Valuation is a critical part of the costing process in health economic evaluations. However, an overview of specific issues relevant to the European context on harmonizing methodological requirements for the valuation of costs to be used in health economic evaluation is lacking. We aimed to inform the development of an international, harmonized and multi-sectoral costing framework, as sought in the European PECUNIA  project.We conducted a scoping review (information extraction 2008–2021) to a) to demonstrate the degree of heterogeneity that currently exists in the literature regarding central terminology, b) to generate an overview of the most relevant areas for harmonization in multi-sectoral and multi-national costing processes for health economic evaluations, and c) to provide insights into country level variation regarding economic evaluation guidance. A complex search strategy was applied covering key publications on costing methods, glossaries, and international costing recommendations augmented by a targeted author and reference search as well as snowballing. Six European countries served as case studies to describe country-specific harmonization issues. Identified information was qualitatively synthesized and cross-checked using a newly developed, pilot-tested data extraction form.Costing methods for services were found to be heterogeneous between sectors and country guidelines and may, in practice, be often driven by data availability and reimbursement systems in place. The lack of detailed guidance regarding specific costing methods, recommended data sources, double-counting of costs between sectors, adjustment of unit costs for inflation, transparent handling of overhead costs as well as the unavailability of standardized unit costing estimates in most countries were identified as main drivers of country specific differences in costing methods with a major impact on valuation and cost-effectiveness evidence.This review provides a basic summary of existing costing practices for evaluative purposes across sectors and countries and highlights several common methodological factors influencing divergence in cost valuation methods that would need to be systematically incorporated and addressed in future costing practices to achieve more comparable, harmonized health economic evaluation evidence.The online version contains supplementary material available at 10.1186/s13561-022-00390-y. The increased health needs and demands as well as the scarcity of resources have resulted in a more prominent role of economic considerations in evidence-based healthcare decision-making . NeverthWhile these unit costs and valuation sources have nonetheless been accepted for use in EEs in the healthcare sector , the appThe importance of harmonization of methodological requirements in relation to costing increases when healthcare interventions influence resource use outside the healthcare sector . These sIn light of the methodological heterogeneity, it often remains unclear if differences in (unit) costs observed between national and international studies are e.g. due to differences in applied costing methodology or differences attributable to the service . From a a) To establish a set of definitions of central economic terms and main concepts to determine the degree of heterogeneity that currently exists in the literature;b) To generate an overview of the most relevant potential areas for harmonization in multi-sectoral and multi-national costing processes for health EEs;c) To provide insights into country level variation regarding EE guidance, based on a case study of six European countries represented in the PECUNIA project.The overarching objective of this current scoping review was to inform the development of an international, harmonized and multi-sectoral costing framework, as sought in the European PECUNIA  project . This reThis scoping review was guided by the approach to map the main concepts, theories, evidence, knowledge gaps and their main sources as recommended by the Enhancing the QUAlity and Transparency Of health Research (EQUATOR) network . The repWith key grey literature and scientific peer-reviewed articles including multiple sectors  and methodological aspects regarding costing not being always clearly identifiable as such, a comprehensive search strategy was required.For the establishment of a set of definitions of central economic terms and main concepts to determine the degree of heterogeneity currently existing in the literature (research aim a)) we screened textbooks and online glossaries. In addition, we screened all sources identified in course of the literature search for research aim b) for definitions. The selection of the economic terms included in the glossary was based on the criteria ‘relevance’ and ‘likelihood for interpretation differences’, determined by the research team, who are experts in the field of health economics. This overview does not make any claim to comprehensiveness , but rather aims to highlight the potential variation and partial contradictions of existing definitions of costing approaches and their components. For the identification of general methodological issues regarding sector-specific valuation issues (research aim b) potentially causing amenable heterogeneity in service use costing, the following approach was adopted: Publications from previous EU projects linked to costing methods were screened . RefFor the identification and analysis of country-specific harmonization issues (research aim c)), six European countries represented in the European PECUNIA project , were selected as case studies for feasibility reasons. Since these countries represent different types of health care systems  the overview is nevertheless expected to provide a fairly complete picture. At the same time, broader international insights were considered where identified. As the development of costing methods and relevant recommendations is often part of the national Health Technology Assessment (HTA) agenda, national websites were considered as most promising to screen for country-specific guidance  manuals, costing guidelines and unit cost programmes).The searches were augmented by additional literature  from the PECUNIA consortium and scientific advisory board (SAB).The search was conducted throughout 2018 (the start of the PECUNIA project) and information extraction limited to publications from the past 10 years (i.e. 2008) to cover timely methods. Full-text publications of peer-reviewed articles and books as well as grey literature were included. In regards to EE guidelines, the latest available publications within the defined search window were considered. In addition, we conducted an update of the search until 2021 to include any major advancements after 2018 (i.e. updates of costing guidelines).Textbooks, online glossaries, peer-reviewed publications and grey literature were selected if they included a relevant definition/description of central economic terms. Relevant health economic and HTA websites, peer-reviewed and grey literature were selected if they included main concepts or general methodological issues regarding sector-specific valuation issues, which are of relevance when conducting multi-country, multi-sectoral health EEs. National EE/HTA guidelines and manuals, available costing programmes/manuals and guidelines, and country-specific health economic and HTA websites were selected in case they included country-specific information and recommendations regarding valuation aspects for EEs.Publications in German, Hungarian, Spanish, Dutch, and English were considered for inclusion.Identified publications were screened for their definitions/descriptions of central economic terms and main concepts. These were collected, compared and organized in a table format. General methodological aspects in respect to sector-specific valuation issues were extracted and grouped according to the specific issue it related to. For the identification and analysis of country-specific harmonization issues, data extraction was conducted using a newly developed and standardized, pilot-tested data extraction form created in MS Excel. Synthesised information on service costing methods recommendations covered: analytic study perspective , costs (to be included in an EE), list of national/regional unit cost lists available, source of valuation (cost data), the hierarchy of data sources stated for valuation, costing methodology , measurement of capital costs, overhead costs and operating costs , sources used to calculate reference prices for various healthcare services (e.g. public statistics), physical units , reporting of costs (any requirement to report costs and quantities/volumes separately), time horizon, discounting, adjustment for inflation/price indexation, specific considerations per field/area of application, periodicity of costing manual, recommended level of guideline adherence (e.g. mandatory).In each stage of the applied methodology, at least two researchers were involved. The searches in the multiple information sources were executed by two researchers . Identified literature was screened against the above pre-defined inclusion criteria by two researchers . Potential disagreements regarding inclusion were resolved by discussion or if needed, by a third reviewer .N = 35), relevant books (N = 2), and grey literature (N = 14) resulted in 51 hits. The search for EE guidelines, unit cost(ing) manuals/programmes/list of unit costs, health economic and HTA websites added 46 hits. This resulted in the inclusion of a total of 97 sources for information synthesis. The overall study selection process is illustrated in the PRISMA flow diagram  resulting from healthcare interventions that affect the education sector could include special education services and the costs for student transport to the education facility , 57. CriA summary of some key costing recommendations from national health economic guidelines of the selected six countries is shown in Table With regards to the adopted analytical study perspective of an EE, the German and the English recommend a healthcare perspective. The Austrian guideline is not specific in this regard. While the Dutch guideline recommends a societal perspective, the Spanish one recommends both the societal and the healthcare perspectives to be applied parallel. The Hungarian guideline states that if costs and outcomes are falling mainly outside of the healthcare system  the societal perspective is suggested in addition to the healthcare perspective. That what is meant by societal is, however, is not always explicitly stated and may vary very much from analysis to analysis.Most guidelines state which costs should be included