In conclusion, common childhood illnesses are associated with significant health-related costs, which can potentially be reduced by targeting perinatal risk factors, including maternal diet during pregnancy, cesarean delivery, preterm birth and low birth weight.Childhood illness is extremely common and imposes a considerable economic burden on society. We aimed to quantify the overall economic burden of childhood illness in the first three years of life and the impact of environmental risk factors. The study is based on the prospective, clinical mother–child cohort Copenhagen Prospective Studies on Asthma in Childhood (COPSAC2010) of 700 children with embedded randomized trials of fish-oil and vitamin D supplementations during pregnancy. First, descriptive analyses were performed on the total costs of illness, defined as both the direct costs  and the indirect costs (lost earnings) collected from the Danish National Health Registries. Thereafter, linear regression analyses on log-transformed costs were used to investigate environmental determinants of the costs of illness. The median standardized total cost of illness at age 0–3 years among the 559 children eligible for analyses was EUR 14,061 . The exposures associated with reduced costs were fish-oil supplementation during pregnancy (adjusted geometric mean ratio (GMR) 0.89 ,  Childhood illness is very common in the general population, particularly in the first three years of life . The majThere are a few known modifiable risk factors that increase childhood illness in general, which can be targeted to reduce costs. Cesarean delivery is one such risk factor, which has increased due to changes in obstetrical practices, medical risk profiles and increasing “maternal request”. Children delivered by cesarean section have an increased risk of a range of immune-related diseases such as asthma, inflammatory bowel disease, leukaemia, and immune deficiencies , which mWe have previously shown in a randomized controlled trial (RCT) that maternal fish-oil supplementation in pregnancy reduces the risk of asthma and lower respiratory tract infections in childhood ,9. LikewThe aim of this study was to estimate the overall economic burden on society caused by childhood illness the first three years of life by using data from a prospective, clinical population-based mother–child cohort study. Secondly, to investigate whether a range of early life environmental risk factors, including the effects of dietary supplements with vitamin D and fish-oil in pregnancy are related to the costs of illness in early childhood in order to identify targets to reduce costs of childhood illness.2010) is a longitudinal, population-based mother–child cohort study of 700 Danish children born from 2009 to 2011. The mothers were included during pregnancy and the children were followed closely from birth with scheduled clinical visits as well as acute visits for any acute airway and/or skin symptoms. The cohort study design and baseline characteristics have previously been thoroughly described [COpenhagen Prospective Study on Asthma in Childhood  were included in the estimation.The costs of illness were investigated as “direct costs”, “observed total costs” and “standardized total costs”: “Direct costs” were defined as the direct medical expenditures associated with contact with the healthcare system, including payments for hospital outpatient services, hospital inpatient stays, emergency department visits, physician and facility payments. Data were retrieved from the Danish National Health Registers  Mothers were assigned to a daily supplement of either 2.4 g n-3 LCPUFA (fish-oil) or control (olive oil) [Two double-blind RCTs were conducted in the COPSACive oil) . (2) Motive oil) ,10. Both2010 study, see http://copsac.com/available-data/ (accessed 20 February 2020). This includes information on a wide range of environmental and constitutional factors collected prospectively [Highly detailed risk factor assessment is a key feature of the COPSACectively . On the ectively ,23,24,25Descriptive statistics of the costs of child illness and the number of days with parental work absenteeism due to child illness were performed. The incidences of disease during the first three years of life were estimated by ICD-10 group. Differences in baseline characteristics between groups were compared with Chi-squared test for categorical variables and Student’s t-test for continuous variables.The effects of the pregnancy interventions with fish-oil and high-dose vitamin D on the costs of child illness were investigated with linear regression models on log-transformed costs of illness , estimated as crude Geometric Mean Ratios (GMR). The effects of the interventions on number of days absent from daycare were investigated with Poisson regression models estimated as Odds Ratios (OR).Thereafter, linear regression models on the log-transformed costs adjusted for the fish-oil intervention were used to investigate associations between environmental factors and costs of illness for each environmental factor. Poisson models adjusted for the fish-oil intervention were used for associations between environmental factors and daycare absenteeism. A multivariate model excluding collinear factors was fitted for all factors, which were significantly associated with more than one type of costs. Additionally, the actual costs were calculated based on the estimates and geometric mean (GM). For parental work absenteeism, the costs were standardized based on average daily salaries in three countries: Denmark, the United Kingdom and the USA.We performed a subanalysis excluding children of mothers who received active fish-oil supplementation, previously shown to be significantly associated with childhood illness , for genA significance level of 0.05 and 95% confidence intervals were used unless otherwise stated. Missing data were treated as missing observations. All statistics were performed with R (version 3.5.1 (2 July 2018)). All results are stated in EUR 2017.2010 mother–child cohort, we excluded children who did not complete the first 3 years of the study, children with less than 90% of the daily diary registrations and children not registered in the National Danish Register, leaving 559 (80%) eligible children for analysis  whose mothers did not receive fish-oil supplementation the costs were slightly higher: observed total costs, EUR 11,987; direct costs, EUR 4372; and standardized total costs, EUR 14,061, respectively. Excluding children (n = 158) whose mothers received high-dose vitamin D supplementation resulted in similar estimates: observed total costs, EUR 11,469; direct costs, EUR 4116; and standardized total costs: EUR 13,553.The crude median number of days absent from daycare due to illness was 24 days (IQR 15–37). When accounting for time enrolled in daycare, the median number of days was 12 days per year in day care (IQR 7–17). In the subgroup of children whose mothers did not receive fish-oil supplementation the median number of days absent was 26 days (IQR 16–39). Excluding children whose mothers received high-dose vitamin D supplementation resulted in a similar estimate of 26 days (IQR 14–41).p = 0.021) and showed a trend of association with lower direct costs , p = 0.09) and number of days absent from daycare , p = 0.07). There were no associations between high-dose vitamin D supplementation in pregnancy and costs or number of days absent from daycare . The baseline characteristics of the four intervention groups are summarized in Fish-oil supplementation in pregnancy was associated with lower total costs during the first three years of life , p = 0.001, whereas sex remained significantly associated only with the total costs: standardized total costs: aGMR = 1.15 , p = 0.006. Due to the strong collinearity between GA and birth weight (r = 0.65), each of these variables were excluded when evaluating the other. Both remained significant for all three types of costs, e.g., aGMR estimates for standardized total cost were 0.94  and 0.98  per week GA and per 100 g birth weight, respectively. There were no significant associations with birth weight when adjusting for GA (data not shown).We performed a multivariate analysis including the variables: cesarean delivery, GA, birth weight, sex, and fish-oil supplementation on the costs of illness . In this model, cesarean delivery remained significant for all types of costs, e.g., standardized total costs: aGMR = 1.24 , p < 0.001; and direct costs: aGMR = 1.25, , p = 0.017. Excluding costs of hospitalizations , p = 0.011; and direct costs: aGMR = 1.17, , p = 0.037) or excluding children born before GA 37 weeks from the analyses did not change the associations , p = 0.003; and direct costs: aGMR = 1.27 , p = 0.013). Excluding children with asthmatic symptoms in the first three years of life also did not change the associations: standardized total costs: aGMR = 1.28 , p < 0.001; and direct costs: aGMR = 1.37 , p = 0.002. Stratifying the analyses by the first, second or third year of life did not alter the results , p = 0.92). A total of 110 (20%) children were born by cesarean delivery, of which 49 (45%) were planned procedures. The association between cesarean delivery and the costs of illness remained significant after adjustment for GA, birth weight and fish-oil supplementation: standardized total costs: aGMR = 1.24 ,  results . There wWe found that the median observed total cost of illness in the first three years of life among 559 unselected, population-based Danish children was EUR 10,932 and the median number of days absent from daycare was 24. Birth by cesarean delivery was associated with 30–45% higher costs and a 17% higher number of days absent from daycare due to illness. Cesarean delivery was significantly associated with all measures of health costs, both direct and indirect, and the association persisted throughout all three study years. Male sex, low GA and low birth weight were also associated with higher costs of child illness, whereas fish-oil supplementation during pregnancy was associated with 12% lower health-related costs in the offspring during the first three years of life. These findings may be useful for developing strategies to avoid or reduce costs of childhood illness.A major strength of the study is the longitudinal, prospective clinical surveillance of a mother–child cohort at the COPSAC clinical research unit. The study is a single-center study with assessments by experienced study-physicians, examining the cohort children and obtaining the clinical history based on standard operating procedures supported by daily diary cards. We only included children with at least 90% of days actively observed. This method assures a high sensitivity, consistency in procedures, uniform case definitions, secure data capture methods, and reduced risk of misclassification.The data source for assessing costs is unique due to the possibility of linking data from national registries to each cohort participant via the Central Person Registration number, which is assigned to all persons with a permanent residence in Denmark. The Danish National Registries contain information on all the population’s contacts with the Danish healthcare system due to the reimbursement system, where the providers are not reimbursed without a