Evidence on immunization costs is a critical input for cost-effectiveness analysis and budgeting, and can describe variation in site-level efficiency. The Expanded Program on Immunization Costing and Financing (EPIC) Project represents the largest investigation of immunization delivery costs, collecting empirical data on routine infant immunization in Benin, Ghana, Honduras, Moldova, Uganda, and Zambia.We developed a pooled dataset from individual EPIC country studies (316 sites). We regressed log total costs against explanatory variables describing service volume, quality, access, other site characteristics, and income level. We used Bayesian hierarchical regression models to combine data from different countries and account for the multi-stage sample design. We calculated output elasticity as the percentage increase in outputs (service volume) for a 1% increase in inputs , averaged across the sample in each country, and reported first differences to describe the impact of other predictors. We estimated average and total cost curves for each country as a function of service volume.Across countries, average costs per dose ranged from $2.75 to $13.63. Average costs per child receiving diphtheria, tetanus, and pertussis ranged from $27 to $139. Within countries costs per dose varied widely—on average, sites in the highest quintile were 440% more expensive than those in the lowest quintile. In each country, higher service volume was strongly associated with lower average costs. A doubling of service volume was associated with a 19%  reduction in costs per dose delivered, (range 13% to 32% across countries), and the largest 20% of sites in each country realized costs per dose that were on average 61% lower than those for the smallest 20% of sites, controlling for other factors. Other factors associated with higher costs included hospital status, provision of outreach services, share of effort to management, level of staff training/seniority, distance to vaccine collection, additional days open per week, greater vaccination schedule completion, and per capita gross domestic product.We identified multiple features of sites and their operating environment that were associated with differences in average unit costs, with service volume being the most influential. These findings can inform efforts to improve the efficiency of service delivery and better understand resource needs.The online version of this article (doi:10.1186/s12916-017-0942-1) contains supplementary material, which is available to authorized users. Better evidence on immunization costs serves several goals—in the context of new vaccine adoption and service expansion, accurate cost estimates are critical inputs for cost-effectiveness and budget impact analyses of proposed policies. Understanding variation in costs across service outlets can also provide insight on site operations and suggest opportunities for improving efficiency. Despite these needs, there have been few empirical studies with sufficient sample size to provide precise cost estimates or describe inter-site cost variation, and studies have commonly used national-level budgeting data to investigate determinants and trends in immunization costs –3. PriorThe Expanded Program on Immunization Costing and Financing (EPIC) Project was designed to fill this knowledge gap, providing detailed data on routine immunization costs and financing in a large, representative sample of immunization sites in six countries  . InformaCountries were selected from a list of countries that had introduced pentavalent vaccine before 2011, and introduced pneumococcal or rotavirus vaccines in 2011 . Countriwww.immunizationeconomics.org and archived on Dataverse [We collated detailed data files from individual country studies and compiled them into a consistent format, with costs organized into standardized cost categories. We used automated tests to identify data anomalies, which were discussed and resolved with country teams. Three sites were excluded due to unresolved inconsistencies. Data cleaning and standardization resulted in minor differences with earlier studies’ country-level results –11. Dataataverse .Total site-level economic costs were estimated from a program perspective using conventional methods, including all activities undertaken to provide routine immunization services to children aged 0–12 months. We focused on the 0–12 month old age group to allow greater standardization across the data collected through individual country-level studies. Site-level overheads were apportioned by direct allocation , and invData on service delivery volume were extracted from routine reporting systems. We used the number of total immunization doses delivered to children aged 0–12 months by each site during the study period as the primary measure of service volume. We also report results using the number of children receiving their third immunization for DTP3 by each site during the study period as an alternate measure of service volume. DTP3 is a conventional proxy for the number of children completing the basic infant immunization schedule.For this study, we estimated the average cost per site, per dose, and per DTP3 for each country, and obtained confidence intervals with a multi-stage bootstrap with resampling at province and site levels. Average costs per dose and DTP3 were calculated as the sum of routine infant immunization costs for all sites in the sample divided by the sum of outputs  for all sites in the sample. This value is equivalent to a weighted average of the site-level cost per outcome, with weights proportional to service volume. This represents an estimate of the cost per outcome assessed at the program level. We also report results as a simple average of the cost per outcome for all sites in the sample. Average cost estimates are reported with and without higher level program support (“above site costs”) included. All estimates were adjusted for survey weighting, with weights constructed as the inverse of the sampling probability for each site .We explored site-level cost variation by regressing the log of total costs incurred at each site against several explanatory variables. We excluded higher level program support (above site costs) from this part of the analysis, as these costs are unlikely be explained by the site-level determinants considered as explanatory variables. We used a Bayesian hierarchical regression model to combine data from different country studies and account for the multi-stage sample design, with country- and province-level random effects. Using a hierarchical regression model allowed for sources of variation at the site, province, and country levels, and provided a framework for synthesizing data across countries. As we used a Bayesian approach, the uncertainty measures included in the Results section  represent posterior probabilities conditional on priors, likelihood, and regression model, unlike traditional confidence intervals. Regression models were estimated using an adaptive Hamiltonian Monte Carlo algorithm , 22. Prolog(doses)), other site characteristics (government ownership (Govt owned), hospital status  as an indicator of health system level, fraction of DTP3 delivered via outreach (Fraction outreach), fraction of resources devoted to management (Fraction mgmt), and the ratio of DTP3 to doses (DTP3 per dose) as a crude quality measure), and features of the operating environment , local antenatal care (ANC) coverage as a measure of healthcare access (ANC4), and wealth level in the local area relative to the national average ). We also included log per capita GDP in 2011(log(GDP)) as a crude index of inter-country price differences and other factors that vary with income level.Explanatory variables included service volume (Model 1 involves intercept plus country- and province-level random effects. Model 2 incorporates Model 1 plus log(doses) and log(doses) squared. Model 3 incorporates Model 2 plus log(GDP) and site characteristics. Model 4 incorporates Model 3 plus features of the operating environment. Model 5 incorporates Model 4 plus country-level random effects for log(doses).We fit a series of progressively more inclusive regression models including these explanatory variables. Staffing index, not available for 10 sites). We also created an index for the average fraction of time staff spent working on immunization  to describe the extent to which staff were committed exclusively to immunization activities, as compared to being spread across multiple service areas. This was used as a measure of economies of scope for labor. Since the definition of a hospital may differ across countries, we used the number of inpatient beds, categorized into 0 beds, 1–9 beds, or 10+ beds, as an alternative indicator of health system level . Finally, we considered the number of days open per week (Days per week) and the distance to the vaccine collection point (Distance), both not available for Honduras. Table Several variables were unavailable for some sites, and we investigated these in secondary analyses using the subset of sites for which data were available. As salary scales were fixed within each country, we used the site-level average salary, normalized to 1.0 for each country, as an index of staff training and seniority  to describe model fit , 25. Simt distribution [We estimated several alternative regression specifications: (1) country-level fixed effects instead of random effects; (2) addition of district-level random effects; (3) a robust regression specification, with residuals assumed to follow a Student’s ribution , to alloDTP3 coverage, reported as DTP3 divided by number of children < 1 years old in the catchment) exhibited substantial measurement error , attributed to inaccurate population estimates. We omitted DTP3 coverage from the main analysis but undertook sensitivity analyses with (1) reported DTP3 coverage top-coded at 100% , (2) an error-in-variables model for mismeasurement of DTP3 coverage, and (3) use of log catchment population (log(Population)) as an indirect approach for investigating the relationship between coverage and costs.We fit several regression models to investigate coverage as a predictor of site-level costs. We anticipated that unit costs of service delivery would be increasing at higher coverage levels, due to high marginal costs of reaching the very last members of the target population. However, the reported coverage measure (In 2011 the study sites spent $6.79 million (2011 US dollars (USD)) providing routine immunization services to children < 1 year of age, recording 1.50 million vaccine doses and 141,000 DTP3. The distributions of site-level costs and service volume were strongly right skewed, with many small sites and few large sites. There was substantial variation in the cost per dose and per DTP3 in each country, with the coefficient of variation in the cost per dose ranging from 0.45 in Moldova to 1.37 in Ghana (mean 0.99 across the entire sample), such that the quintile of sites with the highest cost per dose were on average 440% more expensive than the quintile with the lowest cost per dose in each country. Additional file Table Table Results show a strong relationship between service volume and total costs, evident in the substantial reduction in WAIC produced by including service volume in the regression model. Including variables for site characteristics and features of the operating environment produced additional reductions in WAIC (though insignificant in the latter case). The additional reduction in WAIC associated with inclusion of random effects for log(doses) (Model 5 vs. Model 4) indicates statistically discernable inter-country variation in the relationship between total costs and service delivery volume.As the regressions used log-transformed costs and standardized variables, the regression coefficients are difficult to interpret directly . First dFigure Several predictors  were not available for the entire sample. We estimated regression models for total site-level costs using the subset of sites with data available on these predictors, and in each case identified a statistically discernable positive relationship with costs  in Benin and Uganda to $13.63 in Moldova. Costs per DTP3 ranged from $27 in Uganda to $139 in Moldova. These values are substantially greater than those reported by earlier studies –6, 29, rRegression analyses revealed several predictors with a statistically discernable relationship with costs. In each country, higher service volume was strongly associated with lower average costs, with output elasticity ranging from 1.27 to 2.52 across the six countries, with a mean value of 1.63, and the largest 20% of sites in each country had a cost per dose that was on average 61% lower than that observed for the smallest 20% of sites. For most countries there were many small sites with high average costs, and these small sites exhibited substantial variation in unit costs. The reduction in unit costs associated with higher service volume was only minimal for sites at the upper end of the service volume distribution, and these large sites exhibited only minor variation in unit costs. While this suggests that greater reductions in average costs might be achieved through efforts to improve efficiency in small sites, it is not clear that these sites should be prioritized, as large total cost reductions might be possible with only small reductions in unit costs at large sites.Higher health system level  was associated with higher costs, possibly reflecting differences in available staffing and infrastructure. Outreach-based delivery was also associated with higher costs, possibly due to the additional resources required to deliver mobile services while maintaining infrastructure for facility-based provision. We designed the staffing index to test whether additional productivity of more highly trained and senior staff would counterbalance their higher salaries, yet the positive coefficient on this variable suggests otherwise. Greater distance to vaccine collection and more days open per week (implying lower daily service volume) were both associated with higher costs. A higher fraction of costs for management and greater completion of the vaccination schedule (proxied by the DTP3:doses ratio) were both found to have a strong positive relationship with costs. The positive coefficient on the dedication index implied lower cost for sites where staff are spread across multiple services (rather than being fully committed to immunization services), consistent with economies of scope and a greater ability to adjust staffing with variable demand. Some of these relationships were stronger than others, but they were generally robust to changes in regression specification. After controlling for site characteristics, there was little difference between rural and non-rural sites, despite rural sites having substantially higher costs per dose in crude comparisons. Per capita GDP was found to have a positive relationship with costs per dose. This likely reflects differences in price levels between countries, but it could also be related to the many other factors that vary with country income level. As only six countries were represented in this sample, we were unable to decompose the effect of these country-level factors.The negative coefficient estimated for coverage was unexpected. We expected costs to increase with coverage, reasoning that improving coverage would require attracting progressively harder-to-reach clients, driving up marginal costs. It is possible that for a given service volume, sites with lower coverage have more widely dispersed or immunization resistant catchment populations, leading to higher average costs. Alternately, if staffing and other immunization resources were determined by catchment population size, lower attendance could produce both lower coverage and higher costs per outcome. While mismeasurement of reported coverage dictates that no firm conclusions should be drawn, the importance of coverage for immunization program strategy suggests that further investigation would be valuable.Several relationships described above have been investigated in country-level studies , 13, witThe EPIC Project is the largest study to systematically investigate routine immunization costs. Prior studies of immunization costs have been hampered by small sample sizes, producing noisy average cost estimates and providing little systematic evidence on the variation in costs within countries. By enrolling a large number of immunization sites and using standardized data collection approaches across countries, the EPIC Project provides a unique opportunity to investigate and understand variation in the costs of immunization service delivery as well as generate more precise national cost estimates. This analysis examined many features of sites and their operating environments, but further investigation of these data is possible. The combined EPIC data are freely available for use by other investigators , with thAdditional file 1:Supplementary material for “The cost determinants of routine infant immunization services: a meta-regression analysis of six country studies”. (DOCX 426 kb)