in an EE. The majority of them use a traditional cost component typology referring to health and social care and out-of-pocket expenses as direct costs and lost productivity as indirect costs . The DutThe inclusion of social care costs such as those resulting from the use of respite care and supportive care services is explicitly recommended in four guidelines , while the inclusion of patient (and family) costs  are recommended in four guidelines . In Germany, patients’ out-of-pocket expenses are included as part of the perspective of the Statutory Health Insurance (SHI)-insured community. In contrast, time costs are not standardly considered, but are examined in the course of sensitivity analyses. The Hungarian guideline specifies that the inclusion of cost types should be determined based on the adopted perspective. The inclusion of lost productivity costs is recommended in four guidelines  and are to be reported separately from direct medical costs). In the Hungarian guideline the inclusion of productivity costs is recommended just a complementary element of cost calculation if majority of costs are falling outside of the healthcare sector.There is large variation between the guidelines regarding the suggested valuation approach. All guidelines stress that the valuation method chosen should reflect opportunity costs. In addition, various valuation methods are recommended in the guidelines, varying from top-down micro-costing to gross-costing, including the use of standard unit costs/reference prices, tariffs, market prices, administrative data, and diagnosis-related groups (DRGs). All guidelines name one or more sources, which should preferably be used for the unit costing. A standard cost list containing sets of standardized unit cost estimates is officially recognized and applied by the HTA agency in the Netherlands (Zorginstituut Nederland) . These hThere is limited information in the majority of the guidelines about the valuation and allocation of capital/overhead and operating costs, i.e. costs that cannot be directly allocated. Austria and Spain do not include recommendations on this at all. The Dutch guideline names different methods that can be used to measure overheads such as average fixed costs per unit, the equivalence method, and the mark-up method. Although no specific method is recommended, it is emphasized that each of these methods has different advantages and disadvantages. Based on data from the financial statistics for Dutch hospitals, an estimation of overhead costs was conducted in 2012. The derived percentage for overheads on the directly attributable costs of medical departments was 38%. The percentage for overheads on housing and depreciation costs on the directly attributable costs of medical departments was 6%, adding up to a total of 44% for overheads. This percentage reflects an average with huge variations between individual organisations and services .For the adjustment of unit costs from different calendar years, the Austrian and Spanish guidelines recommend that costs should be adjusted to a common reference year, but they fail to provide further details. The Hungarian guideline states that costs shall be uprated to the same date. The consumer price index (inflation) should be chosen as inflation rate, irrespectively of whether the costs (or savings) arise within or outside the healthcare sector, with the official publications of the Hungarian Central Statistical Office as recommended source. Both the German and Dutch guidelines specify that all costs should be inflated to the present value by using the official price index from national statistics. The Dutch guideline also tackles the aspect of correcting for inflation in case of including costs from different European countries. It is suggested that the Indices of Consumer Prices (HICP) of the European Central Bank, which were specially designed for international comparisons of consumer price inflation, need to be used.For EEs to be able to inform efficient resource allocation based on valid high-quality evidence, it is crucial that both outcomes and costs are assessed rigorously . In contThis scoping review highlights multiple methodological problems challenging the harmonization of service unit costs for (inter)national, multi-sectoral health EEs. For the health and social care sectors, these include ambiguity and feasibility problems regarding the definition and application of cost identification and valuation approaches. Several problems regarding the data source used to develop unit costs were identified: the impact of choice of the costing sources, the applied costing perspective and unavailable proxy measures, as well as the absence of a standard to estimate overhead costs.Moreover, the limited cross-country comparability of labour costs and ambiguity concerning the application of cost types were identified as further difficulties. Challenges in respect to the education and criminal justice sectors concern the valuation of health-related service use in these sectors, unestablished valuation methods, and missing feasibility testing in the international context, as well as difficulties regarding the generation of proxy unit prices. Double counting of costing components also may not only be an issue within the health care sector as previously reported, but also cross-sectorial. For example, double counting of out-of-pocket expenses may occur as part of the unit cost of a health care service and as part of patients’ out-of-pocket costs, which may arise due to lacking transparency in the reported costing components. The analysis of national costing guidelines revealed extensive variation concerning the recommendations with respect to guideline adherence, analytical study perspective, valuation approach and inflation.The early FP6-funded HealthBasket project (2004–2007), which focused on harmonization of costing in the healthcare sector, already concluded one decade ago that “the prerequisite for international cost comparison is mutually accepted methodological guidance (standard costing method) and reasonably good compliance with it” . FurtherAt the same time, some practical limits to the harmonization of costing will remain. Firstly, unit costs are one major ingredient to the valuation aspect in EEs. Limited guidance regarding the calculation of overheads resulting in assumptions are a very much under-discussed, under-researched and under-reported area. Both, extensive sensitivity analysis or transparent overhead calculation as one harmonization aspect for unit costs would be potential ways forward. There are, however, also broader costing issues , such asTo the best of our knowledge, this is the first review on costing methods across several sectors affected by healthcare interventions focusing on a set of selected European countries and healthcare systems. Other publications focusing on differences between EE guidelines do exist e.g. , 80), bu, bu80]),The selected countries included in the PECUNIA Project differ regarding their health care systems with varying feasibility and acceptability of EEs in evidence-informed decision-making. Some countries have established national unit cost programmes/lists , some early stage initiatives . Availabilities of health utility value sets for outcome evaluations and requirements in terms of the study perspective also differ. Nevertheless, the country selection is not necessarily balanced in regards to the aforementioned aspects.Study selection and data extraction was challenging in some cases, especially when slightly divergent information on one topic was identified. However, two researchers were involved in the study selection process and data extraction phase, and with two additional researchers consulted in case of disagreements, which enabled extensive discussions and thorough assessment of the identified material. Due to the broad topic area and expected spread of relevant information between different types of publications, mostly within the grey literature needed to be included. Our search strategy to identify grey literature was very comprehensive and national experts were involved to complement our findings. In addition, a search update was conducted to enhance the review’s timeliness. Although the applied strategy to identify peer-reviewed publications was expected to capture all relevant key topics, as it covered different approaches which were explained in our methods section, it cannot be ruled out that some potentially relevant material has been left out, despite our attempts to be as comprehensive as possible.Several methodological issues were identified that lead to the current heterogeneity in valuation methods in health EEs used across sectors and countries. To address these explicitly in future costing guidelines and tools is a key step towards more comparability and harmonization in national and international health EEs. Additional file 1:Figure 1. Adapted PRISMA flow chart of study selection. Table 2. Preferred Reporting Items for Systematic reviews and Meta-Analyses extension for Scoping Reviews (PRISMA-ScR) Checklist. Table 3. Synthesis of country-specific recommendations regarding valuation aspects from economic evaluation guidelines of six selected European countries.


