In Italy, the West Nile Virus surveillance plan considers a multidisciplinary approach to identify the presence of the virus in the environment  and to determine the risk of infections through potentially infected donors (blood and organ donors). The costs associated with the surveillance program for the Lombardy Region between 2014 and 2018 were estimated. The costs of the program were compared with a scenario in which the program was not implemented, requiring individual blood donation nucleic acid amplification tests (NAT) to detect the presence of WNV in human samples throughout the seasonal period of vector presence. Considering the five-year period, the application of the environmental/veterinary surveillance program allowed a reduction in costs incurred in the Lombardy Region of 7.7 million EUR. An integrated surveillance system, including birds, mosquito vectors, and dead-end hosts such as horses and humans, can prevent viral transmission to the human population, as well as anticipate the detection of WNV using NAT in blood and organ donors. The surveillance program within a One Health context has given the possibility to both document the expansion of the endemic area of WNV in northern Italy and avoid most of the NAT-related costs. West Nile Virus (WNV) is a flavivirus transmitted by ornithophilic mosquitoes between avian hosts . Virus aThe ecologic aspects of WNV infection were first described in the 1950s in Egypt  and it iIn addition, the economic impact of WNV infections might be relevant for health services around the world. Two published analyses, related to the context of the United States, reported costs for hospitalization equal to 778 million USD (US Dollar) between 1999 and 2012, with median initial costs of hospitalization (both direct and indirect) per case between 7501 and 82,542 USD, and median long-term costs (both direct and indirect) between 7025 and 76,747 USD among patients with fever, meningitis, encephalitis, and acute flaccid paralysis .In Italy, the first outbreak of WNV infection was reported in 1998 in the Tuscany region , and theDue to the re-emergence and the following geographical spread of WNV, the Directorate General for Prevention of the Italian Ministry of Health (MoH) issued in the spring of 2010 a national plan for WNND human surveillance that integrated human and veterinary surveillance .Since then, WNV has been repeatedly identified through integrated surveillance programs conducted in Italian regions , includiThe occurrence of the WNV infection cycle is favored by geographic aspects  and climate  . These cEpidemiological surveillance identifies human cases to quantify the disease burden and to identify seasonal, geographical, and demographic patterns of human morbidity and mortality, while environmental/veterinary surveillance monitors local WNV activity in vectors and nonhuman vertebrate hosts in advance of epidemic activity affecting humans. The aim of the surveillance program is to address and to maximize the response of healthcare providers and professionals via the early detection of the virus in the different epidemiological actors, as well as focus the interventions toward target areas and seasonal periods. The WNV surveillance plan considers a multidisciplinary approach to identify the presence of the virus in the environment  and to determine the risk of infections through potentially infected donors (blood and organ donors) .According to the indications coming from the environmental/veterinary surveillance, the analyses conducted to detect the presence of WNV in blood samples for transfusions, as well as all activities related to organ donations, are conducted starting from the date of the first positive sample identified in mosquitoes, birds, or horses, instead of being performed for a fixed period from the beginning of June to the end of November. Such an integrated and multidisciplinary WNV surveillance system, which includes, in addition to humans, wild birds, mosquitoes and horses, has been implemented in Lombardy Region since 2014. The main aims of this surveillance system are (1) early detection of the circulation of WNV in the environment, and (2) mitigation of the risk of viral transmission through blood and organ donations . The detThis One Health approach is based on the collaboration of different public institutions, in a network that connects humans, animals, and environmental health, under the coordination of the working group of the Regional Health and Welfare Unit.There have been few studies establishing the economic efficiency of One Health approaches to disease mitigation . HoweverThe analysis was conducted considering the costs associated with the environmental/veterinary surveillance program, the direct medical costs associated with the screening activity for blood donors and organ donations, and the costs associated with the management of WNV infections including hospitalization.Furthermore, the costs of the program were compared with a scenario in which the program was not implemented, requiring only NAT analyses to detect the presence of WNV in human samples throughout the possible transmission period. The costs considered in the analysis are referred to each reference year.Culex mosquitoes , birds , and horses (equine surveillance).With the objective of assessing the presence in the environment of WNV between May and October, veterinary surveillance was conducted in three areas of activity, targeting different species: 2 areas.Entomological surveillance was performed through fortnightly sampling with attraction traps, between May and October. The number of traps progressively increased from 38 at the beginning (2014) to 44 in total in 2020. They were placed in areas suitable for circulation of the virus vector  and distPica pica), hooded crow (Corvus corone cornix), and Eurasian jays (Garrulus glandarius) were collected and delivered to the laboratory by rangers and hunters. These species are considered pests for crops and, therefore, are under population control programs, authorized yearly by the National Institute for Wildlife (ISPRA). The sampling from passive surveillance mainly involved birds belonging to the orders Accipitriformes, Charadiiformes, Columbiformes, Falconiformes, and Passeriformes.Ornithological surveillance was performed through active surveillance (capture and killing of resident “reservoir” birds of selected species) and through passive surveillance . Carcasses of magpie , a process analysis was performed identifying all the resources involved in the different actions, in terms of human resources, equipment, and consumables for both on-field and laboratory activities .The screening of blood for transfusion has been one of the most important aspects of the WNV surveillance plan, since 2002, when the first WNV infection due to blood transfusion was detected in the United States ,29.Italian national guidelines mandate the screening of blood units between 1 June and 30 November for all blood donations in the provinces in which WNV was detected the previous year .This analysis also considered direct medical costs related to human WNV infection cases confirmed by the Italian National Institute of Health (Istituto Superiore di Sanità—ISS) assuming the Regional Health Service perspective, in terms of inpatient activity and diagnostic activity. Costs were derived from a previously published analysis related to the Italian context . A mean The multidisciplinary approach to WNV surveillance made it possible to identify the viral circulation in the study area early. In The total cost of the surveillance activity for a five-year period was equal to 542,935 EUR. Costs were mainly related to entomological surveillance , followed by ornithological surveillance . Total NAT costs were equal to 10.38 million EUR, while the costs to manage WNV-related infections in terms of diagnostic activity and hospitalization were equal to 705,107 EUR. The costs per year for each activity are reported in Considering the number of NATs for checking blood donations, performed starting from the first positive specimen detected by environmental/veterinary surveillance, mean annual avoided costs of 1,535,451 EUR were estimated due to delayed testing on blood units, compared with the scenario in which NATs were performed throughout the vectorial season, i.e., from 1 June to 30 November, as reported in Overall, considering the five-year period, application of the environmental/veterinary surveillance program allowed a reduction of approximately 7.7 million EUR in costs incurred in Lombardy Region.Application of the above-described surveillance system allowed the Regional Health and Welfare Unit of Lombardy Region to avoid costs representing more than 40% of the WNV monitoring plan costs. This was due to the possibility to postpone the beginning of the blood samples tests until the first positive specimen detected by environmental/veterinary surveillance.Moreover, the sharing of information obtained from epidemiological and environmental/veterinary surveillance has enabled controlling the risk of WNV transmission via blood transfusion.In accordance with White (2001) and Healy et al., (2015) ,34, the Our results also showed that viral activity was generally detected earlier in mosquitoes and birds than in humans . In factIt should be noted that the cost of the personnel involved in the human and equine surveillance is not included for laboratory tests, since data available does not allow to isolate the costs of the tests related to WNV. This is due to the fact that the costs available for personnel (payroll) include a sum of activities performed as part of the normal routine activities expected of the specific role (physician or veterinary). Thus, it is not possible to identify the costs related dedicated to the sole aforementioned activities which are part of the surveillance systems.In this context of West Nile Disease viral circulation control, savings in terms of resources were obtained by not activating NAT screening on blood transfusion or organ donors until circulation of the arbovirus was confirmed at the local  level. Indeed, studies, in some published cases , have deThe costs associated with the management of infected patients vary widely depending on the clinical manifestation of the infection and on the course of the disease, being related to inpatient activity and considering severe cases, long inpatient days, and intensive care unit management.In Italy, a cost–benefit analysis related to the implementation of the WNV surveillance program in Emilia-Romagna Region estimated 1.21 million EUR of costs saved by avoiding tests for blood donations and up to 2.98 million EUR of costs saved by avoiding hospitalization-related infections between 2009 and 2015 . In thisAn integrated surveillance system, including birds as virus reservoirs, mosquito vectors, and dead-end hosts such as horses and humans, was used to identify the location and timing of viral activity in the Lombardy Region. The information gained through surveillance was used to prevent viral transmission to the human population, as well as to anticipate the detection of WNV in blood and organ donors using NAT ,40. At tThe above-described surveillance program highlighted the presence of WNV in Lombardy every year in the period 2014–2018, albeit with different intensities, and it allowed the possibility to both document the expansion of the endemic area of WNV in northern Italy and guarantee an avoidance of NAT-related costs. The latter was estimated to be 7.7 million EUR in the five-year period, with mean yearly avoided costs of 1.5 million EUR.