The workplace has been identified as a priority setting to positively influence individuals’ dietary behaviours. However, a dearth of evidence exists regarding the costs of implementing and delivering workplace dietary interventions. This study aimed to conduct a cost-analysis of workplace nutrition education and environmental dietary modification interventions from an employer’s perspective.Cost data were obtained from a workplace dietary intervention trial, the Food Choice at Work Study. Micro-costing methods estimated costs associated with implementing and delivering the interventions for 1 year in four multinational manufacturing workplaces in Cork, Ireland. The workplaces were allocated to one of the following groups: control, nutrition education alone, environmental dietary modification alone and nutrition education and environmental dietary modification combined. A total of 850 employees were recruited across the four workplaces. For comparison purposes, total costs were standardised for 500 employees per workplace.The combined intervention reported the highest total costs of €31,108. The nutrition education intervention reported total costs of €28,529. Total costs for the environmental dietary modification intervention were €3689. Total costs for the control workplace were zero. The average annual cost per employee was; combined intervention: €62, nutrition education: €57, environmental modification: €7 and control: €0. Nutritionist’s time was the main cost contributor across all interventions, .Within multi-component interventions, the relative cost of implementing and delivering nutrition education elements is high compared to environmental modification strategies. A workplace environmental modification strategy added marginal additional cost, relative to the control. Findings will inform employers and public health policy-makers regarding the economic feasibility of implementing and scaling dietary interventions.ISRCTN35108237. Date of registration: The trial was retrospectively registered on 02/07/2013.Current Controlled Trials:  Chronic diet-related diseases such as obesity, cardiovascular disease, type 2 diabetes and stroke continue to endanger population health worldwide , 2. The The surrounding environment in which an individual lives and works has the potential to influence their health-related behaviour. It is widely accepted that modification of these surrounding environments can promote behaviour change at a population level . The worHowever, uncertainty exists with regards to the effectiveness of workplace dietary interventions , 12. PreThe Food Choice at Work Study (FCW) was a complex workplace dietary intervention trial which assessed the comparative effectiveness of an environmental dietary modification intervention and a nutrition education intervention both alone and in combination versus a control workplace . This stA detailed description of the study design, intervention elements and methods of the FCW study has been published previously . In summParticipants in all workplaces, underwent physical assessments  and 24-h dietary recalls which were conducted by trained research assistants/nutritionists as per the Standard Operating Procedures (SOP) manual for the FCW study . In the The complex interventions were guided by a soft paternalistic ‘nudge’ theoretical perspective . The nutN = 111, Education: N = 226, Environment: N = 113 and Combined: N = 400). The number of employees recruited per workplace was proportionate to the company size. Costs were initially measured for 850 employees, however for the purposes of this study, total costs were standardised for a cohort of 500 employees per workplace. This was conducted for ease of comparison as having the same sample size per workplace will allow employers to use the costs as a benchmark in similarly sized workplaces that are implementing dietary interventions. Similarly, standardising costs over a 1-year period rather than a 9-month period (duration of the intervention) will further increase the comparability of findings for employers.All costs incurred from implementing and delivering the FCW interventions over a 1-year period were measured from the perspective of the employers. A total of 850 employees took part in the FCW study  and the nutritionist at the outset of the study and meetings that were held to discuss the logistics of the monthly group presentations and nutrition consultations.Employee costs: These costs included the time associated with employees attending the individual nutrition consultations during working hours and also lost leisure time for employees attending monthly group presentations and the healthy eating chat table during lunch breaks.Printing and material costs: This category was comprised of costs related to printing material and menu holders for the display of detailed nutrition information in the workplaces.In order to obtain an accurate estimate of costs associated with implementing and delivering the intervention outside of a trial setting, research costs  were excluded from the analysis. Furthermore, the costs incurred through conducting physical assessments were excluded from the total costs but are presented in the results section of this study. A bottom-up approach, using micro-costing was employed to disaggregate the cost of each intervention. The resources consumed in each intervention were identified and the unit costs of the resources were multiplied by the quantities used . The FCWTable These resources were valued in monetary terms using standard techniques –23. For For printing and material costs, cost data were obtained from FCW expense reports which were made available by the FCW project manager. With regards to the implementation and delivery of menu modifications, no extra costs were incurred. All menu modifications (which were recommended by the FCW nutritionists) were within the existing budget predefined by the catering provider for that workplace.A detailed breakdown of the total costs associated with setting up and implementing the nutrition education intervention, the environmental dietary modification intervention, the combined intervention and the control over a 1-year period for a cohort of 500 employees are contained in Table For the nutrition education intervention set-up costs were reported at €1788  and maintenance costs were reported at €26,741 . The total cost of the nutrition education intervention was estimated at €28,529. The average annual cost per employee for implementing and maintaining the nutrition education intervention was estimated at €57.The environmental dietary modification intervention reported set-up costs of €3154  and maintenance costs were reported at €535 . Total costs for the environmental dietary modification intervention were estimated at €3689. The average annual cost per employee for implementing and maintaining the environmental dietary modification intervention was estimated at €7.For the combined intervention set-up costs were reported at €4696  and maintenance costs were reported at €26,412 . Total costs for the combined intervention were estimated at €31,108. The average annual costs per employee for implementing and maintaining the combined intervention was estimated at €62.In the control workplace set-up costs and maintenance costs were non-existent as no intervention elements were implemented in the workplace. Total costs of €21,412 were reported, of which physical assessment costs accounted for 100%. Thus, the cost per employee in the control workplace was estimated at zero.Total costs were higher for the nutrition education intervention when compared to the environmental dietary modification intervention. These higher costs were attributable to the delivery of one-to-one dietary counselling which was the main element driving the maintenance costs in the nutrition education intervention. This element which was not provided in the environmental dietary modification intervention, required substantial investments of both the nutritionist and employees’ time. The cost of this element can be observed in the high nutritionist costs  and high employee time costs  for maintaining the environmental dietary modification. Similarly, the provision of detailed nutrition information and monthly group nutrition sessions incurred additional costs for the nutrition education intervention (€2151 and €2963). These elements included costs associated with printing and materials, nutritionist time and employee time.The environmental dietary modification intervention reported marginal additional total costs when compared to the control workplace (€3689). These additional costs were associated with the set-up (€3154) and maintenance (€535) costs that were associated with the environmental dietary modification intervention. This included the time associated with nutritionists modifying menus, training of catering staff with regards to portion size control and monitoring adherence to menu and canteen modifications. Similar to the control, the maintenance of the environmental dietary modification intervention did not incur printing and material costs.It can be observed that for each intervention, the nutritionist was the main contributor to the costs and accounted for the largest proportion of total costs: nutrition education intervention: 53%, environmental dietary modification intervention: 75% and combined intervention: 55%. In the control workplace, the nutritionist and employees did not incur set-up or maintenance costs as no intervention was implemented. In terms of the other staffing costs, employee time accounted for the second highest proportion of costs for each intervention . Catering and workplace staffing costs and printing and material costs accounted for marginal proportions of the total costs.When physical assessment costs were factored into the analysis, total costs increased substantially for each intervention Table . Total cThis study reports the results of a bottom-up costing study of complex workplace nutrition education and dietary modification interventions. As we are entering into an era where workplace health promotion dietary initiatives are garnering increasing attention, it is imperative that a detailed breakdown of the costs associated with these approaches is reported in a transparent manner. To our knowledge, this study is the first detailed cost-analysis of a complex workplace dietary intervention and therefore the findings can be considered novel. The combined intervention was revealed to be the most expensive intervention to implement and deliver  and the nutrition education intervention  was found to be considerably more expensive than the environmental dietary modification intervention (€3689) to implement and deliver. These findings persisted with the addition of physical assessment costs to total costs with the combined intervention remaining the most expensive intervention. The findings indicate that the implementation and maintenance of environmental dietary modification strategies in the workplace add minimal additional cost to the control when compared to nutrition education strategies.In the analysis of this study, physical assessment costs were purposively made distinguishable from the other categories of costs. As the FCW study was a research study, physical assessments were conducted in order to measure the clinical effectiveness of the different interventions. However, in ‘real-world’ settings such as workplaces, such outcome data would not need to be collected and the interventions could be implemented and delivered without physical assessments being carried out.There is limited available evidence to suggest that workplace health promotion interventions that are based on the provision of nutrition information can result in modest improvements in terms of employee dietary behaviour and weight loss , 15, 16.This study has revealed that the implementation and maintenance of environmental dietary modification interventions is less expensive than nutrition education interventions, irrespective of the inclusion of physical assessment costs. These findings begin to address the paucity of evidence regarding the costs associated with the implementation and delivery of environmental dietary modification strategies in the workplace. There is consensus in the literature that an individual’s surrounding environment has significant capacity to influence their health-related behaviour . AlterinAs previously mentioned, workplace health promotion strategies have become prominent features on organisational agendas both nationally  and inte2 ) were also observed in the combined interventions [A recent study conducted by FCW researchers assessed the effectiveness of the high-intensity complex FCW interventions and provides the strongest evidence to date regarding the effects of these types of interventions. The comparative effectiveness of a workplace environmental dietary modification and an educational intervention both alone and in combination was assessed versus a control workplace . It was ventions . EffectsThe implementation of workplace ill-health prevention initiatives continue to be highlighted as a potential strategy for employers to improve employee health outcomes and to reduce escalating costs that are arising as a result of ill-health, absenteeism and lost productivity . SpecifiA key strength of this study is the use of micro-costing to estimate the costs of each of the interventions. Micro-costing is considered to be the most useful method to use when estimating the cost of a new health technology or intervention. Therefore, a high-level of precision in our cost estimates in the selected workplace settings can be ensured –22. DespThis study offers a unique insight into the costs associated with both implementing and maintaining a complex workplace nutrition education and an environmental dietary modification intervention from the perspective of the employer. Findings will be used to inform an economic evaluation of the FCW interventions. Due to the level of uncertainty in the evidence regarding the cost of workplace dietary interventions, providing a detailed exposition of the costs was of particular importance. An environmental dietary modification intervention incurs marginal additional costs when compared to the control. Nutrition education interventions and combined interventions are more costly owing to the set-up and maintenance costs associated with the education strategies, demonstrating the need for careful consideration when selecting suitable education elements. Accurate cost data can be used to determine the potential scalability of such workplace dietary interventions and inform evidence-based decisions. It is envisaged that the findings can be used alongside studies investigating the clinical effectiveness of workplace dietary interventions to inform both employers and public health policy makers on how to achieve an appropriate balance between improving employee health outcomes and the economic feasibility of implementing workplace dietary interventions.