Chinook salmon in California conservation hatcheries have suffered disease outbreaks and significant mortality due to the fish virus infectious hematopoietic necrosis virus (IHNV) since at least the 1940s. Although steelhead trout in California are also occasionally infected with the same virus they do not typically experience disease. In this study the susceptibility of California Chinook salmon and steelhead trout was defined in controlled experiments that exposed groups of juvenile fish to California IHNV strains (L genogroup). The results confirmed high mortality among Chinook salmon but very low mortality of steelhead trout, despite identical virus exposures. Tests of varying conditions found increasing mortality of Chinook salmon with increasing virus dose, but reduced mortality at higher temperatures or with increased age or size of fish. Among Chinook salmon that survived virus exposure the persistence of virus was detected in one fish 8 months after virus exposure. These findings demonstrate that L genogroup IHNV in California has host specificity for Chinook salmon, and provide an understanding of factors that contribute to disease epidemics in California hatchery Chinook salmon.Oncorhynchus tshawytscha) have experienced disease outbreaks due to L genogroup IHNV since the 1940s, while indigenous steelhead (anadromous O. mykiss) appear relatively resistant. To characterize factors contributing to the losses of California salmonid fish due to IHNV, three populations of Chinook salmon and two populations of steelhead native to California watersheds were compared in controlled waterborne challenges with California L genogroup IHNV isolates at viral doses of 104–106 pfu mL−1. Chinook salmon fry were moderately to highly susceptible (CPM = 47–87%) when exposed to subgroup LI and LII IHNV. Susceptibility to mortality decreased with increasing age and also with a higher temperature. Mortality for steelhead fry exposed to two IHNV isolates was low (CPM = 1.3–33%). There was little intraspecies variation in susceptibility among populations of Chinook salmon and no differences in virulence between viruses strains. Viral persistence was demonstrated by the isolation of low levels of infectious IHNV from the skin of two juvenile Chinook salmon at 215 d post exposure. The persistence of the virus among Chinook salmon used for stocking into Lake Oroville may be an explanation for the severe epidemics of IHN at the Feather River hatchery in 1998–2002.Salmonid species demonstrate varied susceptibility to the viral pathogen infectious hematopoietic necrosis virus (IHNV). In California conservation hatcheries, juvenile Chinook salmon ( Oncorhynchus tshawytscha), kokanee (O. nerka), sockeye (anadromous O. nerka), rainbow trout (O. mykiss) and steelhead trout (anadromous O. mykiss). Infection with IHNV in juvenile fish can cause lethargic behavior and clinical signs including skin darkening, exophthalmia, hemorrhage or ascites. Pathogenesis involves the degenerative necrosis of the kidney and other hematopoietic tissues. IHN disease may result in mortality up to 100% in young salmonids [Infectious hematopoietic necrosis is a severe rhabdoviral disease of farmed, hatchery and infrequently wild salmonids ,2,3,4. Ialmonids , causingThe host range for IHNV includes most salmonids, but the degree of host susceptibility to disease varies among and within species ,7,8,9,10Among IHNV isolates in North America, three genogroups of IHNV, with distinct geographic ranges, are currently recognized: U, M and L for upper, middle and lower regions in the Pacific Northwest of North America . The U gIn California, L genogroup IHNV is most commonly encountered among asymptomatic adult Chinook salmon and steelhead returning to spawn at the three largest hatcheries in the Sacramento River basin, the Coleman National Fish Hatchery , Feather River Hatchery  and Nimbus Hatchery . Periodic epidemics due to IHNV have resulted in losses of juvenile Chinook salmon at the CNFH since 1941 ,4,20,21.An investigation into the possible causes of the 1998–2002 epidemics at the FRH using phylogenetics and virulence studies provided evidence that the virus associated with the epidemics at the FRH was genetically consistent with and no more virulent for Chinook salmon and rainbow trout than historical isolates in the water system . InsteadIn previous studies, viral virulence and variation in host susceptibility have been characterized for IHNV from the U and M genogroups in hosts from their respective northern geographic ranges ,24,25,26Fish from three populations of fall-run Chinook salmon and two populations of steelhead trout native to California watersheds were used in experimental virus challenge studies .−1. The Feather River (FRH) and Nimbus (NH) Chinook salmon populations are from Sacramento River basin hatcheries that have experienced IHN epidemics and are in the geographic range of the LII subgroup of IHNV. Both of these populations are considered to be genetically homogeneous as determined by allozyme, mitochondrial DNA and microsatellite loci analyses [Fish were received as eggs, hatched and reared with a biodiet ration  in 36-L aquaria supplied with 12 °C pathogen-free well water at a rate of 0.2 L minanalyses ,33,34,35analyses ,28,29,33analyses .Novirhabdovirus salmonid, genus Novirhabdovirus, family Rhabdoviridae. Five IHNV isolates used in experimental challenge studies are described in IHN viruses are members of the species Complete isolate designations from fish health agencies or names presented in Kelley et al.  are showThree virus exposure experiments were conducted to determine the kinetics and final levels of mortality after the exposure of various fish populations to selected IHNV strains under varying conditions .4 (low), 105 (intermediate) or 106 (high) pfu mL−1, and actual virus exposure doses were determined directly from the virus suspensions on the day of each challenge [4, 4.6 × 105 and 4.6 × 106 pfu mL−1 for FR04 and 5.7 × 104, 5.7 × 105 and 5.7 × 106 pfu mL−1 for RR98. In Experiment 2, Chinook salmon fingerlings from FRH and IGH were 1260 dd in age and 2.2 g in average weight at the time of challenge. They were exposed to intermediate and high relative concentrations of the FR04 IHNV isolate at three water temperatures, 10 °C, 12 °C and 14 °C, to simulate temperature variation consistent with temperatures at the FRH and IGH. Actual exposure doses of FR04 were 7.0 × 105 (intermediate) and 7.0 × 106 pfu mL−1 (high).Fish were challenged by exposure to IHNV isolates at relative viral concentrations of approximately 10hallenge . The dethallenge , as deschallenge . For Exp4, 1.2 × 105 and 1.2 × 106 pfu mL−1 for FR04 and 2.0 × 104, 2.0 × 105 and 2.0 × 106 pfu mL−1 for CL02.In Experiment 3, steelhead fry from FRH and CNFH were 714 dd in age and weighed approximately 0.68 and 0.67 g, respectively. They were exposed to three doses of IHNV isolates FR04 and CL02. Actual challenge doses were 1.2 × 10For each experiment, duplicate groups of 30 fish were challenged by static immersion in water containing dilutions of cell culture medium with each IHNV isolate at each challenge dose. Duplicate control groups of 30 fish received only MEM-2 without virus. Exposures were conducted by placing fish into 0.5 L  or 1.0 L  of aerated well water with or without virus in 1.7 L plastic containers immersed in flowing well water, to maintain temperature. After 1 h, the fish were transferred to 15 L aquaria maintained in an aerated flow-through system supplied with chilled well water. Cumulative percent mortality (CPM) was recorded daily over a 21 d period post-virus exposure. At 21 d following the initial virus exposure, all remaining surviving fish were euthanized in a 500 mg/L benzocaine solution.All virus exposures were conducted at a water temperature of 12 °C, except Chinook salmon fingerlings in Experiment 2, which were tested at three water temperatures . Fish stocks originally at 12 °C were adjusted gradually (0.5 °C) daily to reach the desired water temperature 3 days prior to virus exposure.4 pfu mL−1). FRH steelhead fry were exposed to a low dose of FR04 or CL02 in mortality Experiment 3 as described above. Lastly, FRH steelhead fingerlings (1.1 g) at 834 dd in age were exposed to a high dose of FR92 or FR69 virus, relative concentration (106 pfu mL−1). After virus exposure, fish were held and sampled at defined time points. For Chinook salmon, at 120 d post exposure (dpe), 4–6 gill filaments, kidney and spleen tissues were collected as separate samples from 30 fish in each Chinook salmon exposure group and respective control groups . A second and final examination of 32 Chinook salmon (28.9 g) occurred at 215–216 dpe with the same tissues sampled as at 120 dpe but also including a portion of the abdominal skin. Skin samples of approximately 0.05 g were taken as slices near the lateral line between the dorsal and pelvic fins and represented an area of approximately 4 × 20 mm. A total of 163 steelhead (22.7 g) were sampled as described above, including skin samples, at 227–248 dpe. Control unexposed fish sampled at the later collection dates included 30 Chinook salmon and 45 steelhead. Samples were processed fresh on the day of collection, using methods described above for determining presence and concentrations of viable IHNV by plaque assay.Longer experiments were conducted to examine the potential persistence of the virus beyond the standard 21 d duration of the mortality studies. Chinook salmon fingerlings originating from NH (5.1 g) and IGH (4.2 g) and 1554 dd in age were exposed to a low dose of FR04 virus concentration , water temperature and fish stock were analyzed using the Glimmix model statistical program . Mortality curves were estimated with the Kaplan–Meier method and compared with a log-rank test with adjustment for sample size by Tukey-Kramer. Data that presented with a Chinook salmon fry (average weight 0.54–0.57 g) from three different populations demonstrated a moderate to high level of susceptibility to two L genogroup IHNV isolates, with cumulative percent mortality (CPM) ranging from 47–87% depending on virus dose .p value < 0.0001) for both the RR98 and FR04 IHNV isolates, with cumulative percent mortality (CPM) increasing in correlation with greater virus doses (p = 0.0562), the NH Chinook salmon appeared slightly more resistant than the FRH fish at the highest virus dose based upon CPM and MDD = 11.4–12.5 and 10.0–10.6, respectively  or CPM between Chinook salmon fry of different hatchery origins exposed to either the RR98 (LI) or FR04 (LII) genotypes of IHNV at each exposure dose. Significant differences in mortality were observed between Chinook salmon fry exposed to different concentrations of IHNV  and with water temperature (p < 0.0001). Moderate mortality (40–58%) following exposure to IHNV was observed among Chinook salmon fingerlings originating from both the FRH or IGH at water temperatures of 12 °C and 10 °C, with the exception of significantly (p = 0.0282) lower mortality (20%) for IGH at the intermediate virus dose at 10 °C (p < 0.0001) at a water temperature of 14 °C (CPM = 10–22%). No mortality was observed in unexposed control groups of Chinook salmon fingerlings over the 21 day period.Two populations of Chinook salmon fingerlings (ave. 2.2 g) exposed to intermediate and high doses of IHNV isolate FR04 at 10, 12 or 14 °C had CPM that ranged from 10 to 58% . The CPMat 10 °C . Mortalip value < 0.0001) were observed between fish exposed to different concentrations of both IHNV isolates  showed a low susceptibility to IHNV, with CPM ranging from 1.7 to 33% after low-, intermediate- or high-dose exposures. There was no significant difference in the susceptibility of either population of steelhead fry (FRH or CNFH) following exposures to LII IHNV isolates FR04 or CL02 at the same virus dose . Signifiisolates . Mortaliisolates . A trend2 to 1.5 × 103 pfu g−1 which was at or near the lower limits of detection of 2.5 × 102 pfu g−1 for the plaque assay employed. Virus was not detected in any of the 163 FRH steelhead sampled at 227–248 dpe  observed in correlation with greater virus dose has been described