at 37 °C and 5 % COThe measured luminescence signal was analyzed using the statistical programming language R. The signal in each well was first normalized by the median value of all the samples from the same plate.high ‐ signal + Stdlow ‐ signal)/.The Z’ factor  was calc2 .For each siRNA, the relative infection index was calculated from the corresponding normalized signals from the MARV and AIV plates with the following equation: relative infection index = logij indicates the value for a particular well at row i and column j. The constant 1.4826 is used to make MAD comparable to standard deviation.Robust z-score was calculated for both normalized signal and relative infection index. First, median absolute deviation (MAD ) was cal


Community-acquired pneumonia is a common cause of patient hospitalization, and its burden on health care systems is increasing in aging societies. In this study, we aimed to investigate the factors that affect hospitalization costs in community-acquired pneumonia patients while considering the intermediate influence of patient length of stay.Using a multi-institutional administrative claims database, we analyzed 30,041 patients hospitalized for community-acquired pneumonia who had been discharged between April 1, 2012 and September 30, 2013 from 289 acute care hospitals in Japan. Possible factors associated with hospitalization costs were investigated using structural equation modeling with length of stay as an intermediate variable. We calculated the direct, indirect (through length of stay), and total effects of the candidate factors on hospitalization costs in the model. Lastly, we calculated the ratio of indirect effects to direct effects for each factor.The structural equation model showed that higher disease severities , use of mechanical ventilation, and tube feeding were associated with higher hospitalization costs, regardless of the intermediate influence of length of stay. The severity factors were also associated with longer length of stay durations. The ratio of indirect effects to direct effects on total hospitalization costs showed that the former was greater than the latter in the factors, except in the use of mechanical ventilation.Our structural equation modeling analysis indicated that patient profiles and procedures impacted on hospitalization costs both directly and indirectly. Furthermore, the profiles were generally shown to have greater indirect effects (through length of stay) on hospitalization costs than direct effects. These findings may be useful in supporting the more appropriate distribution of health care resources. Community-acquired pneumonia (CAP) is a common infectious disease and a major cause of hospitalization that places a heavy burden on health care systems. In JapanIn 2003, the Japanese government began the implementation of the Diagnostic Procedure Combination/Per-Diem Payment System (DPC/PDPS) for reimbursements to acute care hospitals under the public medical insurance scheme. The DPC/PDPS is similar to the US prospective payment system with diagnosis-related groups (DRG/PPS), and was implemented with the aim of reducing hospitalizations and length of stay (LOS) without decreasing quality of care.,5 HoweveThe Japanese MHLW is considering the integration of a “Comorbidity Complication Procedure” (CCP) matrix into the current DPC/PDPS in the near future. The CCP matrix would enable analysts to account for variations in patient severity and comorbidities when investigating the use of health care resources. In order to develop an appropriate CCP matrix for the DPC/PDPS, it is first necessary to ascertain the degree of influence of patient severity, comorbidity, and procedures on medical expenses in each disease category. Several studies have previously reported the hospitalization expenses required to treat CAP.,8 HoweveLOS is a major factor to consider when examining the relationship between patient severity and hospitalization costs because there is a high possibility that LOS acts both as an intermediate variable and an explanatory variable for costs. A previous study has reported that the LOS in CAP inpatients has a high degree of influence on hospital costs. In additA structural equation modeling (SEM) method may be able to clarify the relationship described above. SEMs are applications of traditional multivariate analyses, and a response variable in one regression equation in an SEM may become a predictor in another equation. Variables in an SEM can demonstrate relationships with one another either directly or through other variables as intermediaries.The purpose of our study was to investigate how patient risk profiles and procedures affect hospitalization costs in CAP patients while taking into account LOS variations through the use of an SEM in order to contribute to the establishment of a more appropriate payment system in the future.We obtained patient data from the Quality Indicator/Improvement Project (QIP), which regularly collects administrative claims data from voluntary participant acute care hospitals in Japan. In 2014, there were 388 QIP participant hospitals, which varied in scale, region, and health care provider type.th Revision (ICD-10) codes. Clinical information about the A-DROP pneumonia severity scoring system, which is a modified version of CURB-65, is also included in these data.