A surprisingly diverse bacterial community was discovered in this highly saline, alkaline and nutrient-poor environment, with the bacterial phyla Proteobacteria  and Firmicutes (16.6%) showing dominance. Compared to the surface layer, higher bacterial abundance and diversity values were detected in the deep zone, where more stable environmental conditions may occur. The surface layer was dominated by members of the genera Phenylobacterium, Chelativorans and Skermanella, while in the interior layer the genus Fictibacillus was dominant. The culturable aerobic, haloalkaliphilic bacteria strains isolated in this study belonged mostly to the genus Bacillus and were closely related to the species Bacillus pseudofirmus, B. cereus, B. plakortidis, B. thuringensis and B. pumilus.Soda lime is a by-product of the Solvay soda process for the production of sodium carbonate from limestone and sodium chloride. Due to a high salt concentration and alkaline pH, the lime is considered as a potential habitat of haloalkaliphilic and haloalkalitolerant microbial communities. This artificial and unique environment is nutrient-poor and devoid of vegetation, due in part to semi-arid, saline and alkaline conditions. Samples taken from the surface layer of the lime and from the depth of 2 m (both having pH ~11 and ECThe online version of this article (doi:10.1007/s10482-017-0866-y) contains supplementary material, which is available to authorized users. It can be expected that these environmental conditions favour the selection of microorganisms capable of growing both in the presence of high salt concentrations and in conditions with highly alkaline pH  sodium bicarbonate precipitation from brine saturated with ammonia and carbon dioxide, (II) conversion of sodium bicarbonate into the final product–sodium carbonate, and (III) regeneration of ammonia from ammonium chloride solution. Alkaline soda lime is generated as a by-product of the third stage of process. It has the form of a thick sludge and consists mainly of calcium carbonate (constitutes over 90% of the lime), amorphous calcium hydroxide (represents around 6%), and sodium and calcium chlorides  was chosen by us as a model of a nutrient-poor, semi-arid , which is used in agriculture for de-acidification of soil. The particular pond from which samples were taken contains increased concentration of salts due to the deposition of lime sludge after cleaning a technological line used in wet and dry vacuum salt production.Saline soda lime has been deposited in Janikowo  since 1957. The repository of the lime covers an area of 200 ha, has the capacity of over 13,000,000 Mg and is composed of several ponds, which are separated by causeways. This area rises up to 16 m above the ground level  were placed in sterile glass jars and they were transported to the laboratory in a portable ice bag at 4 °C. In the laboratory, subsamples (10 g) from a particular layer were mixed thoroughly for subsequent microbial and physicochemical tests. All analyses started within 4 h after sample collection.Samples of the saline soda lime were collected in September 2013 from the surface layer of the deposit (depth of 0–20 cm) and from a depth of 1.8–2.0 m using a telescopic Edelman mud auger . The alkaline lime, a product of chemical processes, is more homogenous than soil. Therefore only ten samples were taken: five from the surface layer and five samples from the deep layer. The surface layer was the most recent, compared to the bottom layer, and the age of the two layers was estimated at 1 and 10 years, respectively. For each sampled layer, five separate subsamples were taken in the corners and in the centre of a 2.5 m square using a hand coring device . The suspensions were titrated until the indicator dye was stable  according to Ntougias et al. (−1) and yeast extract (0.2 g l−1), and solidified with agar (1.5%). The cultivation was performed in three parallel repetitions. All plates were incubated up to 21 days at 25° C. The number of bacteria was expressed as colony forming units (CFU) per g dry weight of the saline soda lime.To isolate culturable bacteria and to estimate their number, 10 g of lime sample was added to 90 ml of NaCl solution (a concentration corresponding to the chloride content in a given sample). Mixtures were blended thoroughly, tenfold serial dilutions were prepared, and 0.1 ml of each suspension was spread onto three replicate plates. An isolation medium consisted of the lime extract , yeast extract (1 g l−1), MgSO4 anhydrous (0.01 g l−1), and NaCl (60 g l−1), supplied with the buffer as given above. Then glycerol stocks of isolates were prepared and stored at −80 °C.Isolates differing in appearance, colour, size and shape of bacterial colonies on the medium were chosen for subsequent taxonomic identification. Colonies were subcultured in a medium containing glucose  was placed into glass tubes containing 9 ml of filter-sterilized citric acid solution . Citric acid was added in order to dissolve calcium carbonate present in the samples. For each suspension, three subsamples were stained with acridine orange  for 3 min and then the slides were immediately viewed under a microscope .TaqDNA polymerase (EURx), 0.2 mM dNTP mixture (EURx), 1× Polbuffer B with 1.5 mM MgCl2 (EURx), 0.25 µM of universal primers 27F  following the instructions given by the manufacturer. The 16S rRNA gene was amplified by the PCR in a reaction mixture containing 1 U 3′, Lane ) and 1493′, Lane ), and 1 3′, Lane . NucleotThe taxonomic affiliation of bacterial strains was determined using the EzTaxon online tool , and washed twice with pH 8 phosphate buffer.Environmental genomic DNA was isolated from samples homogenized as follows: 10 g of sample was added to 90 ml citric acid  in order to dissolve calcium carbonate, the suspension was centrifuged  were assigned to Bacteria , however 9.7% of all reads belonged to Archaea. Most archaeal reads were identified in the interior sample  compared to the surface (1.6%), indicating indirectly that Archaea were relatively numerous in the deep layer of the lime. The Archaeal community was dominated by members of genera “An analysis of the distribution of the most abundant bacterial phyla revealed that the saline soda lime samples were dominated by Proteobacteria, Firmicutes, Bacteroidetes and Actinobacteria  were present only in the deep layers of the lime.Differences in overall community composition are presented as an association network of major bacterial OTUs Fig. . OTUs beOchromonas , while the others were related to the genera Mesotaenium  and Picochlorum . Other detected genera included Dunaliella , Chaetoceros (Bacillariophyta) and Euglena (Euglenozoa).A significant portion of the sequences constituted chloroplasts of algae in both samples . They were more abundant in the surface layer of the saline-alkaline lime  than in the interior sample (8%). Chloroplast sequences formed three major clades in the phylogenetic tree presented in Fig. Bacillus, Micrococcus (one strain), and Nocardiopsis (one strain). The closest relative of 18 strains  was Bacillus sp. A73, first isolated from a hypersaline lake in Iran  and seventeen from the interior sample . All of the bacterial isolates stained Gram-positive. Molecular taxonomic identification of the isolates Fig.  revealedThe saline soda lime is an unusual environment created by humans as a result of the chemical processes of soda production. This extreme environment, consisting almost entirely of calcium carbonate, is highly alkaline, saline and nutrient-poor, especially in terms of nitrogen and organic carbon content. Most of the organic matter present in this artificial environment originates from allochtonous airborne material and atmospheric deposition on the lime surface. A comparison of the bacterial diversity observed in this study with that of other studies is difficult because of the lack of papers describing similar environments.The high alkalinity caused by the presence of carbonates, bicarbonates and hydroxides, as well as the high concentration of salts, are thought to be the key factors that shape the structure of microbial communities in this environment. The influence of pH and salinity on bacterial and archaeal community composition in various ecosystems was extensively investigated by other researchers  was low compared to dolomites and soil, where it ranged from 108 to 109 cells g−1  in the Kuyavia region, Central Poland The distribution of the most abundant bacterial phyla in the alkaline, saline soda lime was similar to that observed for submarine ikaite columns  and it was dominant in the surface layer. Members of this genus are strict aerobes or facultative anaerobes and have been isolated from various environments, including highly alkaline, oligotrophic groundwater , which contains species recently reclassified from the genus Bacillus  and Skermanella  within the class Alphaproteobacteria were also among the abundant OTUs found in the surface layer of the lime. These Gram-negative, strictly aerobic bacteria were previously isolated from various environments, including nutrient-poor environments , encompassing moderately halophilic and halotolerant bacteria  in the surface layer of the highly alkaline and saline lime. Its representatives are obligate intracellular pathogens  were still conspicuously present in the saline, alkaline lime deposit, especially in its deep layer.Hypersaline environments are generally dominated by halophilic Archaea, which belong to the order Halobacteriales  and Mesotaenium (Zygnemophyceae). Therefore, eukaryotic algal phylotypes detected in the studied samples may represent novel phylotypes, and most probably belong to genera which have yet to be described. Picoeukaryotic and phototrophic members of the genus Picochlorum, which carry sequences that were found in the saline soda lime, were also reported in various saline environments, both marine  to the genera rns Oren  and the rns Oren .3–104 CFU g−1 dry mas) was within the range observed for weathered limestone , an organic sludge-like by-product of olive oil extraction . The examined artificial environment maintains extremophilic bacterial taxa adapted to thrive in this unusual ecosystem. Most of the detected bacterial and plastid sequences were related to microorganisms found in various alkaline and saline, nutrient-poor environments such as solonchaks, desert soils, limestone, soda lakes and ponds, oceans, saline springs and alkaline ground water or even air. The phylogenetic groups identified in the surface and in the interior layers of the lime were distinct. Higher bacterial abundance and diversity was detected in the deep zone of the lime than in the surface layer, possibly due to both geochemical and temporal stability differences between these two environments. Molecular phylogenetic identification of the isolates revealed a very low diversity of culturable bacteria, which belonged mainly to the genus Supplementary material 1 (DOCX 16 kb)Below is the link to the electronic supplementary material.