correct registration, which strengthens the credibility of the Danish National Registries. The private sector in Denmark is neglectable, particularly regarding children, with no inpatient facilities, thus equalizing the gap in healthcare differences within the population. Still, the costs in the register are average numbers and not calculated for individual hospitalizations; however, we do not expect the numbers to be systematically over- or underestimated.It is a strength that we were able to consider the burden of parental absenteeism as a part of costs related to childhood illness, which are rarely included in cost analyses. We acknowledge that there might be additional intangible costs, such as reduced quality of life, and secondary transmissions of illness from child to parent, which we were not able to include, and the true cost of childhood illness is probably still underestimated.The generalizability of our findings is limited by an unintended recruitment bias towards families with atopic illness, conferring a higher risk of those illnesses in their children. In fact, half of the parents were diagnosed with an atopic illness, which is higher than expected in the population . Still, The level of household income in our study is marginally higher as compared to the general population , which cFinally, the association between fish-oil supplementation during pregnancy and total costs of child illness is specifically strengthened by the RCT study design.We found that fish-oil supplementation from pregnancy week 24 to one week postpartum reduced the costs of illness in early childhood by 12%. This suggests fish-oil supplementation for pregnant women as a simple way to reduce the health-related costs of early childhood illnesses. The estimated health expenditure benefit from fish-oil supplementation of 12% of the median observed total cost corresponds to EUR 1400 per child. When we take into account that 61,397 children were born in Denmark in 2017, this reduction of cost amounts to EUR 85,400,000 yearly. Even though full compliance with such future supplementation strategy cannot be expected from the entire population of pregnant women, this would still have the capacity to significantly reduce the health-related costs of childhood illness worldwide.Children born by cesarean delivery had 30–45% higher costs of illness than children born by vaginal delivery without any differences between planned or acute cesarean delivery. The association between cesarean delivery and increased costs was not only caused by a higher risk of hospitalization perinatally since both total and direct costs of illness without the costs of hospitalizations were significantly associated to the mode of delivery, which persisted during all three years. The association between cesarean delivery and the costs of illness was not mediated by an increased number of infants born prematurely with subsequent increased morbidity, since the associations were still significant when they were adjusted for GA and even when we excluded children born before GA 37 weeks.Cesarean delivery is a known risk factor for childhood asthma (8), and it is therefore plausible that an increased risk of asthma was driving the association between mode of delivery of costs of illness. Other studies have also shown that cesarean delivery is associated with a higher burden of respiratory illness in childhood ,32, whicIt should be noted that our study identifies an association between cesarean delivery and future health costs with no speculations on the exact mechanisms. Our findings could reflect inherent aspects of susceptibility to illness that also increase the risk of cesarean delivery, and that children born by cesarean delivery constitute a preselected group of more vulnerable children. Future clinical studies can hopefully elucidate these associations.The fact that low birth weight and premature birth are also associated with healthcare costs throughout early childhood, independently of delivery mode, is consistent with previous studies demonstrating that preterm infants have a significantly higher financial burden throughout their childhood ,37. The This study provides an overview of the costs of illness in Danish children in general, adding to the current knowledge on the economic burden of childhood illness in affluent countries. The issue about parental absenteeism is regularly a part of the public debate on work–life balance, affecting not only the children and their parents but also the employers, and means by which to alleviate this burden are highly relevant.Common childhood illnesses are associated with significant health-related costs to society. These costs can potentially be reduced by targeting the pregnancy and perinatal period, including supplementing maternal diet with fish-oil, reducing the rate of cesarean delivery by maternal request, and by preventing preterm birth and low birth weight.


Virus disease pandemics and epidemics that occur in the world’s staple food crops pose a major threat to global food security, especially in developing countries with tropical or subtropical climates. Moreover, this threat is escalating rapidly due to increasing difficulties in controlling virus diseases as climate change accelerates and the need to feed the burgeoning global population escalates. One of the main causes of these pandemics and epidemics is the introduction to a new continent of food crops domesticated elsewhere, and their subsequent invasion by damaging virus diseases they never encountered before. This review focusses on providing historical and up-to-date information about pandemics and major epidemics initiated by spillover of indigenous viruses from infected alternative hosts into introduced crops. This spillover requires new encounters at the managed and natural vegetation interface. The principal virus disease pandemic examples described are two  that threaten food security in sub-Saharan Africa (SSA), and one (tomato yellow leaf curl) doing so globally. A further example describes a virus disease pandemic threatening a major plantation crop producing a vital food export for West Africa (cacao swollen shoot). Also described are two examples of major virus disease epidemics that threaten SSA’s food security . In addition, brief accounts are provided of two major maize virus disease epidemics , a major rice disease epidemic (rice hoja blanca in the Americas), and damaging tomato tospovirus and begomovirus disease epidemics of tomato that impair food security in different world regions. For each pandemic or major epidemic, the factors involved in driving its initial emergence, and its subsequent increase in importance and geographical distribution, are explained. Finally, clarification is provided over what needs to be done globally to achieve effective management of severe virus disease pandemics and epidemics initiated by spillover events. Virus disease epidemics and pandemics threaten all types of cultivated plants including those grown to feed the world’s human population and its domestic animals, and others grown for ornamental, fiber or medicinal uses ,4,5,6,7.Development of damaging virus epidemics is favored by the introduction of new crops to parts of the world where they have never been grown before and the adoption of intensive cropping systems both of which lead to new encounters with virulent viruses infecting crops or indigenous vegetation. They are also favored by introduction of vulnerable new cultivars bred for increased yields ,38,39,40Several of the world’s plant virus disease pandemics and major epidemics have resulted from infection with emerging viruses that arose from new encounter situations in which indigenous viruses spread by spillover (= host species jumps) from infected indigenous plants to infect introduced cultivated plants ,19,20,42Pandemics or epidemics occurring in diverse crops and all continents, apart from Antarctica, were documented in a series of reviews written by the late Professor Michael Thresh . These rThis review describes virus disease pandemics and major epidemics that arose from spillover scenarios involving new encounters between indigenous viruses and introduced crops, rather than virus spread from introduced crops to indigenous crops or natural vegetation. It does this by providing historical and up-to-date information on five examples of virus diseases that threaten staple food crops critically important for food security in developing countries, placing special emphasis on the situation in sub-Saharan Africa (SSA). The sixth virus disease example threatens livelihoods in SSA because it devastates production of a valuable food export crop. In addition, brief coverage is provided of several other examples of major virus disease epidemics that arose from new encounters between indigenous viruses and introduced crops important for food security in different parts of the world.In his 1970 review of ‘catastrophic plant diseases’, Klinkowski  emphasizAn emerging virus is usually considered to be “one that causes damaging epidemics but has only evolved or been recognized recently, changed its pathogenesis, increased its host range or increased its geographical distribution” ,55. FurtZea mays), cassava (Manihot esculenta), potato (Solanum tuberosum) and tomato (Solanum lycopersicum) [Selection of local land races of crop plants from wild ancestors commenced more than 10,000 years ago in the worlds’ plant domestication centers ,62. Viruersicum) . In consersicum) ,23,40,52Successful spillover starts with spread of already existing genetic virus variants from a virus infection source plant to the new host plant, and the outcome for each individual variant depends on its relative abilities  to survive once it infects each host, adapt to new hosts or vectors and achieve efficient epidemic spread . A rangeOryza sativa) is a cereal crop domesticated from wild rice in China approximately 10,000 years ago. It soon spread from there to Southeast Asia, the rest of East Asia and the Indian subcontinent, next to the Middle East, Europe and North Africa, and more recently to the Americas and Oceania. Approximately 1000 years ago, it was introduced to East Africa where it was grown in coastal regions. In the second half of the 19th century, it was taken inland to be sown in the rest of East Africa, Central Africa and then taken to West Africa and Madagascar. The inland delta of the upper Niger River was where African rice (Oryza glaberrima) was first domesticated 3000 years ago. It spread gradually from there within West Africa [Asian rice  was first described in 1966 infecting rice plantings in the Lake Victoria region of Kenya in East Africa . This inSobemovirus, family, Sobemoviridae). RYMV infection occurs naturally in cultivated African and Asian rice, the wild rice species O. barthii and O. longistaminata, and the wild grasses Echinocloa colona, Eragrostis atrovirens and Panicum repens [Sesselia pussilla. In addition, it is transmitted by mammals and in irrigation water and soil, but is not seed transmitted to seedlings [The causal agent of RYMD is rice yellow mottle virus . During the period 1983–2012, five further begomoviruses associated with CMD were found in SSA and one in Madagascar. All seven cassava begomoviruses were persistently transmitted by the