Worldwide, the number of cancer survivors is rapidly increasing. The aim of this study was to quantify long-term health service costs of cancer survivorship on a population level. The study cohort comprised residents of Queensland, Australia, diagnosed with a first primary malignancy between 1997 and 2015. Administrative databases were linked with cancer registry records to capture all health service utilization. Health service costs between 2013–2016 were analyzed using a bottom-up costing approach. The cumulative mean annual healthcare expenditure (2013–2016) for the cohort of N = 230,380 individuals was AU$3.66 billion. The highest costs were incurred by patients with a history of prostate (AU$538 m), breast (AU$496 m) or colorectal (AU$476 m) cancers. Costs by time since diagnosis were typically highest in the first year after diagnosis and decreased over time. Overall mean annual healthcare costs per person (2013–2016) were AU$15,889  and highest costs per individual were for myeloma , brain  or liver cancer  patients. Our results inform policy makers in Australia of the long-term health service costs of cancer survivors, provide data for economic evaluations and reinforce the benefits of investing in cancer prevention. Cancer survivorship is defined as the time from cancer diagnosis to the end of life. Different stages of survivorship have been described as acute (diagnosis to treatment), chronic (ongoing), long-term/late survivorship (≥5 years post diagnosis) and cured (disease-free) ,2. WorldBeside the enormous direct cost to the healthcare system, cancer diagnosis and treatment often have long-term health and financial costs for patients and their families. Complex late effects, both physical  and psychosocial , require ongoing medical care and can substantially affect the quality of life of cancer survivors ,10,11,12Previous Australian studies on cancer-related expenditure are limited because they did not estimate long-term healthcare costs for all types of cancer on a population level beyond five years post diagnosis ,17,18,19Cancer survivors are significantly more likely to be diagnosed with other chronic conditions that share similar risk factors, such as smoking, physical inactivity, overweight, poor diet, and increased alcohol consumption . People We investigated long-term health service use  and associated costs for cancer survivors on a population level  from an A detailed description of data collection, data linkage, variables extracted (linkage variables/research variables) and data storage was provided in our Study Protocol manuscript  and is sQueensland is the third most populous state in Australia, with over 5 million residents . Every QEthics approval was obtained from the University of the Sunshine Coast Human Research Ethics Committee  and from the Australian Institute of Health and Welfare (AIHW) Human Research Ethics Committee (EO2017/3/348). Approval for Queensland data extraction and linkage was obtained under the Public Health Act 2005 (grant RD007281).Healthcare in Australia is provided by a mixture of private and public sector funding and relies on co-payment by private health insurance, or directly by the patient . MedicarEach Australian state, including Queensland, routinely records cancer diagnoses in population cancer registries and collects administrative data on hospital and emergency admissions. In order to capture healthcare services provided in primary, allied health, specialist and hospital care, our identified study cohort from the QCR was linked with national and state data from the Queensland Hospital Admitted Patient Data Collection (QHAPDC), Emergency Department Information System (EDIS), Healthcare Purchasing and System Performance (HPSP) data, Medicare Benefits Schedule (MBS) and Pharmaceutical Benefit Schedule (PBS). This allowed us to capture costs directly incurred by hospital or emergency admissions in public hospitals, as well as Medicare-subsidized primary or allied healthcare and prescription pharmaceuticals. MBS and PBS data also captured patient OOP claims data. This research does not include costs incurred by private healthcare provided without Medicare involvement, or funded by private health insurance.Data linkage was conducted by the relevant state  and national departments (AIHW) and transferred to a Secure Unified Research Environment (SURE) after de-identification of the data and assignment of a new, random patient ID; researchers obtained approval to remotely access de-identified records on the password-protected virtual platform SURE .Cancer types were defined using topography codes, based on the World Health Organization’s International Classification of Diseases for Oncology, 3rd edition (ICD-O3)  and the We focused on the most recent data available to ensure relevance of cost estimates, and selected healthcare costs incurred between 1 January 2013 and 31 December 2016. Individuals were included in cost calculations if they were diagnosed with a primary malignancy at any point from January 1997 to the end of December 2015, and incurred healthcare costs at some time between January 2013 and December 2016. All patient costs were included and patients who died incurred partial costs for that year. We used a bottom-up costing approach for the most accurate results. Individual-level patient records for each cost component were aggregated to form the total cost product, including hospital admissions, emergency presentations, medical and allied health services, and pharmaceuticals, as described below. Healthcare costs included all services utilized in the given timeframe, whether or not cancer related. All costs were reported in 2016 Australian dollars (AU$).Hospitalization and Emergency Presentation costs: Healthcare purchasing data comprised individual cost per episode of care for both hospitalizations and emergency admissions between January 2013 and December 2016 and included total cost per episode, being the product of direct and overhead costs.Medical and Allied Health Service costs: These data were extracted from the Medicare Benefit Scheme via Medicare claims records. Benefits paid represent the dollar value received by healthcare providers from the government. Patient out-of-pocket (OOP) costs were calculated as the difference between fees charged by providers and benefits paid. The sum of patient OOP costs and benefits paid made up total cost of Medical Services in our analyses.Pharmaceutical costs: These data were extracted from the Pharmaceutical Benefit Scheme via claim records of drug prescriptions. Net benefit paid is the government contribution towards the cost of prescription medicinal products and, along with patient contribution, was used as the overall pharmaceutical cost.Costs by time since diagnosis: Time since diagnosis was calculated as the interval between the most recent year of data collected, 2016, and year of cancer diagnosis (as per QCR records). Time since diagnosis was calculated in months and rounded to years, to form the following timeframes: 0–1 year, 2–4 years, 5–9 years, 10–14 years and 15–20 years since diagnosis. ‘Costs since diagnosis’ were calculated for all individuals with cost data incurred in 2016, excluding people who died that year.Cost by type of cancer and relative 5-year survival: Types of cancer were categorized into cancer types with low, medium, or high relative 5-year survival rates. These were defined as: low for 5-year relative survival rate of 0–35% , medium for 36–69% survival rate  and high for 70–100% survival rate  [thyroid) ,39.Data were checked for missing values, logical errors and outliers, but no extreme values were excluded to represent the whole range of health service utilization. In the original QCR data, sex was only used as a linkage variable and was not available to researchers. We were able to access sex of the original patient cohort by merging data from QHAPDC, using gender-specific cancer codes  and gender recorded in EDIS. Mean and median costs, cost ranges, standard deviations and cumulative costs were calculated for each cost component per patient per year, as well as per episode of care. SPSS software version 21.0 was used for all statistical analyses and data were illustrated graphically using Microsoft Office Excel version 2108.A total of N = 365,443 individuals were ascertained from the Queensland Cancer Register . Of thesDemographic information for the selected cancer cohort of N = 230,380 individuals is shown in Mean annual healthcare costs, between 2013–2016, were AU$15,890  per person. Distribution of Cancer Cohort by Time since Diagnosis and Vital StatusBased on reference year 2016, the distribution of time since diagnosis by vital status is shown in Costs by Time since DiagnosisMean healthcare costs per person incurred in 2016 by time since diagnosis, hence only for individuals with vital status ‘alive’ in 2016, are shown in Mean annual healthcare costs per person between 2013 and 2016 by type of cancer, and 5-year relative survival, are shown in The total mean annual healthcare expenditure for the cancer cohort between 2013 and 2016 was AU$3.66 billion. Total mean annual healthcare cost from 2013–2016 by type of cancer and health service component are illustrated in Our cost analysis shows mean annual healthcare expenditure by individuals with a history of cancer of over AU$3.66 billion for the period 2013–2016, with the highest mean annual costs per person incurred by those with a history of myeloma , brain cancer , liver cancer  or leukemia . The most recent AIHW report on health system disease expenditure for Australia (2018–19), reported the highest expenditure from non-melanoma skin cancer (NMSC) (AU$1326m), breast cancer (AU$1313m), and prostate cancer (AU$1200m) . Total hIn comparison, another Australian study by Goldsbury et al. used a different approach and estimated excess healthcare costs attributable to cancer (using a control group), which during 2013 was around $AU6 billion Australia-wide  . Their eBates et al. published total healthcare system costs in Queensland for all cancers during the first 12 months after diagnosis (2011–12) of AU$4.8 billion . These aIn the Canadian setting, De Oliveira et al. appraised the economic burden of cancer on a population level in the state of Ontario of CAD$7.5 billion in 2012 , and higAlthough the costs by age group crudely aligned with the prevalence in the cohort, we found that younger individuals aged 0–14 years incurred the highest patient-level costs, although these only made up a small proportion of overall costs due to the small number of individuals. Healthcare expenditure for patients with vital status ‘deceased’ was the highest for 0–19-year-olds. This is likely to be due in large part to the very different mix of cancers that occur in children and adolescents, compared to older people. Leukemias (with one of the highest per person costs) make up almost a quarter of cancers diagnosed in people under 20 years, while breast, prostate, colorectal cancer and melanoma (with some of the lowest per person costs), make up a relatively higher proportion of cancers in older people. In Australia, there is some evidence of the use of expensive cancer drugs at the end of life, despite limited benefit . This isPatient-level healthcare costs over time were nearly twice as high in the first year after diagnosis, compared to medium to long-term. Our data included around 10% of individuals in the first year after diagnosis, 58% between 2–9 years, and over 30% were long-term survivors of 10–20 years. Most deaths were recorded 2–5 years after diagnosis, which may have been attributable to a large proportion of individuals in the category. Given that costs drastically decrease after the first year, it is plausible that our cohort costs were lower compared to previous reports (as discussed above).Limitations of this work were the use of health service data, which was created for administrative purposes rather than research purposes, and not all private hospitalizations were included in this data.Also, the health service data included all types of costs incurred by any health service contact, rather than cancer-specific costs, and due to financial limitations we were unable to include a control group to calculate excess costs due to cancer. Details of cancer diagnoses were retrieved from the Queensland Cancer Registry data, which does not record basal and squamous cell carcinoma or stages of cancer; hence, these were missing from our analyses. Non-melanoma skin cancer (NMSC) cases were only reported occasionally, and costs data reported here should only be seen as an indication of costs per person, but not as a representation of overall costs for NMSC in this cohort. We only analyzed data for patients with available system cost data between 2013–2016, which means many patients from the initial cancer cohort were excluded; nevertheless, we are confident that the distribution of gender and types of cancer is nearly identical between the initial cohort, including all cases, and the latter cohort, as our cohort comparison has shown (details available on request). Nearly 3% of our selected cohort, with system cost data recorded between 2013–2016, showed a date of death prior to the date of health service contact, indicating deceased people receiving health care services. These seemingly implausible scenarios might be explained by government data (MBS/PBS) using ‘data perturbation’ to ensure non-identifiability of individuals, where the date of birth/death in the data is randomly selected from the actual date +/−3 months. As we were unable to account for this intricacy, we had to exclude this small proportion of people from further analysis. Furthermore, the focus of this work was on direct health services costs, rather than overall economic costs. Hence, not all relevant costs were included, such as opportunity costs, productivity costs or carer costs. Also, we only partially included patient out-of-pocket expenditure, reflected in co-contributions to prescribed pharmaceuticals (not available for over-the-counter drugs) and some Medicare services. Given that our data included population data, it was to be expected that some cases showed unusually high costs, but to reflect the full spectrum of health services costs, no cost outliers were excluded. Costs were reported in 2016 Australian dollars and not adjusted for the Consumer Price Index, due to the current instability of the inflation rate.Strengths of this project comprise the use of highly reliable, routinely collected administrative healthcare data from a variety of sources, the inclusion of population data without any limitations on age or type of cancer, starting with the date of cancer diagnosis, and continuous records with long-term outcomes of up to two decades. Given that our sample is very large and population-based, our results are likely to be generalizable to the rest of the Australian population. Furthermore, we applied a bottom-up costing approach to estimate healthcare costs by summarizing individual costs, which is seen as the gold standard in healthcare costing, and to our best knowledge we were one of the first Australian studies to use population data to estimate health service costs of cancer survivors.Future research on long-term cancer survivorship could explore health service costs in other settings on a population level, including developing countries and jurisdictions with different healthcare systems, as well as broader economic costs on a societal level.We captured the whole journey of health service contact and were able to estimate related costs of cancer patients diagnosed and treated in Queensland over a period of 20 years. Our research outcomes form a body of evidence to show potential lifetime cost savings by cancer prevention and will provide a rich data source for future economic evaluations. We also inform policy makers in Queensland/Australia and hence facilitate forthcoming planning on the utilization of healthcare resources, according to the burden of disease, which will potentially lead to more investments in cancer prevention and/or survivorship care.