Rabies is a global fatal infectious viral disease that is characterized by a high mortality after onset of clinical symptoms. Recently, there has been an increase in the incidence of rabies in China. The aim of this study was to investigate the incidence of human rabies and characterize the rabies virus nucleoprotein gene in dogs sampled from Fujian Province, Southeast China from 2002 to 2012.Data pertaining to human rabies cases in Fujian Province during the period from 2002 through 2012 were collected, and the epidemiological profiles were described. The saliva and brain specimens were collected from dogs in Quanzhou, Longyan and Sanming cities of the province, and the rabies virus antigen was determined in the canine saliva specimens using an ELISA assay. Rabies virus RNA was extracted from canine brain specimens, and rabies virus nucleoprotein gene was amplified using a nested RT-PCR assay, followed by sequencing and genotyping.A total of 226 human rabies cases were reported in Fujian Province from 2002 to 2012, in which 197 cases were detected in three cities of Quanzhou, Longyan and Sanming. ELISA assay revealed positive rabies virus antigen in six of eight rabid dogs and 165 of 3492 seemingly healthy dogs. The full-length gene fragment of the rabies virus nucleoprotein gene was amplified from the brain specimens of seven rabid dogs and 12 seemingly healthy dogs. Sequence alignment and phylogenetic analysis revealed that these 19 rabies virus nucleoprotein genes all belonged to genotype I, and were classified into three genetic groups. Sequencing analysis showed a 99.7% to 100% intra-group and an 86.4% to 89.3% inter-group homology.This study is the first description pertaining to the epidemiological characteristics of human rabies cases and characterization of the rabies virus nucleoprotein gene in dogs in Fujian Province, Southeast China. Our findings may provide valuable knowledge for the development of strategies targeting the prevention and control of rabies. Rabies is a fatal disease that is mainly transmitted by the bites of dogs. Globally, rabies is responsible for approximately 55,000 deaths each year . In ChinDuring the past decade, there has been a sharp increase in the incidence of human rabies in Fujian Province, Southeast China –5. This This study was approved by the Ethical Review Committee of Fujian Provincial Center for Disease Control and Prevention (CDC) (permission number: FJCDC-001207), and the study protocol was reviewed by the Institutional Review Board of Fujian CDC. All animal experiments were performed according to the Guidelines for the Care and Use of Laboratory Animals, and written informed consent was obtained from all human rabies cases described in this study.The data pertaining to human rabies cases were captured from the annual reports of Fujian CDC  during the period from 2002 through 2012, and the epidemiological profile of the cases was described.N) gene.Dogs were sampled from Quanzhou, Longyan and Sanming cities, which had the highest incidence of human rabies in Fujian Province, Southeast China Fig. . Table 1Saliva specimens were collected from both rabid and seemingly healthy dogs housed in nine randomly selected villages from each city. For saliva sampling, each dog was housed in a cage, and sterile Q-tips/cotton balls were placed in mouth until Q-tips/cotton balls were thoroughly soaked by the saliva. Saliva specimens were kept in cold storage, immediately transferred to laboratory, pretreated by centrifugation and stored at −20 °C for subsequent tests. The rabies virus antigen was detected in saliva specimens using an enzyme-linked immunosorbent assay (ELISA) kit  following the manufacturer’s protocol. In this study, a rabid dog was defined as a dog that actively attacked humans and animals and presented sympathetic hyperfunction, which showed manifestations of salivation, manic agitation and disturbance of consciousness.N gene was amplified from the brain specimens using a nested reverse transcription polymerase chain reaction (RT-PCR) assay [N gene  dogs were euthanized. Canine brain tissues were collected and stored at −70 °C for the subsequent experiments. Total RNA was extracted from canine brain specimens using a Trizol reagent  according to the manufacturer’s instructions. ne Table .Table 2PN gene sequences of the reference strains obtained from GenBank using the software Bioedit version 7.0.1  and MEGA version 4.0.PCR products were purified with the PCR product extraction kit , and cloned into the pMD18-T vector with a TA cloning kit  following the manufacturers’ instructions. Transformed clones were identified by restriction enzyme digestion and subjected to DNA sequencing. To increase the accuracy, we selected three clones of each fragment for sequencing. The sequence of the three clones that exhibited 100% homology was identified as the target sequence. Sequences were then aligned and compared with the P value <0.05 was considered statistically significant.All data were entered into Microsoft Excel 2007 , and all statistical analyses were performed using the statistical software SPSS version 13.0 . Differences of proportions were tested for statistical significance using chi-square test, and a During the period from 2002 to 2012, a total of 226 human rabies cases were reported in Fujian Province, Southeast China. The cases included 147 men and 79 women, and had a mean age of 34.6 years . The professions included farmers (138 cases), students (29 cases), workers (22 cases), preschool-aged children (14 cases), housekeepers (13 cases), and unknown (10 cases). The human cases were distributed across Fujian Province, including Quanzhou (85 cases), Longyan (67 cases), Sanming (47 Cases), Nanping (11 cases), Ningde (10 cases), Xiamen (3 cases), Fuzhou (1 case), Pingtan (1 case) and Putian (1 case), and 88.1% cases were reported in the three cities of Quanzhou, Longyan and Sanming. The annual number of human rabies cases is shown in Fig. P < 0.01).To investigate the incidence of rabies virus antigen in canine populations, a total of eight rabid dogs and 3492 seemingly healthy dogs were randomly selected from 27 villages of Quanzhou, Longyan and Sanming cities, of nine villages in each city, and dog saliva samples were collected. There were six of eight rabid dogs (75%) with detectable rabies viral antigen, and surprisingly, 165 of 3492 seemingly healthy dogs (4.7%) were positive for rabies virus antigens in saliva samples, suggesting that a considerable proportion of asymptomatic seemingly healthy dogs carries rabies virus  was obtained from these positive specimens with primers N1/N5 and N6/N2. The amplicons were cloned into the pMD18-T vector, and subjected to DNA sequencing .Nested RT-PCR assay detected a 443 bp viral gene fragment in the brain specimens from seven of eight rabid dogs (87.5%) and 12 of 81 seemingly healthy dogs (14.8%), and then, all fragments were amplified with primers N1/N2 and N3/N4. Subsequently, the full-length N gene sequence available in GenBank. Based on sequence alignment and phylogenetic tree analysis, these 19 N genes were characterized as the genotype I rabies virus, although three distinct clusters were found to be geographic region-specific (Groups A to C)  Fig. . SequencAnalysis of the 1960–2014 notifiable surveillance data showed that rabies is still a serious public health concern in China, with a total of 120,913 human rabies cases reported in mainland China, including 19.8% cases observed during the period from 2004 through 2014 . The notN gene is highly conserved within the same subtype, and variations in this region can be used for virus subtyping and genotyping [N genes has been performed in some provinces of China [Rabies virus has a single-stranded, antisense, nonsegmented RNA virus genome (approximately 12 kb) encoding five viral proteins, including glycoprotein (G), nucleoprotein (N), phosphoprotein (P), matrix protein (M) and polymerase (L) . N gene notyping . Nucleopnotyping , and is notyping . Moleculof China –20; howeN gene fragment in the brain specimens of 14.8% (12/81) of seemingly healthy dogs. The results suggest that there are a considerable proportion of false negatives in preliminary screening by ELISA. Indeed, the reliability, reproducibility and diagnostic value of rabies virus antigen detection remain in debate in canine saliva since the complicated components in saliva specimens may affect ELISA assays, and no standardized kits are available in market until now [This study is the first survey on rabies incidence in domestic dogs in Fujian Province, Southeast China. Saliva specimens, which were collected from rabid and seemingly healthy dogs in 27 villages of Quanzhou, Longyan and Sanming cities, were detected for rabies virus antigen by an ELISA assay, and 4.7% of seemingly healthy dogs were positive for rabies virus antigens. Then, dogs with ELISA-positive saliva were sacrificed and brain specimens were sampled for the subsequent nested RT-PCR assay. Surprisingly, nested RT-PCR assay detected a 443 bp viral ntil now –23. In aN genes revealed over 80% nucleotide and amino acid homology between rabies viral isolates form Fujian Province and other strains globally. Our findings demonstrate that seemingly healthy dogs may serve as carriers of rabies virus. Although the virus samples were captured from only three cities of the province, a considerable proportion of infected dogs inhabited in the communities without any obvious symptoms for a period of time, which is inconsistent with previous reports [Sequencing analysis of the 19 full-length  reports –26. NestCanine vaccination has been shown to be the most effective approach for the prevention of human rabies, and it is theoretically possible to eliminate cases of the deadly rabies virus in people worldwide through mass canine vaccination . HoweverN gene, six clearly distinct genotypes were distinguished according to their percentage of amino acid similarity, including the vaccinal and classical rabies viruses of genotype 1, genotypes 2 (Lagos bat virus) and 3 (Mokola virus), genotypes 4 (Duvenhage virus) and 5 (EBL1) and genotype 6 represented by European bat lyssaviruses (EBL) serotype 2 (EBLV-2) [N genes belonged to genotype I. According to their geographical origins, three distinct clusters  were originated exclusively from western, northern and southern Fujian, respectively, which is in agreement with previous studies [To date, there have been seven genotypes of rabies virus characterized . Based o(EBLV-2) . In addi(EBLV-2) . Current(EBLV-2) . In the  studies –36. PhylN gene in the brain specimens of dogs with ELISA-positive saliva samples, showed the low sensitivity (14.8%) of ELISA for the detection of rabies virus antigens in canine saliva specimens. As described above, ELISA detection is affected by multiple factors. Fortunately, the phylogenetic analysis is not affected, since the full-length N gene was amplified from PCR-positive samples. Second, we did not observe the dogs with ELISA-positive saliva samples for a period of time to see the eventual development of rabies or not. Third, we did not detect G gene in the canine samples. Currently, the rabies virus vaccine strains are CTN, 3aG, PV and Flury in Fujian province, and veterinary vaccine strains are ERA and SAD. Since G protein is the major inducer of protective immunity against rabies [The present study has several limitations. First, ELISA-positive saliva samples were not confirmed by RT-PCR. Our nested RT-PCR, which was employed to detect viral t rabies , we cannN gene in dogs in Fujian Province, Southeast China. Our findings may provide valuable knowledge for the development of strategies targeting the prevention and control of rabies. Further extensive surveillance is required to track virus evolution, and infection status in dogs and other carrier species.This is the first description pertaining to the epidemiological characteristics of human rabies cases and characterization of the rabies virus