polyphagous cryptic whitefly complex Bemisia tabaci [Euphorbiaceae or Fabaceae were reported in different parts of mainland SSA [In 1983, the first CMD causal agent was described, African cassava mosaic virus . However, SLCMV is also found in southern India ,80,82. ICBSD was recorded first in 1936 infecting cassava crops in coastal Tanzania . By 1950Ipomovirus; family, Potyviridae) and the closely related Ugandan cassava brown streak virus (UCBSV). The whitefly B. tabaci transmits both semi-persistently [Manihot glaziovii) and the non-cassava wild species Zanha africana and Trichodesma zeylanicum in which CBSV was detected, and the wild cassava species Manihot carthaginensis in which both viruses were found. Whether these species act as virus reservoirs for CBSV and UCBSV spread to cassava crops is unknown but seems plausible [The two CBSD causal agents are cassava brown streak virus , where it was first domesticated from wild tomato species. After spreading north in the Americas in pre-Columbian times, its domestication continued in Mexico. It was taken from there to Europe in the 16th century from where it was later distributed globally . At leasGlobally, the most economically significant tomato virus disease is tomato yellow leaf curl disease (TYLCD) . It occuBegomovirus, family, Geminiviridae) which is persistently transmitted by the whitefly B. tabaci. Tomato is its primary host, but it also naturally infects some alternative hosts sporadically, including common bean (Phaseolus vulgaris), the solanaceous ornamentals petunia (Petunia hybrida) and lisanthus (Eustoma spp.), and several wild tomato species [B. tabaci is a polyphagous supervector that exists as a species complex. Its cryptic species MEAM1 (= biotype B) and MED (= biotype Q) are its most efficient transmitters [B. tabaci transmits TYLCV from infected to healthy plants both locally and, when viruliferous whitefly are blown over greater distances in wind currents. Some strain TYLCV-IL variants may be seed transmitted in tomato [The causal agent of TYLCD is tomato yellow leaf virus  are important for human nutrition and achieving sustainable food production. Their greater use would improve food security considerably not only by improving human and livestock health but also by improving soil fertility through fixing atmospheric nitrogen [Vigna unguiculata), mung bean (Vigna radiata), and soybean (Glycine max), and cool season grain legumes, such as chickpea (Cicer arietinum), faba bean (Vicia faba), field pea (Pisum sativum), lentil (Lens culinaris) and lupin (Lupinus spp.) [Grain legume crops such as peanut  ,133. An Peanut is an important crop that helps to ensure developing country food security. More than 6000 years ago, it was domesticated independently in several locations in South America. It was introduced to SSA in the 16th century . The mosUmbravirus, family, Tombusviridae), groundnut rosette assistor virus  and satellite RNA (sGRV). Aphis craccivora (the cowpea aphid) transmits this tripartite virus complex persistently. It is not seed borne. Presence of GRAV is essential for transmission of GRV and sGRV to occur. Infection with GRAV and GRV but without sGRV fails to elicit any symptoms since sGRV is required for symptom expression [Physalis peruviana and Cassia obtusa are potential alternative hosts for the tripartite virus complex [A. craccivora vectors spread this complex to the peanut crop [A. craccivora vectors and virus reservoirs were most abundant at this stage of the growing season and wide row spacing attracted incoming aphids to land on peanut plants, both of which favored virus spread. For various reasons, GRD control recommendations to sow earlier using narrow row spacing proved too inconvenient to be adopted by the smallholder farmers.GRD is elicited by a tripartite virus complex consisting of groundnut rosette virus  is an evergreen, understory tree indigenous to the Amazonian rain forest in South America. It was introduced to West Africa in the second half of the 19th Century where it was mostly planted in lowland forest areas [Cacao , which is transmitted semi-persistently by several mealybug species. Its most efficient mealybug vectors are Planococcoides njalensis and P. citri. Its alternative natural hosts are five indigenous West African tree species, Adansonia digitata, Ceiba pentandra, Cola gigantean, C. chlamydanta, and Sterculia tragacantha. Its mealybug vectors spread it from infected to healthy trees [C. chlamydanta as the virus source because this tree species is commonly CSSV-infected and colonized by mealybugs, grows in close proximity to cacao plantings and was infected by the same CSSV strain as that found in nearby cacao trees [C. chlamydanta growing near cacao plantings were involved in its emergence, as, where this issue was studied, spread by mealybug vectors possibly occurred to them from infected cacao instead of in the opposite direction [CSSD is caused by cacao swollen shoot virus  and maize rough dwarf virus  spread by their respective hopper vectors from infected Digitaria and other wild grass species. With MSV, its leafhopper vectors Cicadulina mbila and nine other Cicadulina species, and with MRDV, its planthopper vector Laodelphax striatellus. With MSV, this spillover occurred in southern Africa [Maize streak disease (MSD) and maize rough dwarf disease (MRDD) both arose by new encounters at the managed and natural vegetation interface . This wan Africa ,98, but n Africa ,20,94,95n Africa ,20. Currn Africa . Tenuivirus, family, Phenuiviridae) spread by its vector planthopper Sogatodes orizicola from RHBV-infected plants of Echinochloa colona and other wild grasses to rice  arose in northern South America from a new encounter scenario at the managed and natural vegetation interface. It involved spillover of the indigenous rice hoja blanca virus  elicited is considered to have started in the Indian subcontinent. Aphid vectors spread the virus from PRSV-infected cucurbit crop plants already growing there to papaya plants growing in nearby plantations of this introduced tree crop. PRSV’s host adaptation to papaya was attributed to a mutation that enabled cucurbit-adapted PRSV to infect it readily [Instead of becoming infected by indigenous virus spillover occurring directly from local virus-infected native plants, introduced crops can also become invaded indirectly by virus spread from infection reservoirs consisting of infected plants belonging to indigenous crops, crops introduced previously or introduced weeds . For exa readily . Such in readily .Begomovirus, Orthotospovirus and Potyvirus genera [Orthotospovirus, family, Tospoviridae), which is indigenous to the Indian subcontinent and has a wide natural host range. After introduction of the originally South American crop tomato , and at land borders, seaports and airports (border), along with virus eradication and containment programs (post border) . Within regions where a virus disease epidemic is already underway, there is no ‘one-size-fits-all’ approach towards managing its spread within affected farms and fields. What is required to achieve this is devising integrated virus disease management strategies and tactics that suppress its spread most effectively ,38,130. Achieving greater success in managing virus disease pandemics or major epidemics in introduced crops after their initiation by indigenous virus spillover events, requires the strengthening of existing collaborative multidisciplinary research networks developed to address them. Where this is currently absent for a virus disease pandemic currently underway, the creation and fostering of a new collaborative network is warranted. Such multidisciplinary networks require collaboration between developed and developing country researchers, and the participation, amongst others, of specialist plant virologists, entomologists, modelers, agronomists, plant breeders, statisticians, and socioeconomics experts . An examImproving the human food supply by introducing food crops domesticated in one continent to another continent, or to another part of the same continent, has exposed a major drawback to this endeavor. This drawback is the unforeseen development of damaging virus disease pandemics or major epidemics that arise by spillover of indigenous viruses into the introduced crops once they become established in their new surroundings. This review provides graphic examples of the enormous crop damage, gross yield and quality losses, and harm to the dependent human population that result. Most of the affected introduced crop examples described were domesticated in the Americas  and distributed elsewhere in the world as part of the Columbian Exchange, but one of them originated in China (rice), being dispersed from there to other continents. Apart from cacao, these are all staple food crops vitally important for food security in developing countries. Moreover, apart from tomato, all are best suited to growing in regions with tropical or subtropical climates, most of which occur in food insecure parts of the developing world. Tomato not only grows well in such regions but also in warm temperate regions and under protected cropping in cool temperate regions.Historical and up-to-date descriptions are provided of four examples of virus disease pandemics, and two examples of major epidemics, that arose from spillover scenarios involving new encounters between introduced crops and indigenous viruses spreading from infected natural vegetation. Five of these examples concern virus diseases of staple food crops that threaten food security in developing countries. These examples include those caused by CMD, CBSD, GRD, RYMD in SSA, and by TYLVD in all continents, apart from Antarctica. Because it devastates production of a valuable export crop, a sixth example caused by CSSD threatens livelihoods in West Africa. In addition, brief accounts are provided of other major virus disease epidemics arising from spillover scenarios involving new encounters in different parts of the world, namely MSD, MRDD, RHBD and several regional tomato orthotospovirus and begomovirus disease epidemics. For each pandemic or epidemic, the major factors driving its emergence initially, and its sudden increase in importance and geographical distribution subsequently, are explained. All these examples illustrate how spillover event initiated virus diseases epidemics that threaten food security in developing countries, vary greatly. This variation depends upon the characteristics of the causal virus(es), the crop affected, and the diversity of virus transmission modes, disease cycles, epidemiology, agro-ecological production systems and climatic factors involved. Tackling them successfully requires collaboration between policy makers, funding agencies, researchers and extension personnel on intercontinental scales. This scale of activity is needed to obtain a full understanding of each virus–vector–crop pathosystem and its epidemic drivers, and develop effective control measure approaches and extension strategies. Due to the urgent need to feed the growing global population, and address the increasing difficulties in controlling virus diseases of both staple and other food crops effectively as climate change progresses, the importance of the task ahead should not be underestimated.