Begomovirus (Geminiviridae) transmitted by the whitefly Bemisia tabaci cryptic species group. In India and other parts of Asia, the Indian cassava mosaic virus-Kerala (ICMV-Ker) is an emergent begomovirus of cassava causing damage that results in reduced yield loss and tuber quality. Double-stranded RNA-mediated interference (RNAi) is an evolutionary conserved mechanism in eukaryotes and highly effective, innate defense system to inhibit plant viral replication and/or translation. The objective of this study was to identify and characterize cassava genome-encoded microRNAs (mes-miRNA) that are predicted to target ICMV-Ker ssDNA-encoded mRNAs, based on four in silico algorithms: miRanda, RNA22, Tapirhybrid, and psRNA. The goal is to deploy the predicted miRNAs to trigger RNAi and develop cassava plants with resistance to ICMV-Ker. Experimentally validated mature cassava miRNA sequences (n = 175) were downloaded from the miRBase biological database and aligned with the ICMV-Ker genome. The miRNAs were evaluated for base-pairing with the cassava miRNA seed regions and to complementary binding sites within target viral mRNAs. Among the 175 locus-derived mes-miRNAs evaluated, one cassava miRNA homolog, mes-miR1446a, was identified to have a predicted miRNA target binding site, at position 2053 of the ICMV-Ker genome. To predict whether the cassava miRNA might bind predicted ICMV-Ker mRNA target(s) that could disrupt viral infection of cassava plants, a cassava locus-derived miRNA–mRNA regulatory network was constructed using Circos software. The in silico-predicted cassava locus-derived mes-miRNA-mRNA network corroborated interactions between cassava mature miRNAs and the ICMV-Ker genome that warrant in vivo analysis, which could lead to the development of ICMV-Ker resistant cassava plants.Cassava mosaic disease (CMD) is caused by several divergent species belonging to the genus  Manihot esculenta Crantz is a tropical food crop of societal and agricultural importance in Africa, the tropical Americas, and Asia [n = 36) [Begomovirus (Geminiviridae) that are endemic to Africa and Asia [Bemisia tabaci (Gennadius) (Aleyrodidae: Hemiptera) cryptic species complex [Cassava and Asia . Cassava[n = 36)  is heter[n = 36) . Cassavaand Asia ,6. Begom complex ,9,10,11. complex ,12,13. E complex . The Ind complex ,16,17. T complex ,19.MIR genes, and govern gene regulation and cell growth, regulate host–virus interactions, and control gene expression at the post-transcriptional level [MicroRNAs (miRNAs) represent a class of endogenous, small, non-coding single-stranded (ss) RNA regulatory molecule of 20–24 nucleotides (nt). The long single-standard primary miRNAs (pri-miRNAs) are produced by nuclear-encoded al level . The bioal level ,23,24,25al level ,27,28.Artificial microRNA (amiRNA) technology is one of the most effective antiviral defense strategies to protect crop plants against infection by plant viruses. RNAi-mediated gene silencing through amiRNA has been used to develop plants with resistance to diverse plant viral species, among which are cucumber green mottle mosaic virus ,30, riceThe identification, analysis, and experimental validation of mature miRNAs in cassava is an important initial step in evaluating host–virus interactions, which are aided by the use of regulatory, network analysis of the host plant, in the context of environmental stresses, including virus infection ,40,41,42In this study, an integrative computational approach for combatting ICMV-Ker infection was used to predict the presence of target-binding sites of cassava locus-derived mes-miRNAs in the ICMV-Ker genome. The goal is to identify homologous amiRNAs that for expression in transgenic cassava plants to confer resistance to ICMV-Ker. The predicted cassava locus-derived mes-miRNAs were further analyzed for the predicted involvement in cassava host plant–ICMV-Ker begomoviral interactions to identify virus-specific targets of interest. The goal is to express the most promising in silico cassava locus-derived mes-miRNAs in transgenic cassava plants to achieve amiRNA-mediated resistance to the recently emergent, bipartite ICMV-Ker begomovirus in cassava crops in Southeast Asia.Manihot esculenta-microRNA) (mes-miRNA156-mes-miR11892) have been identified (http://mirbase.org/ accessed on 26 May 2019) biological database. The miRBase is the central primary public online repository for miRNAs with a user-friendly web interface to access published miRNA sequences and annotations. The full-length genome (2743 bases) of ICMV-Ker (IN-Ker2-021) (Accession number AJ575819) was downloaded from the National Center for Biotechnology Information NCBI GenBank database (http://ncbi.nlm.nih.gov) (accessed on 26 May 2019) [In a previous study, 175 mature cassava microRNAs (ay 2019) .The in silico prediction of miRNA-mRNA target sites is an initial step towards discovering gene regulatory networks controlled by miRNA. Several computational algorithms are available for predicting potential miRNA target sites in viral mRNA conducive to effective silencing. Each computational algorithmic tool set specific criteria for miRNA prediction. An integrative bioinformatic approach involves the combined use of publicly available in silico algorithms. Among the most widely used are miRanda, RNA22, Tapirhybrid, and psRNATarget. The latter four algorithms were used to screen the ICMV-Ker genome and predict the ‘most effective’ miRNA target sites of the cassava miRNAs . The cashttp://www.microrna.org/) (accessed on 9 June 2019). Target prediction was carried out using the default parameters . The MFE statistical parameter is used to evaluate miRNA-target pair strength.The miRanda is a seed-based scanning computational algorithm, and has been used as a standard for miRNA-mRNA target prediction, following its releases in 2003 . It conshttp://cm.jefferson.edu/rna22v1.0/ (accessed on 20 July 2019) [The RNA22 algorithm is a diverse, web-based algorithm that uses a pattern-recognition based approach, available online ly 2019) . It predly 2019) . Defaulthttp://bioinformatics.psb.ugent.be/webtools/tapir (accessed on 19 July 2020). The standard default settings—score <9 and MFE ratio <0.2—were used for this study.The Tapirhybrid algorithm is a web-based, rapid, and precise plant miRNA target prediction algorithm  used to http://plantgrn.noble.org/psRNATarget) (accessed on 26 May 2019). The miRNA–mRNA target binding sites were predicted using the default criteria: an expectation cut-off value of 7.0 and the mode of inhibition set to ‘cleavage’  (accessed on 6 September 2022), and analyzed with the user-defined default settings [RNAfold is a recently available web-based algorithm that predicts secondary structures from a single-stranded (ss) miRNA precursor. Precursor sequences of cassava consensus miRNAs in FASTA format were preloaded in the RNAfold web server (available online: settings .http://rna.tbi.univie.ac.at/cgi-bin/RNAWebSuite/RNAcofold.cgi) (accessed on 20 November 2022) [RNAcofold is a newly developed algorithm for estimating co-folding free