in most challenge studies with IHNV, e.g., [The current study examined susceptibility of three Chinook salmon and two steelhead populations from California hatcheries to L genogroup IHNV isolates. Fish and virus populations were from the geographic ranges of both subgroup LI and LII IHNV in California and included watershed specific IHNV isolates to investigate potential factors contributing to severe epidemics among Chinook salmon and juvenile steelhead at the FRH in 1998 and 2000–2002. In the current study, the moderate to high susceptibility of three native California Chinook salmon stocks exposed to L genogroup IHNV at approximately 0.5 g in size was consistent with the high incidence of IHNV epidemics observed in juvenile Chinook salmon at California hatcheries where L genogroup IHNV is endemic. In accord with Bendorf et al. , who exaV, e.g., ,11,12,252−4 fold greater). The mortality (CPM = 47–87%) observed for the three stocks of Chinook salmon fry in our study is in the range (CPM = 47–72%) reported for similar aged NH Chinook salmon (0.56 g) to equivalent concentrations of seven L genogroup isolates at 12 °C [5 pfu mL−1), mortality ranged from 17–49% [4 pfu mL−1. Among the selected groups of Chinook salmon from California examined here, virus concentration, not isolate nor fish stock origin, significantly impacted mortality to L genogroup IHNV. These finding suggest that a key element in the epidemics observed at the FRH are increased concentrations of virus in the water supply, rather than host stock or watershed-specific virulence of the virus isolates.In comparison with a prior report that very young Chinook fry (10 days post-hatch) had high initial susceptibility to IHNV CPM = 80–100%) but developed complete resistance by 30 days post-hatch , and ourat 12 °C . Moreove0–100% bum 17–49% ,26. Thism 17–49% . Furtherm 17–49%  are consIn our current study, the susceptibility of FRH and IGH Chinook salmon fingerlings exposed to two concentrations of an LII IHNV isolate was used to evaluate the effects of host age and water temperature  on viral virulence. Chinook salmon fingerlings, aged 1260 dd, from the FRH and IGH demonstrated an increased resistance (CPM = 40–50%), compared to their younger counterparts CPM = 68–87%), following exposure to intermediate and high doses of a LII IHNV isolate at a water temperature of 12 °C. Increased resistance concurrent with the age and size of the host has been previously reported for several species of salmonids including Chinook salmon and is thought to be due at least in part to greater immune competence in older fish –87%, fol,44,45. CF. psychrophilum in the steelhead in our trial may have contributed to an increased susceptibility to IHNV as co-infections with these two agents are commonly involved in mortality during rainbow trout production [Among steelhead fry ~0.7 g) from the FRH and CNFH, even at virus concentrations considered environmentally high , both LI g from toduction . NonethePersistence of IHNV in populations of Chinook salmon or rainbow trout in Lake Oroville upstream of the water source for the FRH has been suggested as a potential source of virus for the epidemics at the FRH in 1998 and 2000–2002 . TypicalOur current study provides further evidence supporting our initial hypothesis that increased levels of stocking of juvenile Chinook salmon in Lake Oroville provided a source and concentrations of virus sufficient to precipitate epidemics at the FRH . The curBendorf et al.  providedBased on the absence of variations in fish host susceptibility or viral virulence and the observation of long-term persistence of IHNV in a small proportion of Chinook salmon, we conclude that the amplification of virus in the water source (Lake Oroville) for the hatchery due to stocking high concentrations of Chinook salmon, some of which could have had subclinical/persistent IHNV infections, was the most likely cause of the observed IHNV epidemics at FRH between 1998–2002. In experimental challenge studies Chinook salmon originating within the geographic range of L genogroup IHNV are moderately to highly susceptible to disease and mortality if exposed to the virus as young fry, but susceptibility decreases with age and at the increased temperature of 14 °C. California steelhead are much less susceptible and appear to be a spillover host when reared in close proximity to the more susceptible Chinook salmon. Despite the divergence of L genogroup IHNV into distinct subgroups LI and LII, there is no evidence for differences in virulence, and Chinook salmon populations originating within each subgroup range did not show specific adaptation to the sympatric viral subgroup. Virulence, host specificity and field prevalence  support


This study aimed to estimate healthcare costs of diabetic foot disease (DFD) in a large population-based cohort of people with type-2 diabetes (T2D) in the Tuscany region .Administrative healthcare data of Tuscany region, with 2018 as the base year.Retrospective study assessing a longitudinal cohort of patients with T2D.Using administrative healthcare data, DFD were identified using the International Classification of Diseases, Ninth Revision, Clinical Modification codes.We examined the annual healthcare costs of these clinical problems in patients with T2D between 2015 and 2018; moreover, we used a generalized linear model to estimate the total healthcare costs.p < 0.0001). Among patients with T2D experiencing DFD, those who experienced complications either in 2015–2017 and in 2018 incurred the highest incremental costs  followed by those with complications in 2018 only  and from 2015 to 2017 .Between 2015 and 2018, patients with T2D experiencing DFD showed significantly higher average direct costs than patients with T2D without DFD (DFD significantly increase healthcare utilization and costs among patients with TD2. Healthcare costs of DFD among patients with T2D are associated with the timing and frequency of DFD. These findings should increase awareness among policymakers regarding resource reallocation toward preventive strategies among patients with T2D.The online version contains supplementary material available at 10.1007/s10198-022-01462-w. Worldwide, diabetes is a major health public health concern, with > 460 million adults living with diabetes, which is estimated to increase to 700 million in 2045. Moreover, in 2019, the healthcare expenditure costs for diabetes were ≥ USD 760 billion [Some of these expenditures result from complications, including cardiovascular events, kidney failure, and foot diseases . Diabetic foot disease (DFD) is among the most severe and costly long-term diabetic complications. The most significant manifestation of diabetic foot problems, such as amputations significantly contribute to both mortality and morbidity among patients with diabetes and exert a considerable financial burden on patients and healthcare systems. Recent findings based on Global Burden of Disease 2016 data indicated that approximately 131.0 million (1.77%) people worldwide presented DFD in 2016, including 105.6, 18.6, and 6.8 million individuals with neuropathy only, foot ulcers, and amputations, respectively [In 2019, diabetes-related expenditure in Europe was approximately USD 161 billion, which accounts for 21% of the global diabetes-related expenditure. A recent review of studies assessing the costs for DFD which includes amputation, ulcers, gangrene, across European countries reported increasing costs over time, especially for amputation, which widely varied across countries . PetrakiDFD results in greater expenditure than major diseases, including cancer, lung disease, and depression , 6. In 2Strategies, including prevention, patient and healthcare staff education, multidisciplinary treatment, and regular close monitoring, can significantly prevent and reduce DFD, especially amputations , 10.In 2019, Italy, which has a regionally based National Health Service (NHS) with universal coverage that is largely free of charge at the delivery point, was among the top five European countries in terms of the number of people with diabetes (20–79 years) (about 3.7 million people with diabetes) (IDF 2019) , 12.Despite the significance of the increasing prevalence and costs of diabetes and its complications, there remain scarce population-based studies on the economic impact of diabetes and, in particular, DFD; moreover, they are often limited to country-level estimates or prospective data collection from selected diabetic centers  , 13, 14.Given these premises, we analyzed the annual costs of DFD in a large population-based cohort of people with type-2 diabetes (T2D) living in the Tuscany region . Specifically, this work aims at estimates healthcare costs for the management of DFD in patients with T2D considering two cost components: state and event costs. Event costs represent the complication costs associated with resources use in the base year that is specific to the acute episode (managed both in an inpatient or outpatient setting) and any subsequent care occurring in the same year. State costs refers to the management costs for years subsequent to the event year and reflect the typical utilization of health care services for the continuous management of complications experienced in the years before.Studies on cost and service utilization for DFD have predominantly estimated direct medical costs in continental Europe, Northern America or Australia using heterogeneous approaches often limited to cost-effectiveness studies comparing different treatment options . We condItaly’s health-care system is a regionally based National Health Service, which provides universal coverage largely free of charge at the point of delivery . TuscanyThe Tuscany healthcare administrative databases contain information regarding all public and private accredited healthcare providers. The individual-level databases used in this study included (i) hospital inpatient data; (ii) emergency care data; (iii) outpatient care data; (iv) drug prescription data; (v) exemption data; and (vi) a registered person database containing socio-demographic information on all residents enrolled in the Tuscan healthcare system, including sex, date of birth, and date of death. Different administrative databases were linked at the individual (patient) level using a unique identifier. The data were anonymized at the Regional Health Information System Office, where each patient was assigned a unique identifier to prevent tracing of the patient’s identity and other sensitive data. This study was conducted in compliance with Italian law on privacy; approval by the conjoint ethics committee of Scuola Superiore Sant’Anna di Pisa and Scuola Normale