Anas and Spatula spp.), we investigated juvenile mallards (Anas platyrhyncos) and blue-winged teals (Anas discors) orally inoculated with A/northern pintail/California/44221–761/2006 (H5N9) and the virus titer relationship to occurrence frequency of SAα2,3Gal in the intestines and bursa. To test the natural variation of free-ranging duck populations, birds were hatched and raised in captivity from eggs collected from nests of free-ranging birds in North Dakota, USA. Data generated from qPCR were used to quantify virus titers in cloacal swabs, ileum tissue, and bursa of Fabricius tissue, and lectin histochemistry was used to quantify the occurrence frequency of SAα2,3Gal. Linear mixed models were used to analyze infection status, species, and sex-based differences. Multiple linear regression was used to analyze the relationship between virus titer and SAα2,3Gal occurrence frequency.Individual heterogeneity in pathogen load can affect disease transmission dynamics; therefore, identifying intrinsic factors responsible for variation in pathogen load is necessary for determining which individuals are prone to be most infectious. Because low pathogenic avian influenza viruses (LPAIV) preferentially bind to alpha-2,3 sialic acid receptors  in the intestines and bursa of Fabricius in wild ducks (In mallards, we found high individual variation in virus titers significantly related to high variation of SAα2,3Gal in the ileum. In contrast to mallards, individual variation in teals was minimal and significant relationships between virus titers and SAα2,3Gal were not determined. Collectively, teals had both higher virus titers and a higher occurrence frequency of SAα2,3Gal compared to mallards, which may indicate a positive association between viral load and SAα2,3Gal. Statistically significant differences were observed between infected and control birds indicating that LPAIV infection may influence the occurrence frequency of SAα2,3Gal, or vice versa, but only in specific tissues.The results of this study provide quantitative evidence that SAα2,3Gal abundance is related to LPAIV titers; thus, SAα2,3Gal should be considered a potential intrinsic factor influencing variation in LPAIV load.The online version contains supplementary material available at 10.1186/s12917-020-02642-7. Wild waterfowl are the natural reservoir for avian influenza viruses (AIV) and a source of infection for domestic poultry –3. HighlAnas platyrhynchos), are observed to shed virus with high heterogeneity, where 20% of the birds shed 80% of the total virus shed by all birds  PC.” Each PC variable accounted for greater than 80% of the variation between the cell types of that particular tissue. PC variables generated from the PCA were used in the MLR models to determine the relationship between virus titer and lectin score.Virus titer and lectin score relationship was determined by assessing three different MLR models for each species using virus titer as the dependent variable. The virus titer variable in the first model consisted of virus titers from cloacal swabs collected on the DPI each bird was sacrificed. The second model used virus titers in ileum tissue, and the third model used virus titers in bursa tissue. Independent variables for the cloacal swab virus titer model consisted of the lectin score variables, the principal components described above (when appropriate), and five control variables: sex, BCS, LPAIV treatment group, body mass in grams at 55 days after hatch, and inoculation age in days. Independent variables for the ileum virus titer model included only the ileum lectin score variables and the five control variables. Only the bursa epithelium lectin score variable and the five control variables were included in the bursa virus titer model.2, and satisfactory residual patterns  was chosen as the best fitting model to the data.To determine the best fitting MLR model for each dependent variable, we followed a consistent procedure. Global linear models were tested for each dependent variable separately. To select parsimonious model fits to the data, we used stepwise variable selection based on the generalized Akaike’s Information Criterion (AIC). We then used variance inflation factor (VIF) scores to identify problematic co-linear predictors from the stepwise-chosen models. Independent variables with VIFs > 3.0 were determined problematic and were removed from the model one at a time until all VIFs < 3.0 . When twAdditional file 1. All analyzed data for LPAIV H5N9 infected and control blue-winged teals and mallards. NA indicates missing data.Additional file 2 Mean virus titer + 95% confidence interval for (a) species, (b) days post infection (DPI), and (c) the interaction of species and DPI for mallard and blue-winged teal cloacal swab samples one to five DPI.Additional file 3 Mean virus titer + 95% confidence intervals for  sex, and  the interaction of sex and days post infection (DPI) for male (M) and female (F) mallard and teal blue-winged teal cloacal swab samples one to five DPI.Additional file 4 Mean lectin scores + 95% confidence intervals for intestinal tissues proximal (duodenum and jejunum), ileum, cecum, and colon for LPAIV H5N9 infected male (M) and female (F) mallards and blue-winged teals.Additional file 5. Mallard PCA results for proximal and cecum lectin scores.Additional file 6. Residual Plots and AIC table for mallard cloaca swab virus titer multiple linear regression model.Additional file 7. Residual plots and AIC table for mallard ileum virus titer multiple linear regression model.Additional file 8. Residual plots and AIC table for blue-winged teal cloaca swab virus titer multiple linear regression model.Additional file 9. Quantitative limit validation methods and results.Additional file 10. Residual plots and AIC table for blue-winged teal ileum virus titer multiple linear regression model.Additional file 11. Residual plots and AIC table for blue-winged teal bursa virus titer multiple linear regression model.Additional file 12. Pearson’s r correlation matrix for mallard lectin histochemistry scores.Additional file 13. RTPCR raw data for virus titer quantities.Additional file 14. Lectin score raw data for mallards.Additional file 15. Lectin score raw data for blue-winged teals.