energy (ΔG) of RNA duplex sequences, based on minimum free energy and base-pairing patterns of miRNA-mRNA target duplex molecules. It is specifically implemented for evaluating the mRNA and miRNA duplex interactions. FASTA sequences of the duplex pair from miRanda analysis were preloaded in the RNAcofold web server (available online: er 2022) .All of the computational biological data sets resulting from the miRNA target predictions were summarized as graphical representations using R-language  .The in silico predictions of cassava locus-derived mes-miRNAs with the potential to target viral ORFs encoded by the ICMV-Ker genome from among the 175 cassava locus-derived mature mes-miRNAs in the ICMV-Ker single-stranded (ss) DNA genome revealed mes-miRNAs derived from MIR genes at specific cassava genomic loci. The predicted ICMV-Ker genome sequences targeted by cassava locus-derived mes-miRNAs based on the miRanda algorithm predicted 21 miRNA-mRNA target pairs, RNA22: 22 cassava miRNAs and 22 loci. The Tapirhybrid algorithm identified 17 cassava genome locus-derived mes-miRNA-target pairs. In contrast, the psRNATarget algorithm predicted cleavable targets for 29 cassava locus-derived mes-miRNAs targeting at 33 locus positions in the ICMV-Ker genome  File S2File S2.The begomoviral AV1 ORF (295-1065) (770 bases) encodes the coat protein (CP), which is essential for encapsidation of the viral ssDNA genome into virions transmitted by the whitefly vector, and for cell-to-cell movement ,59,60. CThe miRanda algorithm predicted binding of one miRNA: mes-miR2950 at nucleotide position 639 . FurtherThe AC1 ORF (1552-2610 nt) complementary strand encodes a replication-associated protein, whereas the AC3 ORF 1062-1466 nt) encodes a replication enhancer protein. These viral proteins are essential for functions, including viral DNA replication, transcription, transmission, cell-to-cell movement, suppression, and gene regulation -1466 nt ,64,65,66The Tapirhybrid algorithm identified ten predicted miRNAs: mes-miR172  from the common locus 1986, mes-miR399e at locus 2561, mes-miR477  from a common locus 1592, mes-miR477h from locus 1796, mes-miR482d from locus 2073, and mes-miR1446a from locus 2053. Additionally, mes-miR530  targeted the overlapping segments of AC1 and AC4 from the common locus 2425 .The psRNATarget algorithm analysis predicted that the ICMV-Ker ACI ORF was targeted by fifteen miRNAs: mes-miR159  from the common locus 1719, mes-miR395  from the common nucleotide locus position 1789, mes-miR399b from locus 2561, mes-miR827 from locus 2486, mes-miR1446a from locus 2053, and mes-miR2275 from locus 1593 . FurtherThe DNA-A genome of begomoviruses possesses a LIR that functions as a bidirectional promoter, essential for replication and expression of the viral genes, AC1 (Rep) and AV1 (CP) ,68. The Among the predicted cassava locus-derived candidate mes-miRNAs, seventeen miRNAs , mes-miR160e, mes-miR395 , mes-miR399e, mes-miR403 , mes-miR477 , mes-miR530a, and mes-miR535c) were predicted by at least two of the algorithms. Of seventeen consensus cassava locus-derived mes-miRNAs, one miRNA (mes-miR2950 at locus 639) was detected by three algorithms . The mes-miR1446a was predicted by all four algorithms, making it the only unique cassava miRNA identified in this study  Table 2 Tables .Of the 175 targeting mature cassava miRNAs, 15 cassava miRNAs  (locus 2261), mes-miR160e (locus 574), mes-miR395  (locus 1789), mes-miR399e (locus 2561), mes-miR403  (locus 1446), mes-miR477  (locus 1592), and mes-miR530a (locus 2425) were identified by consensus between two algorithms . Of the In this study, based on all four algorithms, mes-miR1466a was the top predicted ‘effective’ cassava locus-derived candidate mes-miRNA, and harbored potential target binding sites at the consensus nucleotide position 2053 . The miRA comprehensive, global purview of the predicted host–virus interactions was visualized by constructing a Circos plot (“Circos” software) that integrated the biological data, comprising cassava locus-derived mes-miRNAs and their predicted ICMV-Ker genomic target genes (ORFs). For cassava miRNAs with predicted cassava host plant-viral gene-target interactions, the plot shows the cassava miRNAs and their ICMV-Ker targets by psRNATarget analysis  File S3File S3.Computational predictions of the consensus cassava locus-derived mes-miRNAs (mes-miR1446a and mes-miR2950) were corroborated by secondary structure predictions. The pre-miRNA sequences were manually curated. Characteristic features of precursor miRNAs were evaluated for stability of the respective secondary structure .G) of duplexes  is highly conserved protein among geminiviruses. The AC1 gene is essential for viral replication and transcription of ICMV-Ker . In thisThe cassava-encoded miRNA–ICMV-Ker-mRNA duplex exhibited a low MFE value, which serves as one measure of controlling for false positive results. When combined with results of the ‘four algorithm’ approach to filter false positive targets, the approach was expected to result in a highly sensitive and specific in silico strategy for predicting ‘true’ interactors ,90,91,92While interactions between cassava genome locus-derived mes-miRNAs and ICMV-Ker have been established, the ability of cassava mes-miRNAs to control ICMV-Ker is yet to be fully understood. Studies on the interactions between cassava locus-derived mes-miRNAs and ICMV-Ker are vital; they reflect initial steps in the development of miRNA-based anti-plant viral therapies. Future work will focus on the validation of this promising cassava locus-derived mes-miRNA to develop ICMV-Ker-resistance in cassava plants, including evaluating the role of the predicted consensus cassava locus-derived miRNAs in ICMV-Ker replication. The amiRNA construct exhibited high specificity for base-pairing with the target gene, which would be expected to minimize detrimental off-target effects and contribute to stable and reliable protection in subsequent future generations of progeny plants . FurtherThe ICMV-Ker, which infects cassava crops in Southeast Asia, is an emergent begomoviral pathogen associated with an ongoing CMD epidemic that diminishes yield and vigor in all cassava cultivars presently grown in the region. Here, in silico tools and approaches were implemented for predicting effective target binding sites of mature candidate cassava locus-derived mes-miRNAs in the ICMV-Ker genome. Among the 175 cassava miRNAs investigated, predicted hybridization sites were identified within the ICMV-Ker genome. Among them, mes-miR1446a was identified as a highly promising, naturally occurring biomolecule with potential to modulate virus infection and reduce damage to the plant host. Present and future work is focused on the development of ICMV-Ker-resistant cassava plants to abate the effects of CMD. These results demonstrate the utility of this combined in silico-screen-molecular approach to design amiRNA therapies to manage ICMV-Ker in cassava crops and other emergent begomoviral pathogens that threaten food and fiber crops worldwide.