di Pisa  was also obtained (Delibera no. 20/2020).To estimate the DFD-related costs in the year 2018, we considered all residents living in Tuscany, Italy who entered the T2D cohort from 2010 to 2014, and alive on December 31st, 2018. T2D account for 95% of all patients with diabetes, in the Tuscany region.These selection criteria ensured that the patients’ complications during 3 years before the base year 2018) were related to diabetes  . This T2 were relBased on a previously described methodology , we idenWe calculated the direct care costs in the year 2018 for each patient with T2D by considering cost information obtained from the following administrative databases: inpatient care (DRG tariffs), drugs (net costs), and outpatient and emergency services (outpatient tariffs). All costs are expressed in 2018 euros. Moreover, we considered demographic variables (age and sex) from regional databases. First, we analysed the different healthcare costs for 2018 of T2D patients considering number and type of complications. Specifically, we defined five patients’ groups ranging from patients with no complication (group 1) to patients with two or more complication distinguishing between DFD complications and other diabetes-related complications (group 5). Subsequently, we focused on estimation of DFD costs. Given the low number of zero costs (2.5%), care costs for DFD in the T2D population were estimated using a GLM. To determine the correct model specification, we employed the modified Park test  which deFinally, we calculated the incremental costs for foot complications by subtracting one from the GLM coefficient expressed as incidence rate ratios and multiplying them by the estimated mean annual healthcare costs of a 70-year-old man without foot complications.p value of < 0.05. Moreover, confidence intervals were calculated at 95%. All statistical analyses were performed using SAS for Windows Ver. 9.3  and STATA Software v14.In all analyses, statistical significance was set at a p < 0.001). In addition, compared with patients without a diabetes-related complication, patients with a complication were older . The mean annual total healthcare costs for men were slightly higher than for women  and increasing by age group (see Appendix 2). Table N = 5,972), 730 (12.22%) patients experienced at least one DFD in 2015–2018  did not experience diabetes-related complications from 2015 to 2018; moreover, the average age was 69 years. The results also showed that men were more likely than women to have diabetes-related complications , while T2D suffering any complication resulted in significantly higher hospitalization costs (groups 2–5). Inpatient costs for managing diabetic complications were significantly higher compared to outpatient expenses, thus representing the key driver of complication costs. When comparing patients by number of complications the cost composition is very similar between T2D patients suffering one complication (group 2 and 3) and patients suffering more than one complication (group 4 and 5), so number of complications do not seem to impact healthcare cost composition. However, it is interesting to note that T2D patients with more than one complication including DFD (group 5) registered a higher share of outpatient costs compared to the other groups of patients with complication often due to expenses attributed to the management of events in ambulatory settings, such as foot ulcers and revascularisations. Cost for emergency care is very limited in all patient groups (< 3%).The total expenses for acute, emergency, outpatient, and drugs for T2D patients in Tuscany resulted in 125.1 million euros in 2018. The annual per-capita direct health care costs for the five groups of patients are summarized in Fig. A specific analysis of the 730 patients with DFD revealed that 114 patients had an amputation  in the period of analysis (2015–2018) and the respective annual per-capita costs was calculated at € 12,146 compared to an annual patient cost of € 7,864 for the remaining 616 patients incurred in other DFD excluding amputation. This evidence confirms amputations to be the major costs driver of DF problems.When considering the costs associated to DFD only, the GLM analysis results Table , show thSpecifically, using the mean value for estimation, the annual costs in year 2018 ranged from € 2,334 (95% CI 2.284–2.383) in patients with T2D without DFD to € 10,931 (95% CI 9.525–12.336) for patients with DFD in 2018 who had no history of that complication from 2015 to 2017 , € 3,055 (95% CI 2.493–3.617) for those with the presence of the DFD at some point between 2015 and 2017  and € 17,390 (95% CI 14.987–19.794) for those who incurred in DFD both between 2015 and 2017 and in 2018 , followed by those who experienced DFD in 2018 only  and 2015–2017 .The cost related to complications is the most significant contributor to the costs related to diabetes requiring expensive medical interventions and treatment , 24. ThiIn 2018, the adjusted event cost of DFD (cost related to first experienced DFD) was 4.6 times that of patients without DFD and 3.7 times the adjusted state costs in 2018 . On the other hand, consistent with the findings by Kähm et al. (2018), DFD were likely to affect the total direct health care costs even years after the event, with the state costs in 2018 being 1.31 times higher than those of patients without DFD.Moreover, there were differences among women and men in different age groups in the complication costs; specifically, males and the elderly had significantly higher costs compared with other categories.https://www.diabetes.co.uk/cost-of-diabetes.html—report on costs). A German study using claims data reported that T2D complications significantly affected the total direct health care costs even years after the event, with variations according to demographics and complication type [These findings are consistent with previous reports. In the UK, the direct costs of diabetes in 2010/11 were £9.8 billion, with approximately 80% being spent on complications  . At systThis study had some limitations. First, we only used administrative data sources. Although they are widely available at a reasonable cost, only allowed identification and estimation of direct medical costs and, therefore, underrepresents the economic care burden of DFD. Moreover, administrative data lack information about the clinical aspects of diabetic patients, such as severity and duration of the disease (there is no information about the date of onset of the disease) thus limiting the comparability of individuals by stage of disease. Second, the complications were identified based on the ICD-9-CM codes listed in administrative data, which might have had errors and incompleteness, resulting in the misclassification of patients in each complication group. However, the complications were identified as previously reported .Regarding the strengths, this study covered a homogeneous, large, and well-characterized population with diabetes, with a long-term observation period.Our findings highlight the importance of prevention in terms of value for patients with T2D, society, and the healthcare system, regardless of the health system model. Indeed, implementing preventive strategies to provide optimal care to patients with T2D upon initial diagnosis could reduce the onset and risk of complications. Specifically, the results of this study indicate the need for more efforts toward improving secondary prevention strategies in patients with diabetes to prevent avoidable and costly complications, especially regarding DFD. Shifting money from treating complications to secondary prevention, including implementing more well-equipped outpatient ambulatories, increasing advice and education, and personalized care pathways for patients at a high complication risk, could provide high-value interventions for patients with diabetes, as well as facilitate the sustainability of healthcare systems. This is especially true for amputations, which disproportionally contributes to diabetes-related costs and reduces patients’ quality of life.To promote sustainability and better use of resources, several approaches should be considered, including evidence-based clinical pathways for diabetes care, encouraging healthcare workers working in different setting of care , to be responsible and accountable for patients with diabetes they have in common  also witIn conclusion, the complications of diabetes present a substantial economic burden especially for those related to DFD which significantly contributes to an increased use of healthcare services both hospital and outpatient and associated costs. In addition, healthcare costs of DFD among patients with T2D appear linked with the timing and frequency of DFD. The first experience of a foot complication (event) is per se expensive and higher compared to state costs showing the importance of increasing efforts in avoiding the first occurrence of complications. For many complications the resulting management costs persist after the event, because it has placed the patient in a new health status, and, if not correctly managed, might increase the likelihood of new event. Therefore, significant higher costs (state and event costs) will arise. Efforts to avoid or reduce complications would be beneficial not only to the patient, but also to health care systems. Indeed, findings should increase awareness among policymakers regarding resource reallocation toward comprehensive approaches targeting prevention, primary care and screening, and patient education in the management of their disease.Supplementary file1 (DOCX 12 kb)Supplementary file2 (DOCX 14 kb)Below is the link to the electronic supplementary material.