This research tries to shed light on precise values of logistics costs that so far undisclosed. The values are important for every project planners to evaluate, control and monitoring, and especially imperative for procurement activities. Tender, bidding, and defining the scope of contracts and value can be well organized without time-consuming. This research uses methodology previously developed by Subiyanto (2020a) and discussed only a few particular and directly related to the logistics. The model had been decomposed by utilizing path analysis and the data had been regressed to define variables as the most inflator for logistics. The findings are total logistics costs in Indonesia generally at around 14.6% to total costs of investment. It consists following of insurance cost 0.11%, costs for customs clearance 6.52%, foreign logistics costs 6.62%, domestic manufacture 0.47%, and domestic logistics costs 0.89%. This research is not discussing logistics costs relating to human resources, human travelling, and internal logistics conducted by vendors or contractors as the scope included counted within their procurement contract. These results are important as guidance for project planners, controllers, and other parties to prepare costs more precisely. Negotiating to the banks will be easier as the more precise amount of budgets had been estimated. Due to the important values that have been identified, there will help transparency and governance conducting the projects. Procurement processes involving societies are more objective and better. This is a novelty finding as previous researchers have not decided yet. Projects Management; Logistics Costs; Customs Clearance Costs; Foreign Logistics Costs; Domestic Manufacture Costs; Domestic Logistics Costs; Insurance Costs; Economic Development; Pricing; Logistics; Strategic Management; Scale Construction; Business. Starting projects have been marked by groundbreaking ceremony while closing projects would be marked by the closing ceremony. The same couple of ceremonies but at different times with yearly span depend on the size of projects.While project management has few tasks to make final payments, and gradually part of project teams have been transferred as part of operations’ organization at the new plants; by at the time, the important records, histories, achievements, performances, with its numbers are easily gone and evaporated. Physical documents or electronic data are carefully stored as required as part of the final report for final auditing, but evaluating, assessing or making simply comparison against to previous feasibility study is rarely done.The most critical thing, while a different team has been assigned to carry other projects –even the same type of project- the new team will face hardly to refer results previous projects achieved as reference for the new projects.Again, the new team must hire new other consultants, hire several personal experts, rent different tools, and make preparation as like a new team with a new project. Everything has been reset as like a newest and beginning from zero. The amateur-like behavior is totally unproductive and professionalism is highly under questioning.To disclose numbers; moreover, this case is correlating with contract procurement that is generally not common, unusual, and even more, have been campaigning against the laws. Part of the officers states that the contract is hard to define precise numbers. The numbers will be liquid depends on the market. While supply over the demand will suppress the price, and in contrast, the price will be hike while the demand higher over than supply. This culture-type is retaining advanced information of investment costs and particularly logistics costs that to be addressed in this research.Based on the situation, this paper had risen. The aim of this paper is to disclose several values associated with project costs particularly in the terms of logistics activities. Unit costs for even the smallest logistics must be carefully identified to make a greater accurate level of results of this research. Due to the findings obtained, these results are a novelty in logistics, giving a contribution to the knowledge and can be a foundation for further research future.2Estimating precisely construction costs at the general physical projects are always becoming a major tasks for every project planners. Many subjects have been presented by According to A little bit developed research had been conducted by Due to the development of costs in the project that as important as other departments, the procurement department then took bigger portions to contribute. Even more; good procurement will help to minimize failures of overrun costs of projects that traditionally happen in generally countries . The stuDifferent views with However, to precisely determine costs estimation in the projects are remaining mysterious and so far halted by developing model, simulation, and approximately techniques. The earned value management (EVM) method employed from the interval gray numbers (IGNs) has been tested by In terms of logistics costs in the projects, not many researchers have studied this particular theme. A Linear Programming (LP) has been performed by 3This study elaborates on the methodology based on empirical cases in the heaviest several cement projects in Indonesia. The path analysis type is introduced and carried out to get a comprehensive mapping of costs, all data then have been tested by the regression method to get results which the highest inflator among other variables that must be appropriately concerned or prioritized.To convince of the approach; several 8 cement projects were built within period of 2010–2018 have been assessed to get detail results precisely. Data collection had been taken at the whole project during the time construction, be carefully checked and verified daily and sorted in quarterly. Costs associated and incurred to the interest of logistics have been grouped into the category of logistics costs, while costs associated with other interests have been accounted proportionally to other posts.For data total costs of projects are being differently treated, like nature, due to it accumulated not quarterly separated, then for the case is taken by quarterly accumulated and key-in into the spreadsheet. This technique is to avoid autocorrelation but at the same time keeping data between variables free independently.This research utilizing a model developed by Due this research is observing only to discuss associates with the direct logistics costs incurred in every project; therefore, few variables were chosen are (1) insurance; (2) customs clearance; (3) foreign logistics costs; (4) domestic manufacture costs; and (5) domestic logistics costs. To sort of variables considered in this study are of course expert judgements needed. The expert is the only way to decide each factor directly or indirectly associate with logistics.To calculate logistics costs is by measuring real data taken from the projects. Logistics costs every project has been measured, counted, compared against of the total project costs. The bigger logistics costs is showing more inefficient and vice versa the smallest logistics costs is showing better in organizing logistics costs at the projects.44.1To decomposing The tiers are leveling logistics stages identified at the projects. Variables directly related to logistics such as at the portion of foreign logistics is easily identified, but variables correlated came from domestics manufacture must be carefully justified. Domestics manufacturing costs in the case are logistics associated to perform delivery of goods for projects, manufactured origin from the domestics. Movement costs from the originated place to the project site. In the case for example cost delivery of concrete, rebar, plate-works from workshop to project site, bulky materials. This is particular to discuss delivery or logistics costs that generally counted during logistics tenders.Based-on Figures Besides insurance, costs that directly impacted the projects are costs for customs clearance. This activity is mandatory while dealing with import machinery and common in every country worldwide. The State levied a variety of costs in the form of a variety of taxes or others. In this regard are costs of duty, value-added tax, income tax, and some of them are luxury goods tax. In Indonesia costs of duty are at least 5% to 10%, the value-added tax is 10%, income tax is 2.5% for particular conditionals met, and uncertainty of luxury goods tax to about at least 50% to 500%. This research exempted the luxury goods tax as the costs are not applicable for generally manufacture projects in Indonesia. In this case, the mandatory costs of customs clearance are also taking consideration in this research.Further, the costs of logistics come from foreign logistics costs. This stage is delivery activities loading and unloading goods for projects generally machinery from point of origin ports to the destination ports in the country where the projects are carried on. The costs are service costs to pick-up machinery from certain places according to procurement contract, loading to the hatch of vessels, sailing on the seas, unloading to the destination ports, and to hand-over goods according agreed the terms based on certain incoterms applied. It is therefore the variable of foreign logistics costs is taking into consideration.Besides, Finally, as benchmark primarily studied in this research, the dependent variable of total investment is directly accommodated with a little bit of different treatment. Data on total investment is accumulated based on quarterly; therefore, if we can see tabulation the amount of numbers was increasing but in uncertain multiply. It depends on the amount disbursed according to project progress and correlated with achievement quarterly.The direct relevance variables of this research then could be defined as following and described by following 4.2Indonesia is a distinctive archipelago-type of country. Geographically, at least 17,000 islands throughout at the country, majorly each of islands separated by water or sea. Since the biggest percentage of total population approximately 272 million people (2019) domicile in Java Island, all disparities then occurred in our today's life. This is not surprising as to make an equal distribution in all islands are requiring prodigious resources. The development model is applied by the current government is building project infrastructure as it mandatory required, not as wishing.In terms of executing certain physical projects or cement projects, considering the uncertainty of hurdles or obstacles along the logistics routes; therefore, logistics design must be prior determined. Following Few road-type lines must be accommodating by various types of trucks. A route services from certain container-type direct to the project site. A route to service bulky unidentified goods from the center city to the project site, and a route to prepare heavy machines from jetty point to the project site. Within the area of projects itself, there is a single route to carrying concrete from the point of batching plants