Three isolates APMV/gull/Kazakhstan/5976/2014, APMV/gull/Kazakhstan/ 5977/2014 and APMV/gull/Kazakhstan/5979/2014, were obtained from independent samples during annual surveillance for avian influenza and paramyxoviruses in wild birds from the Caspian Sea coast in Western Kazakhstan, and were initially identified as putative paramyxoviruses on the basis of electron microscopy. Hemagglutination Inhibition Assays with antisera to nine known APMV serotypes (APMV1-9) indicated no relation to any of them. Next generation sequencing of whole genome sequences indicated the three isolates were genetically identical, and had a nucleotide structure typical for all APMVs, consisting of six genes 3’-NP-P-M-F-HN-L-5’. Phylogenetic analyses, and assessment of amino acid identities, suggested the most closely related lineages to be APMV-2, 8, 10 and 15, but the novel isolate had less than 64% identity to them and all other known avian paramyxoviruses. This value was above levels considered to generally define other APMV serotypes. Estimates of the evolutionary divergence of the nucleotide sequences of the genomes of APMVs have shown that novel Kazakhstan APMV strain was closest to APMV-2, APMV-8, APMV-10 and APMV-15, with calculated distance values of 2.057, 2.058, 2.026 and 2.286 respectively, which is above values considered to differentiate other serotypes (observed minimum was 1.108 between APMV-1 and recently isolated APMV/UPO216/Korea). Together, the data suggest that isolate APMV/gull/Kazakhstan/5976/2014 and other two should be considered as the first representative of a novel APMV-20 group, and is the first time that avian paramyxoviruses have been found infecting members of the gull family, extending the known taxonomic host range. Avulavirus of Paramyxoviridae family possessing linear negative-strand RNA. According to the Taxonomy of the order Mononegavirales: update 2017 [Avian paramyxoviruses (APMV) belong to the genus ate 2017 , 4. APMVate 2017 . APMV-10The APMV genome encodes six major proteins: nucleocapsid protein (NP); phosphoprotein (P); matrix protein (M); fusion protein (F); hemagglutinin-neuraminidase (HN) and large RNA polymerase (L) as well as two non-structural proteins V and W . APMV-6 Very little is known about the molecular biology of APMV in wild bird populations, but an understanding of the molecular and pathological characteristics of APMV is of general epidemiological interest, and is important for developing vaccines in the case of emergence of novel pathogenic strains.Until recently, the known APMV serotypes were restricted to APMV 2–9, which were isolated and characterised in the 1970s. Following expansion of viral surveillance initiatives and improvements in sequencing technology, ten novel serotypes have been discovered since 2001. A virus isolated from rockhopper penguins was antigenically and genetically distinct from all known serotypes 1–9 and considered to represent the prototype of a new group, APMV-10 . A furthKazakhstan has a vast territory crossed by large flyways, and hundreds of bird species concentrate in the natural landscapes during migration and breeding periods . Five APAll research components involving live animals were conducted according to regulations under the legislation ‘Rules for conducting biomedical experiments, preclinical  and clinical studies ’, and were approved by the Institute of Microbiology and Virology Local Ethics Committee .Cloacal and tracheal swabs were collected in 2013 and 2014 from live wild birds captured by ornithologists for banding during studies of migratory patterns. Sampling also was made during seasonal hunting for wild birds and from freshly dropped feces of wild aquatic birds in Northern Caspian seashore in Kazakhstan. For freshly dropped gull fecal samples it was not possible to identify the source species.Samples were collected using sterile swabs which were subsequently placed in viral transport medium containing 2000 U/ml penicillin, 2 mg/ml streptomycin, 50 μg/ml gentamycin, 50 U/ml nystatin and 0.5% bovine serum albumin. In the field, samples were kept in liquid nitrogen, and were stored at -70°C after delivery to the laboratory.Samples were first screened by RT-PCR targeting the M gene of avian influenza viruses (AIV). Then AIV-negative samples were inoculated into 10-day-old embryonated chicken eggs (ECE) and incubated 72 hours at +36°C . The pre2. Monolayers with about 90% confluency were infected with 10−3 dilution of 28 HA units of the virus with and without addition of trypsin (1 ug/ml) (Sigma). The cells were inspected every day for a week to evaluate cytopathic effects and hemagglutination activity of the cell culture supernatant.In order to define the virus growth characteristics, its replication was evaluated in two available in the laboratory cell lines: chicken embryo fibroblast (DF-1) and Madin Darby Canine Kidney (MDCK). Dulbecco’s minimum essential medium (DMEM) with 10% fetal bovine serum (FBS) was used and incubated at 37°C under 5% COVirulence of this virus was defined by intracerebral introduction of virus-containing suspension into ten 1-day-old chickens  as described previously .The pathogenicity of the virus was studied in one-day-old and 4-week-old non vaccinated chickens of Kobb 500 line inoculated intranasally. Chickens were inspected every day over one week for manifestation of any clinical symptoms of disease, and virus shedding was tested by PCR from cloacal and tracheal swabs.Antiserum to the novel APMV strain gull/Kazakhstan/5976/2014 was obtained by double immunization of rabbits with purified viral suspension. A first immunization was conducted by 15–20 intracutaneus injections near cervical and popliteal lymph nodes with antigen mixed with complete Freund’s adjuvant. After three weeks a second immunization was conducted intravenously with incomplete adjuvant. Blood was taken 7–14 days after second immunization .Analysis was carried out using a standard HI assay , testingVirus-containing allantoic fluids were centrifuged at 3000 RPM for 30 min. The supernatant was separated and then subjected to ultracentrifugation at 52500g for 40 min. The resulting pellets were diluted in a minimal volume of 0.05 M phosphate buffer. Virus specimens were prepared on formvar film-coated grids with the addition of a layer of evaporated carbon. The specimens were contrasted with 3% phosphotungstic acid solution and then examined in an electron microscope JEM 100 CX .Viral RNA was extracted from 140 μl of hemagglutination assay positive allantoic fluid using the QIAamp RNA Mini kit  in accordance with the manufacturer’s recommendations.Reverse transcription PCR (RT-PCR) assays were performed on the basis of one-step protocol using appropriate RT-PCR kit  according to the manufacturer’s instructions, employing Pan-PMV primers targeting the conservative fragment of L-gene . The cycAfter purification, the PCR products were Sanger-sequenced using the same PCR primers on an ABI 3730xl DNA analyser  using BigDye Terminator Kit v.3.1 Sequencing Kit (Applied Biosystems).For whole-genome sequencing, viral RNA was used as a template for further library preparation using TruSeq Stranded Total RNA with Ribo-Zero Gold  according to manufacturer’s recommendations. Paired-end sequencing was performed on an Illumina HiSeq 3000 instrument. Raw sequence data were assembled and analyzed using CLC Assembly Cell software (Qiagen). Nucleotide and amino acid sequences were aligned and their evolutionary distances were estimated using Mega 6.0 software . Twenty The most appropriate sequence evolution model for use in phylogenetic analyses was identified on the basis of Akaike Information Criterion (AIC) and Bayesian Information Criterion (BIC) ranking via ModelTest, as implemented in MEGA 6.0.G, parameter = 1.3410;+I, 13.6969% sites), with 500 bootstrap replicates, in MEGA 6.0 [A maximum likelihood phylogenetic tree was constructed for concatenated whole genome nucleotide sequences, using a General Time Reversible model with discrete Gamma distribution and Invariant sites  were generated in MEGA 6.0, based on the Le_Gascuel_2008 model . The phyThe complete sequence of APMV/gull/Kazakhstan/5976/2014 is available at GenBank under the accession no. MF033136.During annual virus surveillance, 486 samples were collected from 348 wild birds belonging to 50 species, and APMV positive samples were found in freshly dropped feces collected on the Caspian seashore in Kazakhstan. Samples were inoculated in ECE and after the first passage hemagglutinating agents were obtained. Viruses were isolated from 3 inoculums: APMV/gull/Kazakhstan/5976/2014, APMV/gull/Kazakhstan/5977/2014 and APMV/gull/Kazakhstan/5979/2014. Whole genome sequencing showed the viruses from the three independent samples to be 100% identical and so only APMV/gull/Kazakhstan/5976/2014 was used for further genetic analyses. Purified virus stock of APMV/gull/Kazakhstan/5976/2014 was examined by electron microscopy .The shape of virions had an expressed polymorphism. The size of spherical virions varied between 150–600 nm. The fine structure of virions was similar to other paramyxoviruses. Virions had 10–15 nm supercapsid envelope coated with 8–12 nm long spikes 4.5–5.0 nm in a diameter .The isolated hemagglutinating agents were tested using Hemagglutination Inhibition Assay and the Kazakhstan isolates did not react with reference sera specific to APMV-1 to APMV-9 indicating a different antigenic structure.Kazakhstan isolates showed minimal cross-reaction with APMV-1, 2 and 6 indicating some antigenic similarity between these serotypes. The highest titers were registered with homologous antisera to this virus obtained from rabbits .Further identification of the serotype was made by Sanger sequencing of a 700 bp PCR product of L-gene fragment. A BLAST-analysis confirmed that the isolated virus sequence grouped within the APMV family, but did not match any currently known APMV.8 HA units in ECE, after 3 days incubation at 35°C. However, the virus did not replicate with and without exogenous protease in tested MDCK and DF-1cell lines that were used to determine whether they can support the growth of the virus.Avian paramyxovirus strain APMV/gull/Kazakhstan/5976/2014 yielded a titer of 2The cells were inspected every day during a week and no cytopathic effect and hemagglutination activity of the cell culture supernatant were observed. Additionally PCR was run with supernatant and negative results were obtained, indicating that these cells did not support the replication of the novel gull APMV.After inoculation of the virus into the cerebrum of one-day-old chickens, the obtained ICPI value was zero, without any clinical signs of disease.During every day observation of infected intranasally chickens, no manifestation of clinical symptoms of any disease was observed. All infected chickens looked healthy without any signs of sickness.To