Alternaria fungus can cause notable diseases in cereals, ornamental plants, vegetables, and fruits around the world. To date, an increasing number of mycoviruses have been accurately and successfully identified in this fungus. In this study, we discovered mycoviruses from 78 strains in 6 species of the genus Alternaria, which were collected from 10 pear production areas using high-throughput sequencing technology. Using the total RNA-seq, we detected the RNA-dependent RNA polymerase of 19 potential viruses and the coat protein of two potential viruses. We successfully confirmed these viruses using reverse transcription polymerase chain reaction with RNA as the template. We identified 12 mycoviruses that were positive-sense single-stranded RNA (+ssRNA) viruses, 5 double-strand RNA (dsRNA) viruses, and 4 negative single-stranded RNA (−ssRNA) viruses. In these viruses, five +ssRNA and four −ssRNA viruses were novel mycoviruses classified into diverse the families Botourmiaviridae, Deltaflexivirus, Mymonaviridea, and Discoviridae. We identified a novel −ssRNA mycovirus isolated from an A. tenuissima strain HB-15 as Alternaria tenuissima negative-stranded RNA virus 2 (AtNSRV2). Additionally, we characterized a novel +ssRNA mycovirus isolated from an A. tenuissima strain SC-8 as Alternaria tenuissima deltaflexivirus 1 (AtDFV1). According to phylogenetic and sequence analyses, we determined that AtNSRV2 was related to the viruses of the genus Sclerotimonavirus in the family Mymonaviridae. We also found that AtDFV1 was related to the virus family Deltaflexivirus. This study is the first to use total RNA sequencing to characterize viruses in Alternaria spp. These results expand the number of Alternaria viruses and demonstrate the diversity of these mycoviruses. Mycoviruses are wide distributions in the major taxonomic groups of filamentous fungi, yeasts, and oomycetes ,4,5,6,7.Alternaria spp. can cause essential diseases in cereals, ornamental plants, vegetables, and fruits, and more than 95% of the species reported globally can become facultative parasites on different plants [Alternaria species are the causal agents of pear black spot diseases, resulting in massive economic losses in China [t plants ,18,19,20Alternaria have been found to harbor several mycoviruses. Some A. alternata strains have been found to contain many uncharacterized virus-like dsRNAs [Alternaria have been classed into twelve families. Among these families, the family Chrysoviridae includes Alternaria alternata chrysovirus 1 (AaCV1) and Alternaria solani chrysovirus 1 (AsCV1) [Partitiviridae includes Alternaria alternata partitivirus 1 (AaPV1) and Alternaria tenuissima partitivirus 1 (AtPV1) [Totiviridae includes Alternaria arborescens victorivirus 1 (AaVV1) and Alternaria alternata victorivirus 1  [Botybirnavirus includes Alternaria botybirnavirus 1 (ABRV1) and Alternaria alternata botybirnavirus 1 (AaBRV1) [Mitoviridae includes two mitovirus called Alternaria arborescens mitovirus 1 (AaMV1) and Alternaria brassicicola mitovirus 1 (AbMV1) [Endornaviridae includes Alternaria brassicicola endornavirus 1 (AbEV1) [Alternaviridae, was isolated from an A. alternata strain [Alternaria have been found to harbor Alternaria dianthicola dsRNA virus 1 (AdRV1), Alternaria longipes dsRNA virus 1 (AlRV1), and Alternaria alternata virus 1 (AaV1) which are unclassified dsRNA mycoviruses [Hypoviridae was found to include Alternaria alternata hypovirus 1 (AaHV1) [A. brassicicola and A. solani, respectively. AbFV1 and AsFV1 have been grouped with members of the recently proposed family Fusariviridae [Magoulvirus in the family Botourmiaviridae [Alternaria dianthus strain HNSZ-1 [Hypoviridae, Totiviridae, Mymonaviridae, and a provisional family Ambiguiviridae [Sclerotimonavirus in the family Mymonaviridea [To date, members of the genus e dsRNAs ,22. Myco (AsCV1) ,24,25,26 (AtPV1) ,28. The  ,30. The (AaBRV1) ,32,33. T (AbMV1) ,35. The  (AbEV1) . Alternaa strain . Importaa strain . Moreoveoviruses ,38,39,40 (AaHV1) . In addiiviridae ,43. The n HNSZ-1 . Among tiviridae . Two −ssaviridea ,45Alternaria fungi. For instance, AaCV1 not only restricted the growth of the host fungus but also rendered the host hypervirulent to the plant [A. tenuissima strain [In most cases, the host does not experience any phenotypic changes as a mycovirus infection ,2,5,7. She plant . AaCV1-Aa strain . AaV1 caa strain . AaHV1 aa strain . AdNSRV1a strain .In recent years, metatranscriptomics has been widely used in virus discovery. Many novel viruses have been discovered in fungi, which has significantly promoted the progress of viromics research and has sped up the discovery and understanding of unknown viruses ,57,58,59Alternaria, which are associated with pear spot disease in China. We also identified near-full-length sequences of putative mycoviruses. We isolated a novel −ssRNA mycovirus from an A. tenuissima strain HB-15, which we designated as Alternaria tenuissima negative-stranded RNA virus 2 (AtNSRV2). We also characterized a novel +ssRNA mycovirus from an A. tenuissima strain SC-8, which we designated as Alternaria tenuissima deltaflexivirus 1 (AtDFV1). Through phylogenetic and sequence analyses, we found AtNSRV2 to be related to the viruses of the genus Sclerotimonavirus in the family Mymonaviridae. We also found that AtDFV1 is related to the family Deltaflexivirus. In this study, we identified viruses in Alternaria spp., for the first time, using total RNA sequencing. These results significantly expanded the number of Alternaria viruses and revealed the abundant diversity of the mycoviruses. In this study, we followed a metatranscriptomics approach to identify the mycovirus communities of six species of the genus Alternaria spp. from among isolated strains of pear black spot samples. We collected these samples from the primary among the 10 provinces that produce pears in China [We selected 78 strains of in China . We usedin China . Table 1w/v). Then, we determined the concentration and selected qualified samples for high-throughput sequencing.We prepared total RNA for high-throughput sRNA sequencing. We cultured 78 strains on cellophane membranes that overloaded PDA plates at 28 °C for 7 days. By mixing the mycelia in equal proportion, we ground the samples using liquid nitrogen. We used a TRIzol RNA extraction kit  to extract the total RNA, which we then clarified with chloroform in 2 mL tubes. We used ethanol precipitation to obtain the total nucleic acid fractions. We used 75% alcohol to wash the sample two times, and then dissolved them in water treated with diethylpyrocarbonate (DEPC). The total RNA was detected by agarose gel electrophoresis with 1.2% , where it was constructed and sequenced for cDNA library. First, rRNA was removed, and then double-stranded cDNAs were synthesized using random hexamers (N6). The cDNA ends were repaired, and the A-tail was added and sequenced. The final library was obtained using the polymerase chain reaction (PCR). Then, the quality of the library was tested and sequenced using an Illumina HiSeq XTen platform.A certain proportion of low-quality data is inevitable when obtaining raw data through sequencing. To ensure the reliability and accuracy of the analysis results, we had to preprocess the raw data, which were strictly controlled, and filtered as follows: we first removed the reads using adaptors, and then removed the low-quality reads for clean data. We analyzed the potential sequences of mycoviruses according to the splicing and assembly of clean data.Escherichia coli DH5α. We conduct the sequencing at Sangon Biotech Co. .We isolated the total RNA of 78 strains and used reverse transcription polymerase chain reaction (RT-PCR) to investigate viruses in the fungal strains using specific primers, which were designed based on the assembled contigs . For cDNE. coli DH5α. The sequencing was completed at Sangon Biotech Co. We determined three or more independent clones for each product in both orientations.We used RT-PCR to obtain full-length sequences of the viruses. We determined virus sequences using primers designed from the data obtained from the assembled contigs . We alsohttps://www.ncbi.nlm.nih.gov (accessed on 22 October 2022)). According to the alignment information, the viruses related information  was preliminarily confirmed. We used DNAMAN  to conduct the sequence assembly. We used the ORF Finder website (http://www.ncbi.nlm.nih.gov/gorf/gorf.html (accessed on 22 October 2022)) to predict ORFs. We searched the CDD database (http://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb. cgi (accessed on 22 October 2022)) and the Pfam database (http://pfam.xfam.org/ (accessed on 22 October 2022)) to predict the conserved domains in sequences. We used MEGA 10 and applied the maximum likelihood method to construct the phylogenetic trees, which we tested with 1000 bootstrap replicates. We used MAFFT (http://www.ebi.ac.uk/Tools/msa/maft/ (accessed on 22 October 2022)) to conduct multiple sequence alignments of proteins encoded by the contigs and the reference mycovirus.We calculated sequence similarities using BLAST program on the NCBI database  had a large ORF , which encoded a 4272 aa protein. We found that this predicted amino acid sequence best resembled the RdRp of Alternaria alternata hypovirus 1  and had a 97.89% homology  had a large ORF (72–2048 nt) that encoded a 658 aa protein. We found that a predicted amino acid sequence of the protein best resembled the polyprotein of Alternaria arborescens mitovirus 1 , which had a 91.04% homology. Therefore, this virus was a strain of AaMV1 that belonged to the genus Duamitovirus in the family Mitoviridae  had a 98.04% homology with the RdRp of Neofusicoccum parvum narnavirus 2 , which meant this virus was a strain of NpNV2 and belonged to the genus Narnavirus. The results of RT-PCR revealed that A. tenuissima strain AH-29 harbored this virus and was called Alternaria tenuissima narnavirus 1 (AtNV1) ( (AtNV1) .Botoulivirus, Betabotoulivirus, Magoulivirus, Penoulivirus, Ourmiavirus, Rhizoulivirus, Betarhizoulivirus, Scleroulivirus, Betascleroulivirus, Deltascleroulivirus, Gammascleroulivirus, and Epsilonscleroulivirus are in the family Botourmiaviridae. In this study, eight of the obtained contigs were homologous with numbers of this family, including two magouliviruses, one ourmiavirus, one botoulivirus, one betabotoulivirus, one betascleroulivirus, one deltascleroulivirus, and one unclassified . AtOLV1 was also detected in A. arborescens strain KEL-4-4 and A. gossypina strain SC-16 . According to the BLASTx result, contig 4360 (2324 nt) best resembled the RdRp of Plasmopara viticola associated ourmia-like virus 37  at 94.75% similarity . According to the BLASTx result, contig 2521 (2918 nt) best resembled the RdRp of Penicillium sumatrense ourmia-like virus 1  at 86.38% similarity . AtOLV5 was also detected in A. gaisen strain AH-20 .The BLASTx results showed that contig 73 (8352 nt) was similar to RdRps of Agrostis stolonifera deltaflexivirus 1 , Alternaria alternata deltaflexivirus 1 , Erysiphe necator associated deltaflexivirus 1 , and Triticum polonicum deltaflexivirus 1 . Contig 73 was identified from a We obtained the cDNA sequence of the AtDFV1 segment by combining the contig 73, RT-PCR, and RLM-RACE sequences. The full-length cDNAs of AtDFV1 were 8402 nt, excluding the ploy(A), which had a GC content of 51.4%. According to the BLASTn, this sequence best resembled segment Agrostis stolonifera deltaflexivirus 1, and had a 93.47% homology . We found that the AtDFV1 5′- and 3′-untranslated regions (UTR) were 19 nt and 146 nt long, respectively. The AtDFV1 contains four ORFs (I-IV). ORFs I and II were arranged in a line on the genome, and ORFs III and IV had 55 nt overlaps A.