to the hundred points of constructions or civil foundation.From Figures According to The other obstacles are for example cables that crossing road, banners, billboards, and the most to be early anticipated is the construction of bridges along the roads to the project site. There are two possibilities to strengthen bridges; first by upgrade or make-up construction with additional mechanical supports, or second by using temporary fly-over bridges. The options are both costly, the difference is the first option requires longer construction days while the second option needs a few days as it requires to install and to dismantle.The costs for uncertainty obstacles in this research have been measured to include in the costs of the domestic manufacture section.After data had been processed in the template, therefore the real portion of logistics costs at cement projects in Indonesia can be seen in the following To help easy to make efficiency evaluation between projects based on Easier understanding of graphs can be seen in These findings again proved that better infrastructure is having a substantial contribution to decreasing the total costs of logistics. Java Island particularly has better and much more infrastructure both soft-infrastructure and physical infrastructures to perform all difficult-level of logistics. International seaports in Java Island for example have two, while other islands have none, or at least one. Road infrastructure is much more available in Java Island than in other islands, logistics tools such as vessels, barges, trailers, portable cranes, even more, the multi-axle trailers are also much more ready in terms of quality and quantities. Java Island in general also does not require over than once in multi-modal or transshipment to reach final project site and therefore help decrease the total costs of logistics.4.32) would be absolutely low. This is meaning, the model will be easily interfered with by other variables that outside undefined within the model or unstable model.Costs of logistics in projects based on empirical findings are small, the highest is about 18.33% and the lowest is 16.63% of the total investment costs. It means the major portions of costs will come from other variables than the costs of logistics. The biggest costs within the range of 81.67% to 83.37% must be allocated for procuring machinery, civil construction, fabrication, land clearing, concrete, services, and many more others directly. Due to the reasons by performing regression tests would be no significant impact as the coefficient of adjusted-determination  for surely requires further research future. The authors advised for researchers to take part in these findings deliberately.For project P14 and Tuban#2, both located in Java Island to concern for domestic manufacture. It implies a lot of domestic goods made and therefore demanding accordingly attention. Remain in Java Island, project Bayah located in the most South-western Java Island is dealing with insurance and customs. This project Bayah has a remote area that requires a customs officer at the designed place by its own costs. Therefore the matter of insurance and customs clearance predominantly becomes attention.Further project Banyumas is having a problem with its project site. As the project at southern Central Java with directly exposing to the Indian Ocean which popular with high-risk delivery of high tide. To anticipate, therefore the project does more utilize roads and therefore costs of domestic logistics variable became significant.The project of Maros has different scientific arguments. The project site is in Sulawesi Island which an international seaport Soekarno-Hatta exists. Customs clearance can be performed at the Island and therefore a matter of insurance and customs clearance become significant. Besides as the Island is outside Java Island in general limited in logistics infrastructure, it is not surprisingly higher attention to the variable of domestic logistics costs.Finally, project Train#1 in Kalimantan Island with limited logistics infrastructure and unavailable international seaports. Normally, foreign logistics costs became much more significant along with domestic logistics costs.However, as this paper mentioned earlier, the total logistics costs amount small number in all costs structure for any projects. Without any statistical tested, every researcher will have immediately answer that the results must be not significant and confirmed according to 4.4Cement projects are generally identical during preparation, execution, and closing. During preparation, the teams are seeking much more information in almost all aspects. Every information is treated valuable as the information can be referred for further process, bidding, or even more, big tenders. The logistics costs play an important role as logistics is very important to support project execution. Logistics is taking contribution during delivery project goods since the smallest to the biggest, from the base weight unit of a kilogram to the hundred tons.Learning from several 8 cement projects within the period of 2008–2018 in Indonesia are valuable experiences. Precise data of the projects are gathered, being clustered, for evaluation, and as sources, information to be referred by interested people or others.The paper can disclose total logistics costs for general projects in Indonesia and could be adopted by other projects. This paper knows how much costs to anticipate insurance, customs clearance, to prepare costs for foreign logistics costs, to reserve costs for handling goods originated from domestic manufactured, and to store adequate costs to perform domestics logistics activities. When the standard has been acknowledged, the smoother processes are going to be easy to be maintained. This is the primary point the use of this paper.5In real project execution, determining costs to anticipate goods delivery to and from the project site is generally done by a trial-and-error approach. Try at first then immediately applying amendments when the budgets are nearly achieved. These actions are totally false, as outsiders especially project owners will judge the project team not adequately professional. These are common in generally every project execution and the response is always repeating yearly.To sort all concerning logistics needed for every project, the first to be importantly determined is predicting total goods required for the project. All members of the project team are highly requested to participate in determining the goods needed within each responsibility based on each professional experience. The team starts from the Safety Health Environment (SHE) department, HRD, Engineering, Construction, Procurement, and other organizations within the project, to the Security department.Based on empirical findings at this research the total logistics costs in Indonesia are at about 14.6% of the total costs of investment. It consists following costs of insurance cost 0.11%, costs for customs clearance 6.52%, foreign logistics costs 6.62%, domestic manufacture 0.47%, and domestic logistics costs 0.89%.The number findings are particularly important to perform procurement contracts, to negotiate with suppliers or vendors, preparing budgets for the projects, anticipating taxes, valuable for planners and or for project owners, and also for auditors.Due to the standards is not yet defined clearly to the date, these findings are not excessively said as providing novelty to the discourse related to the nexus between costs designing in every project.E. Subiyanto and Y. T. Suyoto: Conceived and designed the experiments; Performed the experiments; Analyzed and interpreted the data; Contributed reagents, materials, analysis tools or data; Wrote the paper.This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.The authors declare no conflict of interest.https://www.igi-global.com/journal/international-journal-applied-logistics/1151).Supplementary content related to this article has been planned in forthcoming published at International Journal of Applied Logistics (IJAL), ISSN: 1947–9573, 2020, volume 10, issue 2, article 3, titled Assessing Total Logistics Costs: Case Study during the Execution of Cement Projects in Indonesia by author (


Morbillivirus genus, Paramyxoviridae family, representing a serious threat for domestic and wild carnivores [Canis lupus), CDV appears to cause direct mortality in geographically restricted populations even if with different patterns. While in Iberian wolves (Canis lupus signatus), the virus was detected mainly in 2005–2008 years, and the serological data suggest a diffuse population-level immunity [Canine distemper is a contagious infectious disease, caused by canine distemper virus (CDV) belonging to rnivores . In Eurornivores ,3,4,5. Iimmunity , the Alpimmunity ,8. Conveimmunity . With reimmunity . Based oThe phylogenetic analysis of the H gene and the related amino acid sequences represents the most common tool for CDV characterization. According to this method, strains showing the protein H amino acid divergence less than 3.5% belong to a unique lineage , while aNot only the genetic determinants in viral RNA are involved in CD occurrence in wild species, but many different factors, in particular everywhere the anthropogenic pressure is more persistent, must be considered. The size and spatial organization of each population , any potential overlap with domestic species and human activities, the co-existence of multiple hosts and pathogens require a multidisciplinary approach to study the dynamic of infections .Finally, even if no report of CDV transmission from animals to humans is still available, the adaptation of the virus to non-human primates was demonstrated , and the