reveal possible virus shedding polymerase chain reaction with cloacal and tracheal swabs was conducted. All tested chickens showed negative result indicating absence of virus infection.The complete genome sequence of the APMV isolate was characterized using a ‘next-generation sequencing’ approach. Assembly of the reads produced a contig of 15,786 nucleotides, with a GC content of 42%, which is longer that of the genetically closest strains, APMV-2/Yucaipa (14904 bp), APMV-8/Delaware (15342 bp), APMV-10/Falkland Islands (15456) and APMV/Brazil (14952 bp). Review of the annotated genome identified the six genes typical for APMVs: 3’-NP-P-M-F-HN-L-5’ , which eEstimates of the evolutionary divergence of the nucleotide sequences of the genomes of APMVs showed that the novel Kazakhstan APMV strain was also closest to APMV-2, APMV-8, APMV-10 and APMV-15, with values of 2.057, 2.058, 2.026 and 2.286, respectively, compared to 1.108 (lowest) between APMV 1 and recently isolated APMV/UPO216/Korea, and 5.001 (highest) between APMV-4 and 8 .Calculation of the amino acid sequence identities of F and HN genes  showed tThe putative amino acid sequence of the F-gene cleavage site was defined as GEQQAR↓LIG. This motif lacks multiple basic residues, a characteristic that typically corresponds with non-pathogenic variants, which is concordant with results of ICPI test.A maximum likelihood phylogeny of the whole genome nucleotide sequence places the novel gull APMV in a clade with serotypes APMV-2, 8, 10 and 15, lying on a separate branch basal to this group .The close evolutionary relationship with APMV-2, APMV-8, APMV-10 and APMV-15 is also supported the analyses of the 6 individual APMV genes at the amino acid. .Kazakhstan has a vast territory where three large flyways of water birds cross: Black Sea-Mediterranean, West Asian-East African and Central Asian. Numerous places of nesting, moulting and summer concentration of birds play an important role in the maintenance of the populations of more than 150 water bird species inhabiting Central Asia.Historically, five different APMV serotypes have been isolated in Kazakhstan between 2002 and 2013. APMV-1 serotype was isolated in 2005, 2006 and 2009 from wild birds in Central Kazakhstan and in 2013 from avifauna of Southern Kazakhstan. APMV-4 from wild birds was isolated in 2004 and 2009 in South-eastern and Caspian regions. APMV- 6 and 8 were first isolated in Southern and Northern Kazakhstan respectively in 2013. Aside from these, an APMV-2 strain was also isolated during the 1980s from chickens and turkeys in Kazakhstan . RecentlThis study found APMV positive samples during annual screening of gulls from the Caspian seashore in Kazakhstan. The electron microscopy showed the isolated virus to have morphology consistent with paramyxoviruses but it was not possible to differentiate between APMV serotypes on this basis.Traditionally, APMVs were resolved based on their antigenic differences, and nine groups were defined by Hemagglutination Inhibition Assay in the 1970s. Since the isolation of APMV-10, it was suggested to classify APMVs based on genetic data because of frequent cross-reactions in HI assays, and a possible lack of hemagglutinating activity in potential future serotypes, as observed in APMV-5 .In this research the Kazakhstan isolate, as expected, showed the highest titers with homologous antisera, but also minimal cross-reaction was registered with APMV-1, 2 and 6, confirming the priority of the genetic approach.8 HA units of virus, indicating that it is avirulent for them. Experimental intranasal infection of 1-day-old and 4-week-old chicken by virus also the virus was avirulent in chickens.The novel APMV did not cause death of chicken after intracerebral inoculation with 2The virus did not replicate with and without exogenous protease in tested MDCK and DF-1 cell lines, however, the growth characteristics needs to be evaluated with other cell lines.Sequencing of the viral genome revealed characteristic APMV coding regions and the noncoding terminal sequences . The genome size is 15,786 nucleotides that is compatible with the rule of six and it also has 6 genes typical for APMV: 3’-NP-P-M-F-HN-L-5’.The phylogenetic relationship among APMV-2, APMV-8, APMV-10 and APMV-15 is consistent throughout the genome, at both the amino acid and nucleotide level, forming a monophyletic group, suggesting these viruses share a more recent common ancestor than with other lineages. Their close relatedness observed in phylogenetic trees was also reflected in the pattern of nucleotide sequence identities. While there is no unified criterion for differentiating APMV serotypes based on evolutionary divergence, the distance separating novel APMV from the closest lineage, APMV-2 is 2.057, and this divergence is greater than the minimum inter-serotype distances separating other accepted serotypes (minimum 1.108). This, together with the other serological data, suggests that isolated new APMV should be considered as a novel serotype and the voucher strain of a new APMV-20 group .Laridae). The evolutionary divergence of the virus suggests the serotype could be specific for gulls, and it may be persisting in the population,.Finally, to our knowledge this is the first report of an APMV in gulls (avian family


Flavivirus and the most prevalent arbovirus in tropical and subtropical regions around the world. The incidence of dengue has increased drastically over the last few years at an alarming rate. The clinical manifestation of dengue ranges from asymptomatic infection to severe dengue. Even though the viral kinetics of dengue infection is lacking, innate immune response and humoral immune response are thought to play a major role in controlling the virus count. Here, we developed a computer simulation mathematical model including both innate and adaptive immune responses to study the within-host dynamics of dengue virus infection. A sensitivity analysis was carried out to identify key parameters that would contribute towards severe dengue. A detailed stability analysis was carried out to identify relevant range of parameters that contributes to different outcomes of the infection. This study provides a qualitative understanding of the biological factors that can explain the viral kinetics during a dengue infection.Dengue virus is a mosquito borne Aedes aegypti. The etiological agent of the dengue virus is a RNA virus that belongs to the family of Flaviviridae viruses, of which there are four distinct closely related serotypes  [Dengue virus (DENV) has emerged as the most prevalent arthropod-borne disease in humans worldwide, with an estimated 390 million individuals infected per year, leading to approximately 500,000 hospitalizations and 25,000 deaths . Dengue These four serotypes cause a variety of clinical symptoms ranging from asymptomatic infection to dengue fever (DF) to its more severe manifestations, dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). DF is a self-limiting illness with onset of fever along with symptoms such as headaches, muscle or joint pain, and rash –8. Only α/β that has been shown to induce resistance to infection in neighboring uninfected cells and limit the spread of the virus [The immune response to dengue infection plays an important role in controlling the virus. The human immune system is made up of two parts: the innate immune response and adaptive immune response. The nonspecific innate immune response provides immediate protection against an invading pathogen and is the first line of defense. Innate immune response produces type I interferon (IFN) mainly IFN-he virus , 9, 10. he virus , 12. In he virus . Also eahe virus , 15.Interferon produced by infected epithelial cells is important for the activation of the adaptive immune response , 16. TheAs virions enter the body, they infect dendritic cells, macrophages, monocytes, and hepatocytes. As soon as the body learns that the cells are infected with dengue virus, it triggers the innate immune response. When the innate immunity is unable to curb the infection, it initiates the adaptive immune response. Once the adaptive immune response starts fighting the dengue infection, the antigens present on virus particles activate B-cells, which mature into plasma cells which then produce antibodies called IgM and IgG , 17. TheExtensive research on mathematical modeling of dengue epidemiology has been done for the last century –26 but oThis study is an attempt to develop a computer simulation model to reproduce the known dynamics of healthy cells, infected cells, virus, B-cells, and immune response. Both innate and humoral immune responses have been incorporated to the model to evaluate the effect of immune response on viral control. This model is merely a conceptual model to capture the qualitative behaviour of virus dynamics. Thus we can then extend this model to fit quantitative data from clinical experiments. In this study, two viral titer peaks were observed during the course of infection. It was found that the innate immune response is responsible for the first rapid viral decline and for the subsequent second peak in viral load. It is also noted that the humoral immune response is needed to eventually clear the virus from the body. Next we identify the significant parameters and carry out a sensitivity analysis to investigate the virus dynamics with respect to parameter variability.In order to validate the results, we carry out a detailed stability analysis followed by numerical simulations to identify the relevant ranges of key parameters which generates different scenarios of the infection.Experimental studies have shown that higher viremia titer early in the course of infection is associated with more severe disease , 29. PeaHere we develop a model to study the within-host dynamics of dengue infection which incorporates both innate and adaptive immune response. We assume that only one serotype of dengue virus circulates in an infected host and the virus infects monocytes, macrophages, dendritic cells, and hepatocytes in the blood stream. This model is an extension of the model presented in  in whichTo model the effects of innate immune response on dengue viral dynamics, we make the assumption that number of activated NK cells is proportional to the level of IFN as in the recently published dengue model  and inflS indicates susceptible cells , I indicates infected cells, V indicates dengue virus particles, B indicates B-cells, A indicates antibodies, and F indicates interferon. Susceptible cells are produced at a constant rate μ and they die at a rate α. Susceptible cells become infected by virus at a rate aSV, where a is the rate of infection. The infection period is assumed constant given by 1/β. The infected cells are killed by NK cells at a rate proportional to ϕ. Virus replicates in infected cells and bursts out at a rate k and decays at a rate γ. Virus is being neutralized by antibodies at a rate p. B-cells are produced at a constant rate η and die at a rate δ. On coming into contact with virus, B-cells become activated at a rate c. These activated B-cells then transform into plasma cells (denoted by the same variable as B-cells) which produce antibodies (A) at a rate f. Antibodies are destroyed by virus at a rate q and naturally at a rate κ. IFN (F) is produced by infected cells at a rate q1 and decays at a rate d. A schematic diagram of model .Given an initial value problem,The mean