−31), viral RNA helicase , RNA-dependent RNA polymerase , and protein of unknown function . Therefore, ORF I encoded the viral RdRp with a methyltransferase and viral RNA helicase (−10). We also found that ORF III (7290–7823 nt) encoded a protein (P3) that had 177 aa residues and a mass of 18 kDa. According to BLASTp, the P3 protein was exactly the same as hypothetical protein 3 (HP3) of AsDFV1 . We did not find any motifs in the P3 amino acid sequence. We observed that ORF IV (7780–8256 nt) encoded a protein (P4) with 158 aa residues and a mass of 16 kDa. According to BLASTp, the P4 protein was 97.47% similar to a hypothetical protein 4 (HP4) of AsDFV1 . We did not find any motifs in the P4 amino acid sequence. We deposited the corresponding sequences in GenBank (accession: ON263576).We determined that ORF I (20–6241 nt) encoded a protein (P1) that had 2073 amino acid (aa) residues and a mass of 232 kDa. According to BLASTp, the P1 protein had 98.74% homology with the RdRp of Agrostis stolonifera deltaflexivirus 1 (AsDFV1) . The P1 protein also had high homology with the RdRp of some deltaflexivirus see . In addihelicase A. We obshelicase B–D. ORF Alphaflexiviridae, Deltaflexiviridae, and Gammaflexiviridae. We verified the existence of motifs I-VI in AtDFV1 and members of the deltaflexivirus family due to the amino acid alignment with the predicted RdRp .According to the BLASTx results, four contigs were homologous at 96.90–100.00% with the corresponding proteins encoded by Alternaria alternata chrysovirus 1 (AaCV1), which was a betachrysovirus in the family oviridae . The BLAPartitiviridae contains five genera, namely, Alphapartitivirus, Betapartitivirus, Cryspovirus, Deltapartitivirus, and Gammapartitivirus. Contig 11,000 (1393 bp) had 99.05% homology with a coat protein of Alternaria dianthicola partitivirus 1  . Contig 7155 (1808 bp) shared 89.38% similarity with a dsRNA1 segment of Alternaria tenuissima partitivirus 1 . According to the BLASTx result, contig 7155 best resembled the RdRp of AtPV1 with 94.59% similarity .At present, the family YZ32457) . Therefomilarity . TherefoBotybirnavirus presently includes 21 members. BLASTn searches revealed that two contigs were notable for regions with very strong similarity to viral cap-pol fusion protein gene and hypothetical protein gene of Botryosphaeria dothidea botybirnavirus 1 (BdBRV1). BLASTx searches showed that the contig 206 (6162 bp) sequence shared high similarity with the cap-pol fusion protein gene of BdBRV1 , Bipolaris maydis botybirnavirus 1 , and Sclerotinia sclerotiorum botybirnavirus 4 . The BLASTx searches showed that contig 253 (5808 bp) best resembled the hypothetical protein of BdBRV1 , BmBRV1 , and SsBRV4 . Thus, these contigs might represent a virus that was a new strain of BmBRV1. According to the RT-PCR results, this virus was detected in A. tenuissima strains G-21-2 and GZ-2. Therefore, we called it Alternaria tenuissima botybirnavirus 1 (AtBRV1).The genus A. arborescens strain KEL-4-4 and called it Alternaria arborescens dsRNA virus 1 (AaRV1).The BLASTn results revealed that the contig 10,828 (1417 bp) and contig 13,903 (1210 bp) were notable for regions that had a very strong similarity to the sequences of Alternaria longipes dsRNA virus 1 . According to BLASTx, contig 10,828 best resembled the hypothetical protein of AlRV1 with 97.96% homology and contig 13,903 best resembled the RdRp of AlRV1 with 97.84% homology . TherefoTherefore, we determined that dsRNA viruses in the pooled RNA-Seq sample were not new mycoviruses.Mymonaviridea, and two were assigned to the family Discoviridae .The BLASTx searches revealed that contig 52 (8970 nt) shared 56.36% homology with RdRp of Cryphonectria parasitica sclerotimonavirus 1 . We used RT-PCR with specific primers to investigate the tested strains and found that strain HB-15 harbored contig 52 virus . Therefo−11). Furthermore, we detected a type II toxin-antitoxin system motif from amino acids 2812 to 2902  and a L protein N-terminus motif from 2812 aa to 2902 aa  in the hypothetical protein. According to BLASTp, the protein had low homology with the RdRp of Botrytis cinerea negative stranded RNA virus 10 (BcNSRV10) with only 30.71% similarity, and Macrophomina phaseolina negative-stranded RNA virus 1 (MpNSRV1) had only 32.24% similarity. As shown in A. tenuissima strain HB-15 and called this novel virus Alternaria tenuissima negative-stranded RNA virus 4 (AtNSRV4). Contig 192 (6528 nt) contained one large ORF (54–6473 nt) that encoded a 2139 aa protein. According to the motif scan results, this protein contained a conservative Bunyavirus RNA-dependent RNA polymerase domain from 545 aa to 1224 aa. In addition, we detected a Saccharopine dehydrogenase C-terminal domain from amino acids 1938 to 2012  in the hypothetical protein. According to BLASTp, this protein had low homology with the RdRp of Coniothyrium diplodiella negative-stranded RNA virus 1 (CdNSRV1) with 68.79% similarity. As shown in A. arborescens strain KEL-4-4 and called the novel virus Alternaria arborescens negative-stranded RNA virus 1 (AaNSRV1).We found that contig 35  had one large ORF  that encoded a 3410 aa protein. According to the motif scan results, this protein contained a conservative Bunyavirus RNA-dependent RNA polymerase domain from 1886 aa to 2499 aa  were 300 nt and 85 nt long, respectively. We predicted that the full length of the AtNSRV2 had five major ORFs (ORFs I-V). These non-overlapping ORFs were arranged in a line along the viral genome A. We als−6) and a mononegavirales RdRp domain . Based on the multiple alignment of the sequences of the RdRp amino acid of AtNSRV2 and other related viruses from Sclerotimonavirus, we identified four conserved motifs (I–IV)  (GenBank: QJW39407). We also obtained three motifs from the hypothetical protein encoded ORF I, including Family of unknown function , LRRC37A/B like protein 1 C-terminal domain , and LMBR1-like membrane protein . We found that ORF II encoded a protein of 402 amino acid residues and had a mass of 44 kDa. According to BLASTp, it was 47.46% similar to the N protein (nucleoprotein) of the SsNSRV-1 (GenBank: YP_009094314). We discovered that ORF III encoded a protein of 52 amino acid residues and had a mass of 6 kDa. We did not find any significant similarity protein sequences in the BLASTp search. We found that ORF IV encoded the largest protein of 1931 amino acids in length and had a molecular mass of 220 kDa. According to BLASTp, the L protein was 56.48% similar to the RdRp of CpSMV1 (GenBank: QMP84020). The P4 protein of AtNSRV2 also had high homology with the RdRp of other −ssRNA mycoviruses see . The cons (I–IV) C. We fouMymonaviridae and Discoviridae. We found that AtNSRV2 formed a supported clade with the following viruses: Soybean leaf-associated negative-stranded RNA virus 1 (SlaNSRV-1), Fusarium gramineartm negative-stranded RNA virus 1 (FgNSRV1), AtNRV1, BcNSRV3, SlaNSRV2, and CpSMV1 , which were classed a genus Orthodiscovirus in the family Discoviridae. We also included AtNSRV4 in a clade with the members in the family Discoviridae, but we considered it to be part of different groups. Therefore, AaNSRV1 and AtNSRV4 are in two highly supported groups inside the family Discoviridae  was found in Bipolaris maydis and Botryosphaeria dothidea [Sclerotinia homoeocarpa and Ophiostoma novo-ulmi [Helminthosporium victoriae and Bipolaris maydis [Hymenoscyphus fraxineus and H. albidus [Botrytis cinerea and B. fuckeliana [B. cinerea and S. sclerotiorum [S. sclerotiorum and B. cinerea are necrotrophic fungi. They have wide hosts ranges, and their genomes show high sequence identity as well as a similar arrangement of genes [Lycoriella ingenua, which could act as a transmission vector [Nicotiana tabacum, which is a model plant [Viruses are commonly present in longipes ,34,39,40dothidea . Ophiostovo-ulmi , Previous maydis ,63, and  albidus ,65. Anot albidus  and Botrof genes . Given tn vector . Cryphonel plant . Plant-fA. tenuissima strain G-21-2 contained four viruses that belonged to four distinct lineages. Moreover, five strains showed coinfection by various viruses. Plant pathogenic fungal species often experience coinfection with multiple mycoviruses [R. solani isolate DC-17 harbored 17 different mycovirus species that were assigned to eight or more families [Fusarium mangiferae strain SP1 was coinfected by 11 mycoviruses that belonged to three families [Picoa juniperi using high-throughput sequencing [B. dothidea [B. cinerea [Magnaporthe oryzae [R. necatrix [Rhizoctonia solani [Sphaeropsis sapinea [S. sclerotiorum [S. nivalis [We used RT-PCR and confirmed a variety of viruses in each strain. Our results identified 22 strains that were infected by various viruses. Interestingly, the oviruses . For exafamilies . In addifamilies . Sahin equencing . Similardothidea ,74, B. c cinerea , Magnapoe oryzae , R. necanecatrix , Rhizocta solani , Sphaero sapinea , S. sclerotiorum ,81,82, a nivalis . TherefoColletotrichum truncatum, Diaporthe longicolla, Macrophomina phaseolina, R. solani, and S. sclerotiorum [R. solani isolate DC-17 [F. mangiferae strain SP1[P. juniperi were found using high-throughput sequencing [S. sclerotiorum within a single field for three years were investigated using the metatranscriptomic approach [Recently, deep sequencing has been used to identify the diversity of fungal viruses within fungal species from diverse geographic regions ,88,89,90rotiorum . Multiplte DC-17 . Eleven train SP1. Eight nquencing . In addiquencing ,85. For quencing . The divapproach .Alternaria spp. have been reported to have several RNA viruses, including dsRNA viruses, positive-sense ssRNA viruses, and other unidentified viruses [Mymonaviridae [A. tenuissima and A. dianthicola, respectively [Sclerotimonavirus in the family Mymonaviridae. The genome of the virus AtNSRV2 also was obtained. According to BLASTp, the L protein of AtNSRV2 was similar to the RdRp of SlaNRV2 with 55.85% identity. The virus SlaNRV2 was assembled from soybean leaf metatranscriptomes [Discoviridae. More research is needed to address these differences and to confirm their molecular and biological characterization. A lot of −ssRNA mycoviruses were classed into the order Discoviridae [Alternaria strain. viruses ,43,44,45aviridae . Recentlriptomes . AaNSRV1oviridae . This isAlternaria. We excavated at least 21 different mycoviruses. The +ssRNA viruses belonged to the families Mitoviridae, Narnaviridae, Deltaflexiviridae, Hypoviridae, and Botourmiaviridae. The dsRNA viruses belonged to the families Chrysoviridae, Partitiviridae, and a genus Botybirnavirus, and one unclassified virus. The −ssRNA viruses belonged to the families Mymonaviridae of order Mononegavirales, and Discoviridae of order Bunyavirales. We also identified near-full-length sequences of mycoviral genomes that are putative. We isolated a novel −ssRNA mycovirus from an A. tenuissima strain HB-15, which we designated as AtNSRV2. We also characterized a novel +ssRNA mycovirus from an A. tenuissima strain SC-8, which we designated as AtDFV1. Through phylogenetic and sequence analyses, we found AtNSRV2 to be related to the viruses of the genus Sclerotimonavirus in the family Mymonaviridae. We also found that AtDFV1 is related to the family Deltaflexivirus. The results of this study significantly enhanced the number of Alternaria viruses and identified their abundant diversity.In conclusion, diversity analysis of mycoviruses from 78 strains was executed by using high-throughput sequencing technology. The used stains were collected from 10 pear production areas and belonged to six species of the genus