In recent years, non-coding RNAs (ncRNAs) have gained unprecedented attention as new and crucial players in the regulation of numerous cellular processes and disease responses. In this review, we describe how diverse ncRNAs, including both small RNAs and long ncRNAs, may be used to engineer resistance against plant viruses. We discuss how double-stranded RNAs and small RNAs, such as artificial microRNAs and trans-acting small interfering RNAs, either produced in transgenic plants or delivered exogenously to non-transgenic plants, may constitute powerful RNA interference (RNAi)-based technology that can be exploited to control plant viruses. Additionally, we describe how RNA guided CRISPR-CAS gene-editing systems have been deployed to inhibit plant virus infections, and we provide a comparative analysis of RNAi approaches and CRISPR-Cas technology. The two main strategies for engineering virus resistance are also discussed, including direct targeting of viral DNA or RNA, or inactivation of plant host susceptibility genes. We also elaborate on the challenges that need to be overcome before such technologies can be broadly exploited for crop protection against viruses. Due to climate change and alterations in cropping systems, plant viral disease outbreaks will likely have increasingly negative impacts on food production, which will bring new challenges to current agricultural practices worldwide. To advance virus disease management, rapid, efficient, and safe responses are required to minimize viral threats. The use of virus-resistant crop varieties is a traditional and efficient way to reduce losses caused by viral infections. However, conventional antiviral breeding strategies, even augmented with modern molecular techniques such as quantitative trait locus (QTL) mapping, marker-assisted selection, and whole-genome sequence-based approaches, are slow and laborious and require monitoring of large crop populations over multiple generations .Advances in genetic engineering and plant transformation techniques in the early 1980s enabled the rapid production of virus-resistant transgenic plants . Since tIt should be noted that historically, mainstream approaches to engineering plant virus resistance have been driven by a classical view of the central dogma of molecular biology, in which the genetic information contained in DNA is transcribed into mRNA which in turn directs the synthesis of particular proteins, and variations in proteins give rise to different traits. Accordingly, many transgenic plants were generated to express virus-encoded proteins , with the expectation that any resistance obtained would be protein-mediated. However, in many cases, the resistance had been surprisingly proven to be RNA-mediated. This phenomenon was named RNA silencing or RNA interference (RNAi) . This isWhile the small siRNAs and miRNAs constitute important classes of regulatory ncRNA, many other types of ncRNAs are known, and all of these form the majority of the transcriptome, given that only about 2% of transcribed RNAs encode proteins . ncRNAs Thus, while the central dogma of DNA>RNA>protein still holds true, ncRNAs can greatly influence this information flow and are, therefore, now an emerging major new source of next-generation targets which may be exploited in diverse applications, including conferring plant resistance against pathogens and viruses in particular .RNA technologies provide us not only with new sources of resistance but also with powerful tools to manage plant defense responses. With regards to antiviral resistance, RNAi-based approaches have long been used in the form of transgene-induced RNA silencing to target the degradation of diverse viral RNAs or to inactivate virus susceptibility genes in various crops ,10,11,12RdRp), and serve as secondary siRNAs that systemically spread throughout the plant; which is crucial for efficient RNAi-based antiviral defense [Of the different types of ncRNAs, siRNAs have attracted considerable attention due to their direct targeting of viral RNAs for degradation as a part of the natural RNAi-based antiviral immune response ,30. For  defense . RNA sil defense , which sFor DNA-containing plant viruses  the precursors of siRNAs are presumably produced by DNA-dependent RNA polymerase II (Pol II) which mediates bidirectional transcription in both sense and antisense orientations on the viral DNA. Such transcripts can pair virus-specific mRNAs and hence form perfect dsRNAs to be processed by some DCLs into siRNAs .To successfully overcome siRNA defenses and infect plants, viruses have evolved suppressors of RNA silencing (VSRs) which may target key steps in the siRNA pathways by inhibiting siRNA production, sequestering siRNAs, or preventing the spread of RNA silencing signals . Some otThus, siRNA-triggered RNAi represents a robust host defense mechanism against plant viruses which has been proven for numerous viruses in many plant species and crops ,20. WithFundamentally, miRNAs and siRNAs share some similarities in size, structure, and functions. Both are short duplex RNA molecules that may exert gene silencing effects at the RNA level and are derived from double-stranded regions of RNA precursors. They both require DCL and AGO proteins for processing, maturation, and action, although the species of DCL and AGO may be different. However, there are many important differences between miRNAs and siRNAs. A major distinction between them is that siRNAs are processed from foreign dsRNAs  whereas miRNAs are derived from endogenous precursor transcripts containing double-stranded  regions encoded by miRNA genes (MIR genes). miRNAs are derived from longer, primary transcripts termed pri-miRNAs. The pri-miRNAs contain an RNA hairpin in which one of the two strands includes the mature miRNA fragment. This hairpin then is cut out by a microprocessor enzyme complex which includes DCL1 nuclease (a member of an RNase III endonuclease family) and HYL1, a dsRNA binding protein ,39, whicPlantae from mosses to monocots and eudicots and can be grouped in two classes: one which is conserved and ancient, and one which consists of miRNAs that are less conserved and younger [In toto, miRNAs constitute a highly diverse set of molecules which may match nearly all RNAs encoded in a plant genome, thereby controlling almost any aspect of the plant life cycle; playing key roles in growth and development, signal transduction, innate immunity, and responses to various ecological biotic and abiotic stresses [Many miRNAs are evolutionarily conserved across major taxa of the Kingdom  younger ,40. In tstresses .Arabidopsis plants, thus elevating the accumulation of its gene target transcripts MYB33 and MYB65 . This consequently led to the production of severe viral symptoms [With regards to virus infections, information on miRNAs has mainly focused on their role in the induction of viral disease symptoms and antiviral defense mechanisms. It is well documented that some viruses may control accumulation rates of miRNAs involved in plant symptom development . For exasymptoms . Similarsymptoms . It has symptoms . Interessymptoms . Some ofsymptoms . Why theAGO or RdRp genes, which are involved in siRNA biogenesis and activity, are themselves natural targets of various plant miRNAs. For example, the antiviral AGO1 is the target gene for miR168. To prevent degradation of AGO1 transcripts and thereby alleviate AGO1-mediated antiviral defense, plants have evolved a decoy protein gene AGO18 [AGO18 transcripts compete with AGO1 transcripts for miR168 to attenuate the cleavage of AGO1 mRNA. Expression of AGO18 in rice was shown to be activated upon RSV infection, conferring resistance against this virus through partial miR168 sequestration [RdRp1-mediated RNA silencing pathway in rice by targeting RdRp1-inhibiting proteins [Many components of the antiviral silencing machinery such as ne AGO18 . AGO18 tstration . miR444 proteins .N gene encoding the resistance TIR-NB-LRR protein gene that mediates hypersensitive response against tobacco mosaic virus (TMV). Overexpression of these miRNAs decreases levels of TIR-NB-LRR transcripts and attenuates N-mediated resistance to TMV, suggesting that miR6019 and miR6020 play an important role in controlling resistance to TMV [It has also been shown that some miRNAs can target genes that trigger antiviral resistance . For exae to TMV ,49.AGO18, resulting in increased AO activity and elevated ROS accumulation; rendering plants more resistant to the virus [TCP21 and JA concentrations in rice had elevated abundance after infection by rice ragged stunt virus, leading to a more virus-susceptible phenotype [General plant responses contributing to antiviral resistance are often orchestrated by the rapid production of reactive oxygen species (ROS) and several phytohormones such as salicylic acid (SA), jasmonic acid (JA), and auxin ,51. It hhe virus ,53. miR3henotype . Recentlhenotype .As exemplified by these case studies, miRNAs are currently regarded among the most important gene regulators. As discussed, these small RNAs are involved in many pivotal aspects of plant-virus interactions and are emerging as the next generation targets for engineering plant virus resistance.7 G cap, 3′ poly (A) tail, and exon-exon junctions [Arabidopsis under stress conditions [Another group of non-coding RNAs playing important roles in many biological processes is lncRNAs. LncRNAs are defined as transcripts longer than 200 nt with little or no coding potential. Similar to coding mRNAs, most lncRNAs are transcribed by Pol II. They may also have typical mRNA-like structures such as 5′munctions ,56. In anditions ,58. In gcis and that of distant genes in trans. Cis–acting regulatory lncRNAs modulate the expression of the neighboring target genes either by recruitment of Pol II or through the event of getting transcribed, which results in local chromatin conformational changes that facilitate downstream mRNA transcription [Based on genomic origin, lncRNAs can be classified as intergenic, intronic, or exonic regions in sense or antisense orientation . Besidescription .Trans-acting lncRNAs may act as scaffolds to facilitate the recruitment of protein(s) and the formation of RNA protein complexes. Such interactions are usually mediated by specific lncRNA domains and may regulate gene expression either through sequestering proteins and preventing them from reaching their target (as a decoy) or by delivering them to specific target sites (as a guide) [a guide) ,9. LncRNa guide) . For exaa guide) . The subThe repertoire of biological processes and mechanisms of action of the lncRNAs are rapidly growing. Plant lncRNAs are known to play pivotal roles in the regulation of flowering time, modulation of reproductive organ development, leaf development, auxin signaling, photomorphogenesis, and responses to biotic and abiotic stresses .It is conceivable that lncRNAs may also play important roles in plant-virus interactions. Indeed, it has been shown that both RNA-containing tobacco rattle virus  and DNA-The ELENA1 lncRNA identified in another recent study was shown to take part in controlling plant defense by modulating the SA pathway . It was Nicotiana benthamiana with a CTV isolate deficient in LMT1 production (CTV-LMT1d) led to an elevated SA accumulation  encodes its own subgenomic lncRNA, LMT1 . Infectisistance  and a cosistance . This stsistance . FurtherCircular RNAs (circRNAs) are a group of endogenous non-coding ssRNAs that have a closed-loop structure . CircRNAArabidopsis, miPEP171b from Medicago [Brassicaceae [Despite their definition as non-coding RNAs, a growing number of reports have suggested the existence of stably expressed and functional peptides (or microproteins) translated from lncRNAs . These pMedicago , and miPsicaceae . These pThere is a growing body of evidence that SEPs play an important role in animal cell immunity , suggestAs mentioned above, other classes of non-coding RNAs include rRNAs, tRNAs, snRNAs, and snoRNAs. There are no doubts that some functional crosstalk may exist between various activities of these RNAs and virus infections. A striking example of such interplay is that sophisticated functional mimics of tRNAs (transfer RNA-like structures) are found at the 3′-ends of the genomes of some plant positive-strand RNA viruses , which mPioneering work published in 1986 by Powell Abel et al.  demonstrTo address these hurdles and public concerns, another approach termed “spray induced gene silencing” (SIGS) has been developed whereby RNA silencing-based antiviral protection is induced by spraying plants with exogenous dsRNA (or hpRNA), complementary to viral