Multiple sclerosis (MS) is a chronic, inflammatory disease of the central nervous system, commonly diagnosed during young adulthood. The proportion of direct and indirect costs of MS vary across settings. The International Multiple Sclerosis Study, involving 1152 patients with MS from 19 countries, reported the average annual costs per patient to be €41,212, with direct medical costs of €21,093, direct non-medical costs of €2110, and €16,318 marked as indirect costs. However, there are no precise data on the economic burden of MS in Slovakia. Therefore, the main objective of this study is to assess the economic impact of MS in Slovakia by identifying and measuring the direct medical costs and indirect costs of this disease.We conducted a retrospective prevalence-based cost-of-illness analysis for MS in Slovakia sourced from the third-party payer and societal perspective. Patient co-payments and out-of-pocket expenses were not included in our study. We analysed all available costs and healthcare resources utilised in a 6-years period, from 2015 to 2020. For each year, all costs (in euro) were specified as total and the average annual cost per patient.The estimated total economic burden of MS in Slovakia in 2020 was €57,347,523, with direct medical costs estimated to be €53,348,337 and indirect costs standing at €3,999,186. The total annual cost per patient in 2020 was €6682. Over the 6 years, the total diagnostic and treatment cost of patients with MS was estimated to be €283,974,236. With an average year-by-year increase of 5%, the total direct costs of MS had significantly grown during the examined 6 years. The total cost due to the MS-associated loss of productivity in these 6 years was €16,633,798. The average year-by-year increase of indirect costs of MS was 20%.Our study revealed the substantial health and economic burden of MS, with the average annual cost per patient to be approximately €6,682 in 2020. We provide the first extensive assessment of the burden of MS on Slovakian patients, the healthcare system, and society. It indicates the need for a detailed analysis of the employment of patients with MS to assess disability and work performance and the development of allied health policies.The online version contains supplementary material available at 10.1186/s12913-022-08883-6. Multiple sclerosis (MS) is a chronic, inflammatory disease of the central nervous system, commonly diagnosed during young adulthood . Signs aGlobally, females are twice as likely to develop the disease than males , 7. The Since MS is typically diagnosed between 20 and 50 years of age, it affects education, career, and family life, with negative consequences on patients and their caregivers’ working activity , 10. A sThe proportion of direct and indirect costs of MS vary across settings. Encompassing 1152 MS patients from 19 countries, the International Multiple Sclerosis Study stated the average annual costs for each patient to be €41,212, with direct medical costs standing at €21,093, direct non-medical costs of €2110, and indirect costs to be €16,318 . IndirecSo far, limited information on the prevalence, incidence, and cost of MS in Slovakia has been published. Some estimations were made in the Atlas of Multiple Sclerosis in 2008 and by GBD 2016 Multiple Sclerosis Collaborators , 33. We Evidence of MS costs estimated from payers and societal perspectives are available for most European countries, but no current data for Slovakia has been published so far. Therefore, we aim to assess the economic impact of MS in Slovakia by identifying and measuring the direct medical costs and indirect costs of this disease.This retrospective prevalence-based cost-of-illness analysis for MS in Slovakia was conducted based on the third-party payer and societal perspective. From the third-party payer perspective, the analysis included all direct medical costs paid by the insurance companies. The analysis from the societal perspective also considered the costs of decreased or lost productivity of the patient with MS. Patient co-payments and out-of-pocket expenses were not included in the study. We analysed all costs and healthcare resources used in a 6-years period, from 2015 to 2020. All costs were expressed as total and the average annual cost per patient in a respective year. All estimated costs were expressed in euro. No discounting of costs was applied.http://www.nczisk.sk/Statisticke_vystupy/Tematicke_statisticke_vystupy/Neurologia/Pages/default.aspx). Information on the age-sex structure of the population, and the subdiagnoses were obtained from Institute for Health Analysis .MS was defined according to the WHO ICD-10 classification as ICD code G35. Patients were divided based on disease severity. Data on the prevalence of MS in Slovakia over 2015–2020 were obtained from the National Health Information Center (www.vszp.sk).General Health Insurance Company .Privately-owned Dovera health insurance company (www.union.sk).Union health insurance company .Institute of Health Analysis  for the selected period.While calculating the indirect costs generated by patients with MS in Slovakia between 2015 and 2020, we analysed the aggregated data on days spent on paid sick leave from work due to MS and the number of patients with disability due to MS provided by the Social Insurance Agency  inpatient care costs , (ii) pharmacotherapy, (iii) diagnostic and medical procedures. Costs of medical treatment included all drugs reimbursed for patients with MS in Slovakia separately or together with hospitalization. Costs of outpatient diagnostic and medical procedures included: initial and differential MRI examinations, cerebrospinal fluid examination, visual evoked potentials, PET scans, complete biochemical blood or cerebrospinal fluid tests, differential diagnostic tests, and rehabilitation costs. Our analysis has not assessed direct non-medical costs, e.g., formal care in homes and care homes or informal care, due to the lack of such data in Slovakia. We excluded premature mortality costs due to relatively small and difficult to assess impact of MS on mortality.Indirect costs included in the analysis were: costs of short-term absence from work, long-term absence from work, and early retirement. Productivity loss due to absenteeism was estimated as the number of days spent on paid sick leave from work due to G35.XX diagnosis multiplied by the national average salary in the years 2015–2020  higher than in 2015. The average year-by-year increase in the direct cost of MS was 5%. The major components of medical expenditures were medicines that remined about 91% of direct costs each year. At the same time, costs associated with hospitalisation, diagnostic and medical procedures constituted about 9% of medical costs. The total costs of loss of productivity loss due to MS over the 6 years were €16,633,798. These costs increased significantly during the explored 6 years. In 2020, the cost of productivity loss due to MS was €3,999,186, resulting in €466 annual cost per patient, which was 143%  higher than in 2015. The average year-by-year increase of indirect costs of MS was 20%. The key driver of costs of MS were direct costs. In 2020, the estimated costs of medical care remained at about 93% of the total costs of MS. The proportion of direct costs in total costs was constant during the analysed years and accounted for about 95%.This study is the first prevalence-based analysis estimating the economic burden of MS in Slovakia. Using the societal perspectives, we have estimated that the annual costs of MS in 2020 in Slovakia were approximately €57.3 million. Costs of medical treatment and productivity loss due to absenteeism and disability represented 93% (€53.3 million) and 7% (€4.0 million) of the total costs, respectively. The main cost driver was the pharmacotherapy of patients with MS, corresponding to €48.5 million .As it has been mentioned before there is inadequate amount of published data on MS prevalence in Slovakia. Moreover, published estimates are not up to date and are inconsistent. Estimations provided in the Atlas of Multiple Sclerosis, a world compendium of data on MS epidemiology, showed a yearly incidence of 7.5 per 100 000 people and 8,500 people living with MS in 2008 in Slovakia . The GBDPrevious population-based studies focusing on different geographic regions indicated that females are twice as likely to have MS as males , 7. ThisIn the analyses of different European regions, Paz-Zulueta and colleagues estimated total annual costs per patient with MS from a societal perspective in 2015 to be €44,589 in Northern Europe , €47,619 in Western Europe , €36,978 in Southern Europe  and €15,205 in Eastern Europe , with average total annual cost per patient in Europe being €40,303 . Latest The interpretation of MS cost differences between Slovakia and other European regions is challenging due to the lack of comprehensive, updated systemic data on Slovakian patients with MS. Nonetheless, one of the probable reasons for these differences could be the underestimation of the actual number of Slovakian patients with MS due to the lack of a national registry and published epidemiological surveys. The current analysis is based only on the published data from the National Health Information Centre. However, this aggregated data may not include patients during the diagnostic process or those misdiagnosed. Moreover, this analysis includes only formal state-granted disability and does not cover any informal expenses, costs of sequelae, and out-of-pocket payments. Thus, the underestimation relates also to disability costs and medical costs.Most of the published data on MS costs demonstrate a significant impact of direct medical costs on total costs generated by patients with MS. This is in line with other studies for neurological disorders where medical costs represent a significant part of the total costs of illness –40. In oMS causes immense indirect costs due to productivity loss. Comparing to other MS cost studies, we observed lower productivity loss costs resulting from sick leave and disability pension, ultimately contributing to lessening the total MS cost. These costs were nearly €4.0 million in 2020 — 2.5-fold higher than in 2015. As described by Paz-Zulueta and colleagues, indirect costs comprised about 23% (range 9–36%) of total MS costs . Recent In conclusion, our study revealed the substantial health and economic burden on Slovakian patients with MS. The increase in costs is evident from €43.2 million in 2015 to €57.3 million in 2020, with the average annual cost per patient estimated to be €6,682 in 2020. Pharmacotherapy was the main cost driver accounting for about 85% of the total cost (€48.5 million in 2020). This study provides the first extensive assessment of the burden of MS on Slovakian patients, the healthcare system, and society. It indicates the need for a detailed analysis of the employment of MS patients aiming at assessment of disability and work performance and the development of related health policies.Additional file 1. Average salary in industry in Slovakia (2015-2019 year).Additional file 2. Disability lump sum rates in Slovakia (2015-2019 year).