[All participant hospitals provide data to the QIP in the Japanese DPC data format, which is a case mix classification system for reimbursements under the public medical insurance scheme. The DPC ese data. The typeTotal hospitalization costs were calculated as the aggregate of all fees for health care services incurred during hospitalization. ServicesThe primary endpoint for the analysis was total hospitalization costs. Baseline patient and hospital characteristics were first presented as descriptive statistics, including means, standard deviations, medians, interquartile ranges, minimum values, and maximum values. We drew box plots to illustrate the unadjusted relationships between patient states and total hospitalization costs. In addition, Spearman correlation coefficients were calculated to provide insight into the unadjusted relationships of each variable .SEM analysis was conducted using LOS as an intermediate variable. We defined 3 path effects for the SEM: “Direct effects”, referring to the effects of the candidate factors on hospitalization costs that bypass LOS; “Indirect effects”, referring to the effects of the candidate factors on hospitalization costs through LOS; and “Total effects”, referring to the sum of the direct and indirect effects. Lastly, we calculated the ratio of indirect effects to direct effects for each factor, and compared which effects had a greater impact on total hospitalization costs using a chi-square test.All statistical analyses were performed using SPSS software, version 20  and STATA 13 statistical software  for Windows. Statistical significance was set at P < 0.05.The collection and analysis of DPC data from the QIP hospitals were approved  by the Ethics Committee of Kyoto University Graduate School of Medicine. This study was conducted in accordance with the Ethical Guidelines for Epidemiological Research established by the Japanese national government, which stipulates the requirements for protecting patient anonymity. Based on these guidelines, our study satisfied the necessary conditions to waive the need for informed consent, and the ethics committee approved that waiving.We analyzed 30,041 CAP patients from 289 hospitals. Patient and hospital characteristics are shown in In this study, we explored the factors associated with increased hospitalization costs in CAP patients using Japanese administrative data. Using 30,041 patients from 289 hospitals, SEM analysis demonstrated that A-DROP scores, BI scores, CCI scores, mechanical ventilation use, and tube feeding were statistically associated with increased hospitalization costs, regardless of whether LOS was included as an intermediate variable. Furthermore, the candidate variables  were more strongly associated with increased hospitalization costs through the LOS path.At present, there are no multi-center studies that have evaluated the hospitalization costs to treat CAP in adult patients using validated patient risk profile scores such as A-DROP scores, BI scores, and CCI scores. To the best of our knowledge, there has only been one published report that evaluated CAP costs by patient profiles: Sato et al.  reportedOur study is also the first to report a dose-response relationship between the BI and LOS, although several studies have previously explored LOS prediction factors in pneumonia patients.–13 The BOur results also showed that the direct effects of age had a negative dose-response relationship with hospitalization costs, although age has previously been shown to be a factor of severity. With refOur study suggests that the most severe patient profiles or procedures may impact on hospitalization costs through the prolongation of LOS. The candidate variables  were associated with total hospitalization costs more strongly through a LOS path (indirect effects path) than when bypassing the LOS path (direct effects path). Previous reports have noted the substantial influence of LOS on health care costs in CAP patients.,23 BeforThe impact of LOS as an intermediate variable on hospitalization costs varied greatly among the candidate factors. In This study has several potential limitations. First, our sample may not necessarily be representative of all CAP cases in Japan, as we did not include patients whose major diagnosis was either respiratory failure or sepsis with a secondary diagnosis of pneumonia. Our study sample had a relatively low average A-DROP score (1.49 points), which may have been due to the exclusion of these severe patients. The non-inclusion of these patients could therefore potentially confound our results.Second, there are only 4 coding slots for comorbidities in the Japanese DPC database. Therefore, the reported comorbidities in this study may be lower than the actual incidences. However, a previous study that reported the distribution of CCI for pneumonia in Denmark suggests that the CCI range of our sample was not inordinately low. In any cThird, total hospitalization costs in this study were calculated by the sum of all fees of each medical practice, the prices of which are unique and determined by the advisory committee of the MHLW in Japan. This may reduce the direct generalizability of our results to other countries due to differences in fee schedules. However, these findings provide insight into the possible applications of disease severity information in reimbursement systems for both research and reform purposes, irrespective of national or regional systems.Finally, there is an advanced analytical method in which hospitalization costs (as a response variable) are analyzed with a gamma distribution and a log link function using a regression model. In our sIn this study of 30,041 CAP patients in Japan, we analyzed the patient profiles, procedures, and hospital characteristics that impact on hospitalization costs using Japanese administrative claims data incorporated with clinical data. Our results showed that pneumonia severity, physical disability, comorbidities, use of mechanical ventilation, and tube feeding had both direct and indirect effects on hospitalization costs. Furthermore, it showed that patient profiles had stronger indirect effects on hospitalization costs through LOS than direct effects. Our findings may have applications in supporting the more appropriate distribution of health care resources in the future.S1 Tablea: Barthel Index data were missing in 4,100 of the 30,041 pneumonia patients.All pairwise correlation coefficients were calculated using all available data. * Correlation is significant at the 0.05 level. ** Correlation is significant at the 0.01 level. Abbreviations: THC, Total hospitalization costs; LOS, Length of stay; BI, Barthel index; CCI, Charlson comorbidity index; MV, Mechanical ventilator; TF, Tube feeding; PPB, Physicians per bed; NPB, Nurses per bed. BI(PDF)Click here for additional data file.S2 TableaHospitalization costs were analyzed using a gamma distribution with a log link. bLength of stay was analyzed using a Poisson distribution with a log link. Log likelihood = -381619.15.Abbreviations: CI, confidence interval. (PDF)Click here for additional data file.