RNAs . In addiPMMoV, pepper mild mottle virus; TEV, tobacco etch virus; AMV, alfalfa mosaic virus; PSTVd, potato spindle tuber viroid; CEV, citrus exocortis viroid; CChMVd, chrysanthemum chlorotic mottle viroid; SCMV, sugarcane mosaic virus; PRSV, papaya ringspot virus; CymMV, cymbidium mosaic virus; CMV, cucumber mosaic virus; ZYMV, zucchini yellow mosaic virus; BCMV, bean common mosaic virus; ToLCV, tomato leaf curl virus; PVY, potato virus Y; PPV, plum pox virus; PSbMV, pea seed-borne mosaic virus; LDH, layered double hydroxide clay nanosheets.It is also worth noting that the antiviral RNAi-based resistance mechanism induced by the exogenous dsRNA may be further enhanced via pattern triggered immunity (PTI). PTI is a general plant response that is triggered by a conserved microbe—or pathogen—ssociated molecular patterns  as the first step of plant defense . RecentlAltogether, these scientific achievements create new opportunities for practical applications of dsRNA-based approaches for crops. Recently developed breakthrough technological platforms for large-scale cell-free or bacterial production  and purification of dsRNA at as low a cost as $0.5–1/g , greatlyAnother approach to engineer virus resistance using SIGS is to silence virus susceptibility genes in host plants. Among the known susceptibility genes required for successful infection with some plant viruses, such as potyviruses, are those encoding the translation initiation factors eIF4E, eIF(iso)4E, and eIF4G . InactivAlthough this has not yet been experimentally verified, one can suggest that exogenous dsRNA designed to silence a particular host susceptibility gene target can be applied to intact plants without the need for plant transformation. In addition, modification of individual cultivars is not required due to the sequence similarity of targeted genes; however, only a few such genes have been identified. Therefore, finding novel universal and effective targets among host genes and non-coding RNAs is a challenge for further development and marketing of the SIGS technology.Until now, only exogenous dsRNA has been used in SIGS as a silencing trigger. In the future, the arsenal of RNA triggers for SIGS could be significantly expanded by adding next-generation tools such as artificial miRNAs (amiRNAs), artificial trans-active siRNAs (atasiRNAs), and target mimics of several kinds . A corolThe miRNA mimic technology (miR-Mimic) is another novel approach for gene silencing. miRNA mimics are artificially synthesized dsRNA molecules imitating miRNA duplexes. Once delivered into the cell, these RNA fragments mimic natural miRNAs which bind to the target mRNA molecules and block their expression either by degradation or by translational repression .Although the exogenous applications of ami/atasiRNAs or miRNA mimics have not been reported so far, we believe that these types of RNA molecules will be assessed and exploited in SIGS technology in the future.As mentioned above, CRISPR-Cas technology has emerged as an expanding toolbox with which to generate antiviral resistance in various crop plants. In this regard, two of the most common approaches using CRISPR-Cas to control virus diseases is either by using it to directly target and inhibit virus machinery or by mutating “susceptibility” genes in host plants , the designed sgRNAs were shown to be able to directly target various specific regions of the viral DNA genomes and consequently reduce or abolish disease symptoms caused by such geminiviruses as WDV ), tomatoThe discovery of CRISPR-related sgRNA-guided endonucleases specifically targeting RNA molecules such as FnCas9 and Cas13 , opened These examples show that direct manipulation of viral DNA or RNA genomes by CRISPR-Cas may represent new technologies to engineer antiviral resistance. However, this approach requires persistent maintenance of the CRISPR-Cas components in the plant which thus far can only be carried through their transgenic expression. Due to GMO regulations, this may preclude or significantly limit practical applications of CRISPR antivirals.To mitigate GMO-related barriers in engineering virus resistance, several other CRISPR-Cas approaches avoiding the use of GMO have been developed, which target host plant genes involved in regulating virus susceptibility. In the first approach, CRISPR-Cas components are initially expressed transgenically to modify a host “susceptibility” gene leading to virus resistance, and these transgenic components are then segregated out following sexual reproduction . AlthoueIF4s and coilin genes were previously used for the development of resistance in HIGS experiments  which can affect virus susceptibility have been exploited for CRISPR editing to engineer virus resistance within a rather narrow range of viruses, such as potyviruses and rice tungro spherical virus ,118,119.e above; ,106).As discussed, both RNAi and CRISPR-Cas have been successfully used as powerful tools for engineering plant virus resistance. However, the question arises as to which of these seemingly competing technologies is the right option for a particular application. There are several technical and methodological aspects to be addressed before making a choice:GMO. As noted above, some CRISPR applications, in particular, direct targeting of plant viruses, require the permanent presence of CRISPR-Cas components in plants which is only possible in transgenic plants, whereas SIGS RNAi-based technology is transgene-free.Plant regeneration. To inactivate host plant virus “susceptibility” genes, the constant presence of the Cas protein and sgRNA is not needed and hence, the use of transgenic plants can be avoided either by eliminating the Cas9/sgRNA transgenes or by delivering editing reagents as mRNAs or RNPs. However, the associated steps for the regeneration of whole plants from the edited cells with consequent identification of the edited lines are usually time-consuming, technically demanding, and costly. Moreover, many crop plants and varieties are recalcitrant to regeneration. So far, CRISPR transgene-independent techniques are only feasible within a limited range of plant species and varieties. In contrast, the RNAi-based SIGS approach does not require regeneration steps at all.Individual cultivars. With CRISPR technology, each cultivar should be edited and tested independently whereas RNAi tools developed for targeting conserved gene sequences could be applicable to many cultivars.Time. Preparation of RNAi tools (weeks) is significantly faster than production and selection of edited plants (months or years).Nature of phenotype. Existing (transgene-free) CRISPR technologies induce permanent and irreversible gene edits in plants, whereas RNAi induces transient knockdown of gene expression. In many cases, transient perturbation of gene transcripts may be preferred, particularly when the complete knock-out of those genes that are essential could be lethal , but partial loss of them would not be. For example, virus “susceptibility” genes such as coilin genes (and others) possess many important cellular functions, and therefore their expression should be modulated in a temporary manner during virus attack only.This analysis suggests that RNAi engineering of antiviral resistance may currently offer a number of significant advantages over CRISPR. While CRISPR technology has been a game-changer that led to remarkable progress in the development of new methods for targeted therapy of human diseases , the appOne of the major problems in topical applications of naked dsRNA molecules is their instability in the environment, which is typically manifested in a short antiviral protection window when applied against virus infections . TherefoAnother concern is the capacity of RNAi compounds to penetrate cells, particularly where the large complexes which protect the RNA from degradation may significantly reduce permeability. Plants have evolved stringent security control systems (barriers) which govern specific signaling and transport pathways that prevent foreign molecules from penetrating and transporting through the plant tissues. The cuticle and cellulose cell wall are the first barriers typically encountered which are inhibitory to cell entry . The nexVarious factors, such as climate change, decreases in agricultural land and global population growth as well as hurdles associated with GMO and current chemical pesticide regulations, have promoted the development of new biosafe and eco-friendly technologies for crop protection against pests and pathogens. Among them are RNA-based biocontrol technologies, such as RNAi and CRISPR. Although both of them are still in the research and development pipeline, they are well anticipated to be exploited for engineering plant virus resistance in agricultural practice. In spite of the popularity and great capabilities of the CRISPR-Cas system, RNAi approaches seem to be preferable at least for direct targeting plant viral pathogens. Commercial expectations surrounding RNAi applications are rising, with great interest towards sprayable RNA-based techniques from agribiotech giants such as Bayer Crop Science, Monsanto (now acquired by Bayer) and Syngenta, and many other newly developed start-up companies . This trIn addition, we are well aware that the emergence of CRISPR-Cas editing tools will significantly strengthen the arsenal of applications for genetic crop improvement. Unprecedented advances have been achieved by using CRISPR-Cas technology in basic plant biology. CRISRP-Cas has been proved to be a vital tool for plant functional genomics, including the generation of CRISPR/Cas mutant libraries for developing genome-wide mutation screens . AnotherHowever, there are some common technical challenges and societal problems that must be addressed for both RNAi-SIGS and CRISPR-Cas technologies before they take a certain unique place in engineering plant virus resistance.Identification of novel host susceptibility targets among coding or non-coding RNAs that should be inactivated or modified by RNAi or CRISPR would be a crucial next step to further develop basic engineering strategies for virus resistance. It could also be useful to expand a potential list of gene targets by identifying gene/ncRNA targets in genomes of insect vectors to prevent transmission of plant viruses. RNAi has recently been successfully used to control insect pests .Another critical factor to be considered is the off-target effects of both RNAi and CRISPR technologies. Exogenously applied long dsRNAs can produce many different siRNA species with a higher chance of causing off-target host gene regulation . HoweverThe rapid progress in RNAi and CRISPR applications also desperately requires the improvement of protective delivery platforms that allow efficient uptake and dissemination of RNA silencing or CRISPR editing components by plant cells. Cooperative efforts between plant biologists, geneticists, and nanotechnologists may provide a real game-changer in such crop protection technology.Finally, clear and standardized regulatory guidelines are required for the development of non-transgenic externally applied RNAi and CRISPR tools to enable them to progress to agricultural markets.