This study aimed to estimate respiratory disease hospitalization costs attributable to ambient temperatures and to estimate the future hospitalization costs in Australia. The associations between daily hospitalization costs for respiratory diseases and temperatures in Sydney and Perth over the study period of 2010–2016 were analyzed using distributed non-linear lag models. Future hospitalization costs were estimated based on three predicted climate change scenarios-RCP2.6, RCP4.5 and RCP8.5. The estimated respiratory disease hospitalization costs attributable to ambient temperatures increased from 493.2 million Australian dollars (AUD) in the 2010s to more than AUD 700 million in 2050s in Sydney and from AUD 98.0 million to about AUD 150 million in Perth. The current cold attributable fraction in Sydney (23.7%) and Perth (11.2%) is estimated to decline by the middle of this century to (18.1–20.1%) and (5.1–6.6%), respectively, while the heat-attributable fraction for respiratory disease is expected to gradually increase from 2.6% up to 5.5% in Perth. Limitations of this study should be noted, such as lacking information on individual-level exposures, local air pollution levels, and other behavioral risks, which is common in such ecological studies. Nonetheless, this study found both cold and hot temperatures increased the overall hospitalization costs for respiratory diseases, although the attributable fractions varied. The largest contributor was cold temperatures. While respiratory disease hospitalization costs will increase in the future, climate change may result in a decrease in the cold attributable fraction and an increase in the heat attributable fraction, depending on the location. Global temperature has risen markedly by 1 °C since industrialization began around 1750, largely due to human activities and the increases in greenhouse gas emissions, e.g., from energy production, transportation, agriculture and industries . With inStudies found that both hot and cold temperature exposure can increase the risk of respiratory diseases ,25,26. AAustralia has had a universal health care scheme (Medicare) since 1984, which covers all costs of public hospital services. The Australian state and territory governments cover about 68% of the overall cost of health care in Australia; the other 32% of health care costs are shared by individuals (17%), private health insurers (9%) and non-government organizations (6%) . In AustThe present study aimed to examine respiratory disease hospitalization costs attributable to non-optimum ambient temperature, quantify the attributable fractions from hot and cold temperatures and estimate the associated future hospitalization costs in two Australian cities—Sydney and Perth in the context of climate change. The results will help local and regional government, health authorities and communities to have a better understanding of climate change attributable hospitalization costs; plan, evaluate and optimize current climate change adaptation strategies; and better direct medical resources to respond to respiratory diseases in the context of climate change.This study was conducted in two cities in Australia—Sydney and Perth. Sydney is located on Australia’s east coast and has a humid subtropical climate with warm summers and cool winters  . It is tRespiratory disease hospitalization costs: Daily respiratory disease (ICD-10-AM: J00-J99) hospitalization costs from public hospitals over the study period from July 2010 to June 2016 in Sydney and Perth were obtained from the Australian National Independent Hospital Pricing Authority (IHPA). The daily respiratory disease hospitalization costs were aggregated by the date of admission for statistical analysis. The patient hospitalization costs included all costs, including prescription medications, treatment and medical examination for respiratory health conditions incurred during the hospitalization period.Meteorological data: Daily minimum (Tmin) and maximum (Tmax) temperatures were obtained from the Australian Bureau of Meteorology. Daily mean (Tmean) temperature was calculated by the average of Tmin and Tmax. Tmin, Tmean and Tmax are the averages calculated from 17 weather observation stations in Sydney and 13 weather observation stations in Perth . Daily mIn addition, population data for the baseline period 2010s and projection periods 2030s and 2050s were obtained from the Australian Bureau of Statistics (ABS) for the two cities . For theThe methods for statistical analyses were described in our previous studies on assessing the effects of temperature on healthcare costs in Adelaide, Perth and Sydney ,21,22. Tdf) per year for time [bs] in Sydney and 4df per year for time [bs] in Perth were included in our models [tY is respiratory disease hospitalization costs on day t; α is the intercept; cb(Tempt) is the cross-basis natural cubic spline function for daily mean temperature with both response and lag dimension applied from the DLNM; bs is the B-spline with degrees of freedom per year multiplied by the 6 year study period, adjusted for seasonality and long-term trend in Sydney and Perth [time is in days; dow is the day of the week on day t; and pubhol is a binary variable representing public holidays on day t.In order to assess the shape of the exposure–lag–response relationship, a DLNM model with gamma distribution was fitted simultaneously to estimate the possible non-linear relationship and lagged effects of temperature on hospitalization costs ,38,39. Tr models . The locnd Perth ; time isdf for time from 4df to 8df per year, and the maximum lag days of 14 days to 7–21 days, to compare and best capture the effects of temperature on hospitalization costs. Residual analysis and autocorrelation tests were conducted to evaluate the goodness of model fit and autocorrelation. In order to ensure the hospitalization costs were comparable across the different years, consumer price index (CPI) data were obtained from the Australian Bureau of Statistics [The optimum temperature for respiratory disease hospitalization costs was identified via an overall cumulative exposure–response curve in the first stage, and the outcome below or above the optimum temperature was assigned as the temperature effect due to exposures ,39. The atistics , and theAC) and attributable fractions (AF). The AC and AF were calculated using the method of Gasparrini and Leone [AC and AF are defined as:x is the daily mean temperature exposure on day t; tC is the daily hospitalization costs on day t. t − l after l days have elapsed. The effects of projected daily mean temperatures above the current observed range were estimated using Monte Carlo simulation (n = 1000) [After the baseline exposure–response relationships between daily temperatures and hospitalization costs were obtained from the first stage analysis, future respiratory disease hospitalization costs were estimated based on the baseline model and projected temperature changes in the 2030s and 2050s. The future temperature effects on hospitalization costs were estimated under three temperature scenarios  for the periods 2030s and 2050s. Hospitalization costs and fractions attributable to temperature with reference to the optimum temperature of minimum relative risk were calculated to show temperature attributable costs (nd Leone . The AC  = 1000) ,45. Esti = 1000) .All statistical analyses were performed using R software  with the packages “dlnm”, “tsModel” and function “attrdl” ,46.For the estimation of future hospitalization costs for respiratory diseases under three RCP scenarios  over the periods of 2030s and 2050s , the estdf of the B-spline for the calendar year from 4 to 8 per year  for respiratory diseases than Perth (AUD 709.3 million), which cannot be entirely explained by the population size of the two cities . This could be due to Sydney being a larger multicultural city with a higher number of communities of new Australians, who might not be able to develop their adaptation behaviors to the Australian climate, and higher numbers of people without or not willing to use air conditioners due to electricity costs ,48,49, wWe found there were overall non-linear relationships between ambient temperature exposures and respiratory disease hospitalization costs in Sydney and Perth. Both cold and hot temperatures can increase hospitalization costs, with a higher risk at lower temperatures in both cities. This is consistent with other studies exploring the impact of temperature on morbidity due to respiratory diseases, suggesting more cold-related respiratory diseases than heat-related respiratory diseases ,19,27,51We found an acute heat effect on hospitalization costs on the day of heat exposure in Perth but not in Sydney. The observed differences between the two cities could be due to the different climates and population adaptation. The respiratory disease optimum temperature identified in Sydney, which has a higher relative humidity of 60–70% across the year , was 27.The study estimated AUD 493.2 million and AUD 98.0 million in hospitalization costs for respiratory diseases associated with ambient temperature exposures over the baseline study period in Sydney and Perth, respectively. The cold attributable fraction was as high as 23.7% in Sydney, compared with 11.2% in Perth. The results for both cities indicated that most of the hospitalization costs for respiratory diseases were attributed to cold temperatures. The possible explanatory mechanism could be that cold temperatures are more likely to cause pathophysiological responses that affect the immune system and increase the susceptibility to respiratory diseases, e.g., pneumonia caused by viruses and bacteria kills more people than other respiratory diseases ,58,59,60The estimation of the future hospitalization costs attributable to temperature for respiratory diseases showed a substantial increase from about AUD 500 million and AUD 100 million at the baseline period in Sydney and Perth, respectively, to approximately AUD 700 million and AUD 150 million by midcentury, despite its declining attributable fractions to non-optimum temperature. The possible explanation could be the future burgeoning population by the middle of this century, with up to 9.1 million population in Sydney and 3.7 million population in Perth . In addiThis study is the first to examine the temperature effects on hospitalization costs for respiratory diseases over a long time period in Australia. We employed a DLNM model to control for confounding effects, including seasonality, long-term trends, day-of-week and public holidays. We estimated both cold and hot temperature attributable hospitalization costs for respiratory diseases in cities with different climates and demographics. We also projected such hospitalization costs in the future in the context of climate change. In addition, the comparison between temperature effects in Sydney and Perth may suggest an acclimatization effect. The observed heterogeneity of the temperature effect on hospitalization costs for respiratory diseases may also indicate a need to conduct location-specific and disease-specific studies to explore the temperature effect on population health outcomes in the future.Several limitations of this study should be acknowledged. First, we assumed the baseline exposure–response relationships for the estimation of future respiratory disease hospitalization costs would not change under different climate change scenarios in the future. However, human behavioral adaptations to changing climate in the long term may play a certain role in mitigating the effect of temperature on respiratory diseases. This may change the impact of temperature changes on respiratory disease hospitalization costs. Second, this study did not consider individual-level exposures, personal indoor and outdoor daily activity patterns, local air pollution levels and other behavioral risks because of the data availability issue, which may alter the temperature effect on hospitalization costs for respiratory diseases. Furthermore, technological progress and innovation may impact the emission of greenhouse gases, which could make a difference in such projection. Third, the hospitalization costs for the respiratory disease may reflect only severe and acute episodes, which may underestimate the overall burden, including those who attended the emergency department, and costs due to lost productivity for days off work. Fourth, the hospitalization costs were adjusted based on the consumer price index up to the second quarter of 2016, which may not reflect the future healthcare costs due to economic fluctuations, including inflation, deflation or discounted cash flow. Fifth, age could affect respiratory health risks and hospitalization costs. However, due to restricted data availability, only aggregated daily respiratory disease data were obtained. Lastly, the respiratory disease hospitalization costs were gathered from two cities in Australia, which may limit the generalizability of research findings to other areas and regions. Subsequent studies to explore costs in sub-tropical and inland locations would be warranted. In addition, respiratory disease hospitalization costs due to emerging infectious diseases and pandemics such as COVID-19, avian flu, SARS, and MERS in the future may not be particularly temperature-sensitive but cannot be projected.Cold temperatures are associated with increased hospitalization costs for respiratory diseases with delayed effects in Sydney and Perth and are responsible for most of the temperature-attributable economic burden of respiratory disease hospitalizations. The cold attributable fraction is likely to decline due to global warming from the current 23.7% to 18% and 11.2% to 5.1% by midcentury in Sydney and Perth, respectively. Additionally, the hospitalization costs for respiratory diseases attributed to heat are estimated to gradually increase in the future. The estimated overall hospitalization costs attributable to temperature will increase from currently about AUD 500 million to AUD 700 million in Sydney and AUD 100 million to AUD 150 million in Perth by the middle of this century. Interpretation of the results should be made with caution as other factors such as individual-level exposures, local air pollution levels, and other behavioral risks were not considered. Nonetheless, the findings highlight the significant impact of cold temperatures on the economic burden of respiratory diseases currently in Australia and also may suggest that overall temperature attributable respiratory disease hospitalization costs are likely to increase substantially in Australia in the context of climate change.