virus count and the standard deviation of the virus count are visualized using MATLAB software package and the algorithm used is given as follows.Algorithm for the Sensitivity AnalysisInput the parameter values except the parameter for which the sensitivity analysis is carried for.Set up the initial conditions.p = (v − u)∗rand + u p = generates random numbers ∈. Create 1000 random values of the parameter within a specified interval.N = number of time intervals h = (tmax − t0)/N for i = 1 : number of random numbers do for n = 1 :  N  do  i     for each tn+1 = tn + h     yn+1 = yn + h∗f     end for  end for y)  compute mean(y)  compute sd(Create an iterative loopa)), we solve the model numerically for randomized values of a. Using explicit Euler method, we can analyze how sensitive the system reacts to fluctuations in a. Here we use initial conditions  =  and the parameter values used are μ = 20, α = 0.05, β = 0.5, κ = 0.009, γ = 0.5, p = 0.007, η = 10, δ = 0.049, c = 0.001, q = 0.9, f = 0.8, k = 2, q1 = 0.8, d = 0.7, ϕ = 0.002.To analyze the sensitivity of the model with respect to the rate of infection , the virus count converges to some nonzero value.For very low values of the infection rate, a.By looking at Figures f. Smaller production rates generated an endemic equilibrium in the virus count and high fluctuations in the standard deviation. Thus both innate and antibody immune responses were unable to curb the infection. In this case R0 > 1 and from f values. For moderate and high values of f, the mean virus count displayed two peaks and one peak, respectively, and the standard deviations around the means displayed a predictable pattern and converged to zero ultimately. Higher production rate of antibodies eliminated the second peak as antibody mediated immune response alone controlled the infection. For these two cases, R0 was computed and was less than one (The same pattern of behaviour was observed with respect to antibody production rate, than one  leading k. The human immune system is an enormously complex interrelated system. When the virus burst rate k is small, there is only a less virus count. Since B-cells are activated by virus, the number of B-cells is proportional to the virus count. Therefore for low k values, there is only a fewer number of B-cells, thus less antibodies. Hence the first peak is controlled by the innate immune system and the antibodies would eventually lead to virus clearance, whereas when k is large, there is a higher viral count and proportionally a higher antibody count. The first peak is controlled by innate immunity and by antibody mediated immune response and this high antibody count eliminates the second viral peak. This phenomenon can be seen in Figures k values between 1 and 1.8, an oscillatory behaviour in the virus count is observed. The reason can be explained by A different sensitivity pattern was observed for changes in virus burst rate, k is large enough, from When R0. Then a detailed numerical analysis was done to identify the relevant parameter values that leads to infection free or infectious states.In this study, a computer simulation model based on an ODE dynamical system was developed to understand the dynamics of dengue virus. A sensitivity analysis of some key parameters was carried out to better understand the development of severe disease and what role model parameters play in severe dengue disease. Also the stability of the infection free steady state and endemic steady state of the model were discussed with respect to the basic reproduction number, Two viral titer peaks were observed in our simulation model and was able to show that cytolysis of infected cells by NK cells during innate immune response is responsible for the rapid viral decline after the first peak and for the subsequent second peak. Humoral immune response is needed for the second viral decline and to eventually clear the virus.a, f, and k was carried out to predict the viral load changes and to analyze the dynamics of immune responses. As the infection rate a is increased, the number of peaks in the viral load would increase leading to a virus persistence state. In this case, it was observed that the basic reproduction number R0 > 1. Oscillations in the standard deviations of the mean virus count for all different values of a implied that the model is highly sensitive to changes in the parameter a.Sensitivity analysis of the parameters f increased, the number of virus titer peaks and the mean viral load decreased. This implies that the antibody dynamics has a significant effect on the viral load. Also for high f values, R0 < 1 and the virus count converged to zero. The fluctuations in standard deviation around the mean, for different values of f, displayed a predictable behaviour.As the antibody production rate k values, only one peak was observed in the viral load. As the burst rate is high, the virus count is high and since activated B-cells are proportional to the virus count, there is a high B-cell count and proportionally a high antibody count. Thus the viral load is controlled by IFN and antibodies and because of high immune response, the second peak is entirely eliminated. However since this situation displays a high initial viral peak and excess activation of immune response, it can represent a severe dengue condition. For intermediate k values, we observed oscillatory behaviour in the virus count, IFN, and antibody count. k increases, the virus count would lead to an endemic state, which is a false statement. This explains the fact that human body is an interrelated system and increasing one parameter may not lead to an adverse effect.As the virus burst rate is increased, the number of virus titer peaks decreased. For high


Mosquitoes are classified into approximately 3500 species and further grouped into 41 genera. Epidemiologically, they are considered to be among the most important disease vectors in the world and they can harbor a wide variety of viruses. Several mosquito viruses are considered to be of significant medical importance and can cause serious public health issues throughout the world. Such viruses are Japanese encephalitis virus (JEV), dengue virus (DENV), chikungunya virus (CHIKV), and Zika virus (ZIKV). Others are the newly recognized mosquito viruses such as Banna virus (BAV) and Yunnan orbivirus (YNOV) with unclear medical significance. The remaining mosquito viruses are those that naturally infect mosquitoes but do not appear to infect humans or other vertebrates. With the continuous development and improvement of mosquito and mosquito-associated virus surveillance systems in China, many novel mosquito-associated viruses have been discovered in recent years. This review aims to systematically outline the history, characteristics, distribution, and/or current epidemic status of mosquito-associated viruses in China. Culicidae. There are approximately 3500 species of mosquito in the world, grouped into 41 genera. Mosquitoes carry a wide variety of viruses, and these viruses can be classified into two categories: mosquito-borne viruses and mosquito-specific viruses. Mosquito-borne viruses are arboviruses, which can replicate within the mosquito but can also be transmitted biologically to vertebrates and infect vertebrate cells. Most of the mosquito-borne viruses are pathogenic viruses responsible for disease outbreaks in human and animal populations, such as dengue virus (DENV), chikungunya virus (CHIKV), yellow fever virus (YFV), Zika virus (ZIKV), and Akabane virus (AKV). The majority of mosquito-borne viruses are distributed within the families Peribunyaviridae, Flaviviridae, Togaviridae, and Reoviridae. Mosquito-specific viruses, as insect-specific viruses (ISVs), can infect mosquitoes naturally and replicate in mosquito cells in vitro, but do not replicate in vertebrate cells or infect humans or other vertebrates .DENV is a member of the genus et al.et al.et al.et al.et al.et al.et al.DENV was first isolated from human blood samples in Japan, in the year 1943  resulting in high mortality  serogroup of the genus Culex tritaeniorhynchus is the most important vector for JEV in China. However, other mosquito species have also been implicated as vectors of JEV, such as Aedes vexans, Culex modestus, Culex pipiens pallens, Culex theileri, Anopheles sinensis, and Armigeres subalbatus  and a variety of avian species including ducks, geese, chickens, pigeons, and sparrows in Southeast Asia or China  in China was reported in Shandong and Yunnan provinces, and the results showed a high percentage of positives for TMUV by both RT-PCR and virus isolation assays , with different species recognized as primary vectors in different areas  usually present mild symptoms such as fever, skin rashes, muscle and joint pain, and headache. But recently, the infection was reportedly associated with microcephaly in infants and neurological disorders such as Guillain–Barré syndrome (GBS) , Puerto Rico (2015), and Yap Island (2007)  from Culex gelidus collected in Malaysia in 1955  have been detected in serum from fever and headache patients in Jiashi County in Xinjing with a 13.0% positive rate, and in local livestock in Geermu city in Qinghai  consists of three negative-stranded RNA, designated large (L), medium (M) and small (S) segment, and the viruses can be classified into seven major genetic lineages  and Liaoning virus (LNV) within the family Reoviridae .In nature, BAV can be detected and isolated from pigs, cattle, and ticks  results indicated that the positive rate of anti-BAV IgM antibodies was 11.4%. However, no fourfold or greater BAV specific antibody response was detected by neutralization test in serum from the acute or convalescent phase of illness   was first isolated from a pool of Alphavirus and the family Togaviridae  (Japanese isolate) showed that these viruses exhibit high identity, and up to 5.5% amino acid sequence divergence was calculated between the original Malaysian isolate of GETV and the Chinese isolates  isolated from Alphavirus, family Togaviridae, which can cause polyarthritis and arthralgias. RRV was first isolated from Aedes vigilax captured in the Australian Ross River region in 1959  reported, which was isolated from brain tissue of bat in Hainan province. Experimental infection showed that HBb17 could replicate in mosquitoes and cause mice to die  in China was isolated in 1986 from the blood of a patient with fever in Yunnan Province  from A. vexans in Chaoyang city in Liaoning, China, in 2008  was first isolated from Culex pipiens mosquitoes collected in Shandong, China, in 2006 . A larger survey for the distribution and phylogenetic analysis of CxFV in China was conducted during 2004–2012  VN180 isolated from Vietnam  in China from samples of Culex pipiens quinquefasciatus in Shenzhen, Guangdong, in 2011   and represented a novel variant of this species  from one pool of mosquitoes (Culex tritaeniorhynchus) collected in Mangshi City, Dehong Prefecture, southwest of Yunnan in China in 2013 (Wang J et al.MSV is a newly discovered virus in the genus Culex tritaeniorhynchus rhabdovirus, etc., belonging to various viral genera were found by deep sequencing of Chinese mosquito samples from Hubei and Zhejiang in recent studies, which suggests that more extensive cell culture efforts are highly likely to yield additional viruses (Li et al.et al.The relative distribution of mosquito-associated viruses in China is shown in Fig.