Non-human primates (NHP) share a close relationship with humans due to a genetic homology of 75–98.5%. NHP and humans have highly similar tissue structures, immunity, physiology, and metabolism and thus often can act as hosts to the same pathogens. Agriculture, meat consumption habits, tourism development, religious beliefs, and biological research have led to more extensive and frequent contact between NHPs and humans. Deadly viruses, such as rabies virus, herpes B virus, Marburg virus, Ebola virus, human immunodeficiency virus, and monkeypox virus can be transferred from NHP to humans. Similarly, herpes simplex virus, influenza virus, and yellow fever virus can be transmitted to NHP from humans. Infectious pathogens, including viruses, bacteria, and parasites, can affect the health of both primates and humans. A vast number of NHP-carrying pathogens exhibit a risk of transmission to humans. Therefore, zoonotic infectious diseases should be evaluated in future research. This article reviews the research evidence, diagnostic methods, prevention, and treatment measures that may be useful in limiting the spread of several common viral pathogens via NHP and providing ideas for preventing zoonotic diseases with epidemic potential.  Over the past few decades, the frequency of interaction between non-human primates (NHP) and humans has increased, breaching the natural boundaries between humans and animals and increasing the risks of zoonotic transmission and pandemics . Humans Global public health faces increasing challenges, as “new infectious diseases are emerging constantly, and old infectious diseases are emerging one after another” . The COVGiven the diversity of these pathogens and their ability to infect hosts, bidirectional transmission may also occur . HaltingNew zoonotic and fatal pathogens with high epidemic potential and mortality have threatened global health security for centuries. Therefore, it is necessary to systematically understand infectious pathogens carried by NHP and their research status, diagnostic methods, prevention, and treatment measures to prevent outbreaks of human-infectious diseases .Macaca fascicularis, Cercocebus atys, Pongo pygmaeus, and Pan troglodytes can be infected with MPXV, whereas among the New World NHP, Callithrix jacchus is susceptible to MPXV infection [The MPXV is a zoonotic orthopoxvirus that causes mpox disease, which is similar to smallpox but with a milder rash and lower mortality. The presence of lymphadenopathy in MPXV is a striking clinical feature that distinguishes it from smallpox . MPXV canfection .https://www.cdc.gov/) accessed on 6 January 2023  belongs to the family Herpesviridae and the genus Herpes simplex virus in the Alpha-herpesviruses subfamily . Rhesus Simian cytomegalovirus (SCMV) belongs to the Herpesviridae family of beta-herpesvirus. Rhesus monkeys are the only host of SCMV, and humans and other animals are not susceptible to infection with this virus . AlthougSimian immunodeficiency virus (SIV) can cause immunodeficiency in macaques. African primates are the natural hosts of SIV. Experimental infection of non-natural hosts, such as rhesus monkeys, led to the suppression of the host immune responses, subsequently resulting in simian-acquired immunodeficiency syndrome . SIV is Del-taretrovirus genus [Pan paniscus) housed in zoos in primate centers in Europe and the United States. NHP is also a natural host for STLV-I [Macaca radiata), and then determined the complete sequences and performed phylogenetic analysis. Interestingly, Mra18C9 lacks three auxiliary open reading frames; therefore, to determine whether deletion of the auxiliary protein is specific to this strain, the complete STLV-1 genome available from GenBank was analyzed. The results showed STLV-1 commonly lacks one or multiple secondary open reading frames [Simian T-cell leukemia virus type 1 (STLV-1) was first discovered in Japanese macaques in 1982 and classified as belonging to the us genus . Accordius genus . Notablyus genus . In contr STLV-I . Howeverg frames . Akari eg frames . Co-infeg frames . STLV ing frames . A commeg frames . EpidemiBeta-retrovirus. Depending on the host species, virus, sex, and various environmental factors, the symptoms of infection may be absent, mild, or even severe enough to produce fatal simian-acquired immunodeficiency syndrome [Propithecus coquereli) was reported in 2021. The complete genome sequence of the retrovirus was identified, characterized, and named as prosimian SRV type Ⅰ [Simian type D retrovirus (SRV) is an enveloped, single-stranded RNA virus belonging to the genus syndrome . Owing tsyndrome . Two cassyndrome . A novelV type Ⅰ . AdditioV type Ⅰ . Zhu et V type Ⅰ . Togami V type Ⅰ . Human iV type Ⅰ ; however+ and CD8+ T cells, as well as in B cells in vivo, and it was previously thought that peripheral blood mononuclear cells are the sites of latency of foamy viruses [SFV is also a simian retrovirus and has been reported in 1–4% of people working with NHP in zoos, primate centers, and laboratories and in approximately 24% of staff who have been bitten or scratched by gorillas or chimpanzees . SFV co- viruses .EBOV infection can lead to Ebola hemorrhagic fever, which is one of the most virulent zoonotic infectious diseases, with a mortality rate of as high as 90% . In OctoSHFV is a severe and highly lethal infectious disease that can infect captive macaques. The disease is only transmitted among macaques and is characterized by hemorrhage and fever. Other species of monkeys are chronically infected with the virus but do not develop the disease. The clinical signs and disease course closely resemble those of other hemorrhagic fever virus infections, such as Marburg, Ebola Zaire, and Lassa virus infection in macaques . The RNASHFV causes lethal hemorrhagic fever and induces a strong inflammatory response in macaques but causes asymptomatic persistent infection in baboons. Margo et al. isolated SHFV, which induces hemorrhagic fever in Japanese macaques, from persistently infected baboons . In addiSHFV can be clinically diagnosed based on characteristic clinical symptoms, pathological changes, and epidemiological characteristics combined with serological, histopathological, and molecular biological tests. The feeding and management of monkeys and environmental disinfection should be strengthened to prevent mechanical transmission by personnel, equipment, rodents, insects, and other vectors. Currently, there are no specific and effective vaccines and treatments for SHFV infection .SADV can produce various symptoms, including pneumonia, pharyngitis, gastroenteritis, and conjunctivitis. It is mainly transmitted via the fecal–oral route and primarily infects primates, such as macaques, vervet monkeys, African green monkeys, chimpanzees, and baboons . AdenoviSPV is a single-stranded DNA virus recently identified in cynomolgus monkeys with severe anemia , althougUnder normal circumstances, animals mostly have a latent infection without corresponding clinical symptoms and do not require treatment. However, blood transfusions can be performed for patients with clinically severe anemia or in experimental animals with severe anemia due to immunosuppressive therapy. General treatment and supportive therapy such as fluid replacement and antiviral methods can be used to treat the infection. SPV is an important pathogen in surgically manipulated cynomolgus monkeys, particularly in those that are immunocompromised. Clinically asymptomatic SPV infections are readily transmitted in the environment once colonies are introduced, and the latent virus can be transmitted through organs . SPV canDue to the emergence of acquired immunodeficiency syndrome as well as the recent outbreak of monkeypox, it is imperative to understand the risks of the potential transmission and spread of emerging infectious diseases from NHP to humans. In case of unclear threats, it is necessary to determine which behaviors increase exposure, the types of pathogens that have crossed the species barrier, the importance of this risk to health following infection or frequent contact with NHP, and the appropriate measures. To this end, the habitats of NHP should be protected, and policies and regulations should be issued to restrict the consumption and trading of meat from NHP and to strengthen the inspection and quarantine of experimental animals. Furthermore, self-protection must be emphasized for susceptible animal husbandry managers, tourists, clinical veterinarians, and researchers. Additionally, public health professionals, national authorities, and the media should more readily divulge information to the general public to raise awareness. Active monitoring and health assessments of NHP populations are also necessary. The diagnosis of NHP viruses relies on the combined use of multiple detection methods; there are no effective vaccines or specific therapeutic drugs for most NHP viruses. The pathogens are not well-understood, and the existing data are limited and outdated. To effectively reduce the risk of zoonosis transmission, it is essential to strengthen prevention, monitoring, and international cooperation. Additionally, research and the development of specific vaccines and drugs should be strengthened. Moreover, further studies are urgently needed to determine the mechanisms of replication, infection, and transmission of pathogens of infectious diseases in NHP to prevent outbreaks in humans. In summary, our review of the updated research evidence, diagnostic methods, prevention, and treatment measures, may be useful for limiting the spread of several common viral pathogens by NHP and providing ideas for preventing zoonotic diseases with epidemic potential.