Origanum vulgare, Thymus vulgaris, and Rosmarinus officinalis on Cucurbita pepo plants infected by zucchini yellow mosaic virus or tomato leaf curl New Delhi virus. Treatments were applied either concurrently or after virus inoculation to ascertain an inhibition or curative activity, respectively. Symptoms were observed and samplings were performed weekly. Virus titer and expression levels of phenylalanine ammonia lyase gene (PAL) were measured on treated and untreated infected plants by real-time PCR. PAL gene plays an important role in plant defense response as it is involved in tolerance/resistance to phytopathogens. Results indicated that treatments were effective against tomato leaf curl New Delhi virus whether applied simultaneously with the inoculation or after. A major inhibition was observed with O. vulgare essential oil and hydrosol, resulting in 10–4-fold decrease of virus titer 3 weeks after treatment. Curative activity gave maximum results with all three essential oils and T. vulgaris and R. officinalis hydrosols, recording from 10–2-fold decrease to virus not detected 4 weeks after treatment. An induction of PAL gene expression was recorded at 12 d.p.i. and then was restored to the levels of untreated control. This allows to hypothesize an early plant defense response to virus infection, possibly boosted by treatments. Plant extracts’ composition was characterized by gas chromatography-mass spectrometry. Phenols were largely main components of O. vulgare and T. vulgaris extracts , while extracts from R. officinalis were based on monoterpene hydrocarbons  and oxygenated monoterpenes (hydrosol).In the last decades, the interest in biological activity of natural compounds has been growing. In plant protection, essential oils have been reported to exhibit antiviral, antimycotic, and antiparasitic activities, and are regarded as promising for the formulation of safe antimicrobial agents. Attention has also been focused on hydrosols, the by-products of hydro-distillation of essential oils. Their production is easy, fast, and cheap, and they seem to arise less concern for human health than essential oils. Plant viruses represent a major concern for agricultural crops since no treatment compound is available for virus control. This work was aimed at evaluating the antiphytoviral effectiveness of treatments with three essential oils and corresponding hydrosols extracted from  Apiaceae, Asteraceae, Lamiaceae, Myrataceae) particularly rich in these secondary metabolites  are complex mixtures of low molecular weight and volatile compounds that are formed and stored in aromatic plant families  which are all very expensive, time-consuming, and do not suite all plant/virus pathosystems . Further experiments involving C. quinoa-CMV pathosystem  in C. pepo was investigated both in vitro and in vivo , Thymus vulgaris (TV), and Rosmarinum officinalis (RO) could be effective for the control of ZYMV and tomato leaf curl New Delhi virus (ToLCNDV) in Cucurbita pepo L. plants in vivo. ZYMV, a member of the genus Potyvirus, family Potyviridae, is a ssRNA (+) virus efficiently transmitted by aphids in a non-persistent manner. It affects all members of the Cucurbitaceae family, including pumpkin, squash, vegetable marrow, zucchini, melon, watermelon, cucumber, and gherkin (Geminiviridae) that was identified for the first time in 1995 in Asia (Bemisia tabaci (Gennadius) in a persistent manner.Based on these findings, we hypothesized that EOs and HSs could have a potential antiphytoviral activity  gherkin . It indu gherkin . ToLCNDV in Asia , from wh in Asia , becomin in Asia . The virC. pepo. Several studies have established that plants were able to set up a defense response upon virus infection by using a wide range of regulation mechanisms PCR.The EOs and HSs antiphytoviral activity, foreseen in our hypothesis, was assessed by comparing the relative virus titer in systemically infected treated and untreated plants in order to ascertain if, and to what extent, treatments were able to decrease virus titer in a systemically infected leaf tissue of chanisms . The phechanisms . Under tEuropean Pharmacopeia. Fresh leaves (2 kg) of aerial parts from each plant species were used for distillation. HSs were separated by decantation, and the EOs were treated twice with diethyl ether  to eliminate the water through a separation funnel. EO/diethyl ether layer were dried with anhydrous sodium sulfate  and the solvent was evaporated. EOs yields were 3.48, 0.26, and 0.43% for OV, TV, and RO, respectively. EOs were stored at −20°C and HSs at 4°C, both in the dark in glass vials.Plants of OV, TV, and RO, which are used to produce EOs and HSs, were cultivated and harvested in Latium, central Italy at the agricultural experimental field Centro Appenninico del Terminillo “Carlo Jucci” in Rieti. EOs and HSs were produced by steam-distillation for 1 h using a Clevenger-type apparatus according to the procedures described in the Mentha suaveolens EO as previously described (Gas chromatography/mass spectrometry (GC-MS) analyses of EOs and HSs were performed using a Thermo Scientific gas chromatograph Focus GC, equipped with a fused silica capillary TG-SQC  and a mass spectrometer 700 ITQ MS as detector. n-Hexane (97.0% grade) and all standard references  were supplied by Sigma Aldrich  with the exception of piperitenone oxide, which was in-house purified from escribed . Prior tescribed . When avescribed .Cucurbita pepo “Tullio” were sown in 12 cm plastic pots containing soil “Completo”  and maintained in a greenhouse  with watering as required. The obtained plants were grown in an insect-proof greenhouse under the same conditions. Experimental plants were selected 3 weeks after sowing, when they had two fully expanded cotyledons. Care was taken to ensure that the experimental plants were as uniform in size as possible.Seeds of C. pepo “Tullio” under the greenhouse conditions above described. Systemically infected young leaves were ground with chilled 0.1 M pH 7.4 phosphate buffer (1:5 w/V) in an extraction bag  to prepare virus inocula for the following experiments.ZYMV and ToLCNDV isolates from the Research Centre for Plant Protection and Certification (CREA-DC) collection were propagated in the plant host inoculum was mixed with EO or HS solution in 0.1 M pH 7.4 phosphate buffer , the above trial on ToLCNDV was repeated, extending the period of observation, and after the first collection time point at 5 d.p.i., three more samplings were performed on a weekly basis. Each sampling involved a new leaf , and as above, biological replicates were pooled and downstream analyzed as a single sample.inoculum. In the literature, such activity is often addressed to as “curative” since the treated plants have already started the infection process. In this regard, host plants were inoculated in the same conditions described in section “Treatments Applied at the Same Time With Inoculation of ZYMV or ToLCNDV” , but no treatment was mixed with the inoculum. After 5 h, treatments were applied by smearing the leaf surface with the EO or HS solution described in section “Treatments Applied at the Same Time With Inoculation of ZYMV or ToLCNDV” . Three biological replicates were used per treatment and controls were represented by (i) treatment with ribavirin ; (ii) infected no-treatment ; and (iii) healthy (mock-inoculated with phosphate buffer). When the first new leaf, i.e., the second true leaf, was expanded (5 d.p.i.), plants were sampled as above described. Biological replicates were pooled and downstream analyzed as a single sample. Additional samplings were performed as described in section “Treatments Applied at the Same Time With Inoculation of ToLCNDV (Time Course).”Based on the results obtained from the above-described trials, in which the treatments were applied concurrently with virus inoculation, further experiments were run only on ToLCNDV to evaluate the effect of the tested plant extracts post Total RNA of samples collected from the ZYMV trial was extracted using RNeasy Plant Mini Kit . Total nucleic acid (TNA) of samples collected from the ToLCNDV trials (in treatments together with or after inoculation) was extracted using DNeasy “Mericon” Food Kit . Both kits were used according to the manufacturer’s instructions. Extracts were checked for purity and concentration by NanoDrop™ spectrophotometer .Total RNA samples  were treated with 2 U DNase I  per 1 μg RNA, at room temperature for 15 min. Then, 1 μl of 50 mM EDTA was added, and the mix was incubated at 65°C for 10 min in a CFX96 Touch PCR System  to inactivate DNase and stop the reaction.Total nucleic acid samples  were reverse transcribed to cDNA in order to convert plant transcriptome into DNA substrate suitable for real-time PCR. The reaction was carried out in a final volume of 19 μl using 5X first-strand buffer (Invitrogen), 5 μM random hexamers , 10 μM dNTPs, 100 U M-MLV , and 2 μl TNA. The reaction was incubated for 45 min at 42°C, 3 min at 94°C in a CFX96 Touch PCR System  before 1 μl RNase cocktail mix  was added to remove residual unreacted RNA.® real-time RT-PCR assay using the species-specific primers and probe designed on the coat protein (CP) gene by ® RT-PCR Master Mix and 40X TaqMan® RT Enzyme Mix , 300 nM of each primer, 50 nM probe, and 1 μl DNase-treated RNA.The relative quantification of ZYMV was carried out on total RNA, treated as described above with TaqMan® real-time PCR assay using the species-specific primers and probe designed on the A genome of ToLCNDV by ® RT-PCR Master Mix, 100 nM of each primer and probe, and 1 μl of RNase-treated cDNA.The relative quantification of ToLCNDV was carried out with TaqMan® real-time RT-PCR assay using primers designed by The relative expression of PAL was quantified with SYBR GreenAll real-time (RT-)PCR assays were performed on a CFX96 Touch RT-PCR System . The threshold was automatically set by the instrument.C. pepo was used as housekeeping for both assays using previously published primers and probe  and the results were expressed as mean ± standard error (SE).The virus relative titer and PAL expression level were determined using the method of ΔΔCt . The elond probe . All samPlant extracts (EOs and HSs) from OV, TV, and RO were characterized by GC-MS in order to assess their specific composition, which is known to strongly depend on environmental and plant characteristics.C. pepo plants experimentally inoculated with either ZYMV or ToLCNDV. Using real-time PCR, both the relative virus titer and the relative expression of PAL were measured on treated plants with respect to control plants . Symptoms were also recorded on the inoculated test plants in order to observe possible phenotypical modification and symptom intensity and variability induced by treatments. Preliminarily, symptoms on control plants were evaluated and resulted as expected. In all experiments, the inoculated no-treatment control plants displayed symptoms of the relevant infection. Healthy no-treatment control plants coherently showed no symptoms at any time. They did not display any signal in real-time PCR assays for virus presence and were not included in relevant figures , thymol (1.41%), and piperitenone oxide (1.62%). As expected, due to the polarity of its phenolic moiety, a large amount of carvacrol (91.95%) was also found in the respective HS. Thymol was also present in HS and EO in similar amounts. Thymol was the main constituent of TV EO (68.02%), but in this case, p-cymene and carvacrol were also found in fairly high proportions . As in the case of OV extracts, the main component of TV HS was the same of the corresponding EO (57.35%) and compounds bearing oxygenated polar groups as oxygenated monoterpenes and phenolic compounds were found in similar or higher proportions . The highest number of identified compounds (26) characterized RO EO. Its composition was described in terms of a main constituent α-pinene, accounting for 32.55% composition, followed by 7 compounds at medium concentrations ranging from 4.86 to 9.35% that belong to monoterpene hydrocarbons, oxygenated monoterpenes, and sesquiterpene hydrocarbon classes, and 13 minor components (0.12–3.57%). In the corresponding HS, oxygenated monoterpenes were main components .The relative virus titer was determined by the ΔΔCt method based on the Ct values obtained by real-time PCR amplifications and considering the EF-1α gene as the housekeeping gene and the infected no-treatment group as the control.