In addition to covering spherical beads, VP1 pentamers covered cubic magnetite beads, as well as the distorted surface structures of liposomes. These findings indicate that VP1 pentamers could coat artificial beads of various shapes and sizes larger than the natural capsid. Technology based on VP1 pentamers may be useful in providing a capsid-like surface for enclosed materials, enhancing their stability and cellular uptake for drug delivery systems.The icosahedral capsid structure of simian virus 40  consists of 72 pentameric subunits, with each subunit formed by five VP1 molecules. Electron microscopy, immuno-gold labeling, and  Its capsid is 45 nm in diameter and is formed by 72 pentameric subunits, each composed of five VP1 major capsid proteins . VP1 pentamers are arranged in a T = 7 d (triangulation number 7 dextro) icosahedral lattice, with the carboxyl terminus of VP1 mediating inter-pentameric contacts that hold the capsid together Simian virus 40 (SV40) is a small non-enveloped DNA virus of the in vitro of VLPs isolated from these cells with dithiothreitol (DTT) and ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid (EGTA) results in their disassembly into VP1 pentamers in vitro into VLPs under appropriate conditions in vivo. Other VLPs can also be utilized to modify natural nanoparticles, allowing their use as contrast agents for medical imaging When expressed in insect cells using recombinant baculovirus, VP1 self-assembles into virus-like particles (VLPs) (45 nm in diameter), similar to the natural assembly of SV40 virus capsids Capsid subunits of various viruses were used to encapsidate artificial beads. For example, the capsid protein of a plant virus, red clover necrotic mosaic virus (RCNMV), was used to encapsidate gold nanoparticles. In that study, the surface of gold nanoparticles was immobilized with an RNA sequence to which RCNMV capsid protein binds specifically, resulting in RCNMV capsid protein being concentrated on the surface of the particles This study shows that various artificial beads could be coated with VP1 pentamers, with no particular limitation of the sizes and shapes of coated artificial beads. These results indicate that VP1 coating technology is applicable to a variety of materials, providing capsid-like surface conditions and enabling high dispersibility and stability even in body fluids. Highly dispersible and stable materials coated by VP1 pentamers may be promising materials for diagnosis and treatment of various illness/diseases.22.1VLPs consisting of wild-type VP1 were prepared as described 2.2Polystyrene beads (Polybead@Carboxylate 0.10 Micron Microspheres) were purchased from Polysciences, Inc. Silica beads (silica-coated CMNPs) were prepared using the seeded polymerization technique with sol–gel reaction. Briefly, a solution of tetraethyl orthosilicate (TEOS) in ethanol was added to the CMNPs in water. Silica coating was initiated by rapidly injecting an aqueous ammonia solution into the CMNP–TEOS mixture so that the water concentration of the dispersion medium was 11.0 M (1:4 (v/v) water/ethanol) and stirred overnight at 4 °C. The concentration of TEOS was varied from 2.5 to 44.1 mM, resulting in iron and ammonia concentrations of 3.6 mM and 0.9 M, respectively. Double-distilled water was added to the mixture, followed by evaporation to remove ethanol and ammonia. The resulting mixture was dialyzed against double-distilled water to obtain purified silica beads. CMNPs were prepared as described 2.3n-octylamine (190 g) containing oleic acid (6.14 g). The mixture was gradually heated to boiling with vigorous stirring and maintained at that temperature for 30 min. The mixture was cooled to room temperature, and a large excess of 2-propanol was added, followed by centrifugation at 7500 rpm for 1 h and redispersion in 1-octadecene. For citric-acid coating, prepared magnetite particles (180 mg) were washed with 2-propanol and suspended in toluene (16 ml). After washing, the suspension was mixed with 4 ml dimethyl sulfoxide (DMSO) containing 0.3 g of thiomalic acid and sonicated at 35 kHz for 4 h under a nitrogen atmosphere. The resulting black precipitate was washed with 2-methoxyethanol and dispersed in an aqueous solution of sodium citrate. The dispersed mixture was then sonicated at 35 kHz for 1 h. The resultant magnetite beads were precipitated by adding 1,4-dioxane and separated using a magnet. Finally, citric-acid-coated cubic magnetite beads were dispersed in distilled water and dialyzed for 3 h. The average length per side of ten randomly selected cubic magnetite beads on electron micrographs was approximately 30 nm. The average diameters of uncoated and VP1-coated cubic magnetite beads in solution were determined using ELSZ-2 .To synthesize cubic magnetite beads, the iron oleate complex was dissolved in tri-2.4N-[1-propyl]-N,N,N-trimethyl ammonium chloride (DOTAP) in water (5 μg/ml) was added to 400 μl of PLGA in acetone (25 mg/ml) to generate a water in oil (W/O) emulsion. To this mixture was added 5 μl of 2% polyvinyl alcohol (PVA) in water under vortex to generate a water in oil in water (W/O/W) double emulsion. The latter was centrifuged at 15,000 rpm for 10 min at 4 °C, and the supernatant was removed. The resultant pellet was resuspended in 2 ml of water and stirred overnight at 4 °C to produce purified PLGA beads. The average diameter of PLGA beads (200 nm) in solution was determined using ELSZ-2 .Ten microliters of 2.5sn-glocero-3-phosphoethanolamine (DOPE) (3:1 molar ratio) were diluted in chloroform at a lipid concentration of 5 mg/ml. To prepare a lipid film, chloroform was allowed to evaporate under nitrogen flow. The film was rehydrated in Tris–HCl buffer by intermittent vortexing, and extruded through a polycarbonate filter  using a Mini Extruder (Avanti Polar Lipids).DOTAP and 1,2-dioleoyl-2.64:1. Each mixture was dialyzed in coating buffer, containing 20 mM MOPS–NaOH (pH 7.0), 150 mM NaCl, and 2 mM CaCl2, for 16 h at room temperature. To remove free VP1 pentamers, the sample was centrifuged at 2,0400 × g for 15 min at 4 °C. The pellet fraction containing VP1-coated beads was washed twice and resuspended in coating buffer. For transmission electron microscopy (TEM), the samples were negatively stained with 2% ammonium molybdate and examined under a TEM  as described Purified monomeric VP1 pentamers were mixed with artificial polystyrene, silica, PLGA, or citric-acid-coated cubic magnetite beads, or cationic liposomes, at a ratio of approximately 102.7Artificial beads, either uncoated or coated with VP1 pentamers, were incubated with anti-VP1 antibody for 1 h at 37 °C, followed by incubation with protein-A-conjugated colloidal-gold  for 1 h at 37 °C. The samples were negatively stained and examined under a TEM, as described above.2.8Uncoated and VP1-pentamer coated artificial beads were measured with ELSZ-2  and dialyzed against buffers of pH 5, 7, and 9 using mini-dialysis units with a molecular weight cutoff of 3500 Da .33.1in vitro was approximately 45 nm Our previous studies showed that the diameter of the particles induced by DNA-mediated self-assembly of VP1 2 for 16 h at room temperature. After dialysis, the mixture was washed once, and a portion of the resuspended sample was mounted onto a collodion-coated copper grid and negatively stained with 2% ammonium molybdate for EM . VP1 pentamers were mixed with polystyrene beads and dialyzed against a physiological buffer containing 20 mM MOPS–NaOH (pH 7.0), 150 mM NaCl, and 2 mM CaCle for EM A and B. To confirm that the VP1 pentameric structure occupying the entire surface of these beads was composed of VP1 proteins, the dialyzed sample on the EM grid was incubated with anti-VP1 antibody, followed by immuno-gold labeling . As expe3.2ζ potential of polystyrene beads before and after VP1 coating was analyzed. The ζ potential is the potential difference between the buffer and the stationary layer of fluid attached to the dispersed bead. Therefore, when polystyrene beads are covered with VP1 pentamers, the ζ potential of the beads would be moderated and closer to that of wild-type VLP. To verify this, the surface potential of VLP was assessed at pH 5, 7, and 9. This surface potential was 10 mV at pH 5, declining to −30 mV at pH 9, indicating that the electron charge on the functional groups of VP1 was altered by pH  were covered with VP1 pentamers, however, their ζ potentials between pH 5 and pH 9 ranged from −5 to −25 mV  and approximately 200 kDa Finally, coating of artificial materials with VP1 pentamers may improve their mono-dispersibility and stability. VP1-coated CMNPs were monodispersed in physiological and serum containing buffers, whereas uncoated CMNPs clustered and formed large multimeric structures 5This report showed that monomeric SV40 VP1 pentamers coat a variety of artificial beads, regardless of size and shape, due to their self-reassembly activity. VP1-coated materials may not only have a capsid-like surface but be mono-dispersible and stable, which may enhance their retention in body fluids and cell-targeting properties when used for drug delivery.M.K., K.D., H.F., Y.K., K.I., T.I., T.E., and M.H. performed the experiments. M.K., M.H., and H.H. conceived and designed the experiments. M.K., M.M., Y.Y., and H.H. wrote the manuscript. All authors discussed the results and commented on the manuscript.The authors declare no competing financial interests. Correspondence and requests for materials should be addressed to H.H.