–ΔΔCt) for the experimental trial vs. ZYMV and ToLCNDV described in section “Treatments Applied at the Same Time With Inoculation of ZYMV or ToLCNDV.” The ZYMV relative virus titer  on ZYMV-infected plants, independently of the treatment type. Conversely, only mild yellow mosaic was observed on treated plants inoculated with ToLCNDV, whereas severe symptoms  were only observed in the infected no-treatment control .Based on these results, further experiments were performed on ToLCNDV, which proved to be more affected by EOs and HSs treatments than ZYMV, both in virus titer decrease and symptom development.C. pepo plants over time was investigated by means of an extended trial involving repeated weekly samplings until 26 d.p.i. as described in section “Treatments Applied at the Same Time With Inoculation of ToLCNDV (Time Course).” –2 to 10–1. At 19 d.p.i., treatments with either OV EO or OV HS were still very effective in reducing virus titer . Virus titer in other treatments  was comparable to that measured at 12 d.p.i. In the final sampling, i.e., 26 d.p.i., an increasing of virus titer ranging from 101 to 102 -fold was recorded in all treatments with respect to no-treatment infected control. EOs generally recorded increases of lower extent than HSs. Ribavirin treatment confirmed not to be a suitable positive control since an increase of virus titer was observed at all sampling times. The evolution of symptoms expression during time is shown in The evolution of ToLCNDV relative titer in leaves of treated –1-fold decrease for OV EO and HS and ca. 10–3-fold decrease for RO HS. At 19 d.p.i. in all treatments except TV HS, a decrease of virus titer was observed. In RO HS, as in the previous sampling, the decrease was most remarkable, with the virus target not detected at all in the samples. In the final sampling at 26 d.p.i., all treatments showed a virus titer lower than the control: fold changes ranged from 10–1 for OV EO and HS and TV EO to 10–2 for RO EO and TV HS. The most prominent effect was again observed in RO HS treatment, in which the virus was not detected. Pictures of the plants of these experiments at 19 and 26 d.p.i are shown in The experimental trial described in section “Treatments Applied After Inoculation of ToLCNDV (Time Course)” was designed to evaluate the effectiveness of treatments when applied after virus inoculation . In this case, at 5 d.p.i., ToLCNDV titer was higher in all treatments with respect to no-treatment infected control, except for TV HS. Here, we observed a viral titer reduced to one-third of the control . In the The relative PAL expression was determined by the ΔΔCt method based on the Ct values obtained by real-time PCR amplifications and considering the EF-1α gene as the housekeeping gene and the infected no-treatment group as the control. In this work, PAL represents the first gene analyzed to investigate the antiviral mode of action performed by EOs or HSs in plant defense response.Phenylalanine ammonia lyase gene relative expression levels were measured at 5 d.p.i. on treated plants compared to the infected no-treatment control for the two experimental trials on ZYMV and ToLCNDV described in section “Treatments Applied at the Same Time With Inoculation of ZYMV or ToLCNDV.” The transcription levels of PAL were significantly upregulated in treated leaves infected with ZYMV . In HS tIn ToLCNDV-infected plants  no signiThe evolution of PAL expression over time in all treatments when applied at the same time of inoculation is shown in Phenylalanine ammonia lyase gene expression levels were also studied during the time when treatments were applied 5 h after inoculation  the number of local lesions in Chenopodium amaranticolor infected by ZYMV . The respective HSs from the same plant species were also included in experiments in view of the recent literature supporting biological activity of HSs as antimicrobials and antivirals  were selected considering the great impact they have on C. pepo crops, causing diseases responsible for significant economic losses. Moreover, the two viruses are considerably different in some features which could be relevant for the liability of treatments, i.e., symptomatology, taxonomy, geographical distribution, genome structure and organization, and transmission mode. Resistant varieties are currently used for the control of ZYMV in C. pepo, but resistance breaking (RB) viral strains are becoming more and more frequent. For ToLCNDV, being an emerging pathogen, no direct control measure is yet available. Hence, for both viruses, alternative control strategies are needed. In this study, we hypothesized that the above mentioned EOs and HSs exert an antiphytoviral activity against ZYMV and ToLCNDV in C. pepo. We aim at demonstrating the effectiveness of such treatments in terms of virus titer and symptoms development. Further, we explore a possible mechanism of action by the quantification of PAL expression levels. These findings provide quantitative data on the effectiveness of EOs and HSs in virus control, improving the knowledge on this important topic. Our data could represent a basis for future scientific research on the mechanism of action of such treatments, and the formulation of a possible remedy which is needed in C. pepo cropping.The use of natural products from plant sources for the treatment of plant viruses is a topic of growing interest and application. Previous studies demonstrated that EOs and HSs are effective in reducing the number of local lesions due to virus infection in experimental hosts for a number of plant/virus pathosystems . Furthertivirals ,b. C. peThe qualitative and quantitative EO compositions reported in this work are overall in fair agreement with literature data, considering the intrinsic variability of these extracts due to a number of factors influencing the biosynthesis and metabolism of EOs components. Exogenous factors  and endogenous features  are all aspects reported to strongly affect EOs composition . In OV EC. amaranticolor as described above. We hypothesize that such huge difference in activity by the same treatments against different plant or virus species could be related to the mechanism of action and nature of either the host or the virus. The poor results on ZYMV were, however, in agreement with previous literature. Particularly, that a number of EOs, including RO EO, did not display any activity against TMV in vitro at concentration of the same order of magnitude of our study  of the two types of extract. Hence, this could result in diverse viability of treatments on plant tissues.inoculum. An effective decrease of virus titer was observed only after the second sampling time, regardless of treatments. Particular attention should be paid to the remarkable effect of RO HS. Particularly, in plants treated with such extract, the virus was not detected by real-time PCR at 19 and 26 d.p.i. Additionally, tested plants did not show any symptom. The results of such time-course observations suggested that the treatments’ effect on symptoms was probably longer lasting than previous studies reported. In literature, the observation was limited to one single time point, to the few hours immediately after treatment, or within a maximum period of 7 days (Virus infection evolution and plant response were also investigated in treatments applied after inoculation. The effects of treatments were generally delayed but longer lasting compared to treatments simultaneously applied with virus f 7 days .Besides the assessment of virus titer, the quantification of PAL expression levels was also accomplished in order to verify whether this gene is involved in the plant defense response stimulated by treatments. PAL is the entry enzyme of the phenylpropanoid pathway, and phenylpropanoid compounds are well known as key elements in plant defense against many microbial pathogens . Hence, inoculum, this quantification showed that TV extracts were effective in increasing accumulation of PAL transcripts, especially at 12 d.p.i. TV EO has recently been reported to trigger such effect. For this elicitor activity, it has been proposed for sustainable plant protection against pathogens (When treatments were simultaneously applied with virus athogens . Our resIn the assessment of curative activity, we recorded the highest response in terms of PAL transcripts in all treatments except TV HS. The effect was limited to 5 d.p.i., while following observation points led to transcript levels similar to the control. In view of the above, we speculated that at least one part of the defense response mechanism triggered by EOs and HSs against ToLCNDV involved a modulation of expression of PAL gene.Carvacrol and thymol were the main constituents of EO and HS extracted from OV and TV, while RO extracts displayed a more complex chemical composition, mostly monoterpene hydrocarbons in EO and oxygenated monoterpenes in HS. Antiphytoviral assays showed a decrease of ToLCNDV titer, while no or adverse effects of treatments were recorded against ZYMV. The application of treatments after inoculation, rather than simultaneously, led to a longer-lasting effect on the virus titer. In such conditions, the level of PAL transcripts also recorded a more prominent increase than in the co-inoculation of virus and treatment, although limited to the first sampling time. In our experiments, the best performance combination of treatment, mode of application, and virus was displayed by RO HS applied after ToLCNDV inoculation. In such conditions, the virus was not detected on systemically infected leaves by real-time PCR at 19 and 26 d.p.i.C. pepo plants under greenhouse conditions. Further investigation is needed to evaluate the applicability of such treatments for practical use in greenhouse and in the field.Overall, our results support the feasibility of using OV, TV, and RO EOs and HSs as potential treatments for ToLCNDV-infected The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.AT and LT: conceptualization. AT, LT, and RR: methodology. AT, LD, and PN: formal analysis and data curation. AT, LD, FS, MS, GD, SF, VV, and RR: investigation. AT: writing—original draft preparation and visualization. AT, LD, LF, LT, MS, FS, and RR: writing—review and editing. AT and RR: supervision. All authors have read and agreed to the published version of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.