Dengue is a mosquito-borne viral infection which has been estimated to cause a global economic burden of US$8.9 billion per year. 40% of this estimate was due to what are known as productivity costs . Although productivity costs account for a significant proportion of the estimated economic burden of dengue, the methods used to calculate them are often very variable within health economic studies. The aim of this review was to systematically examine the current estimates of the productivity costs associated with dengue episodes in Asia and to increase awareness surrounding how productivity costs are estimated.We searched PubMed and Web of Knowledge without date and language restrictions using terms related to dengue and cost and economics burden. The titles and abstracts of publications related to Asia were screened to identify relevant studies. The reported productivity losses and costs of non-fatal and fatal dengue episodes were then described and compared. Costs were adjusted for inflation to 2017 prices.We reviewed 33 relevant articles, of which 20 studies reported the productivity losses, and 31 studies reported productivity costs. The productivity costs varied between US$6.7–1445.9 and US$3.8–1332 for hospitalized and outpatient non-fatal episodes, respectively. The productivity cost associated with fatal dengue episodes varied between US$12,035-1,453,237. A large degree of this variation was due to the range of different countries being investigated and their corresponding economic status. However, estimates for a given country still showed notable variation.We found that the estimated productivity costs associated with dengue episodes in Asia are notable. However, owing to the significant variation in methodology and approaches applied, the reported productivity costs of dengue episodes were often not directly comparable across studies. More consistent and transparent methodology regarding the estimation of productivity costs would help the estimates of the economic burden of dengue be more accurate and comparable across studies. Dengue is a widespread mosquito-borne viral disease, with over 40% (2.5 billion people) of the world’s population at risk of infection . The incThe aim of this review is to systematically examine the current estimates of the productivity costs associated with dengue episodes in Asia, and to gain a more comprehensive understanding regarding the variation in the methodology used to calculate these costs. We also discuss more generally many of the areas of debate surrounding the quantification of productivity costs, how productivity costs can be applied within cost-effectiveness analyses, and make recommendations for future research. These findings will help improve future research and consistency regarding the quantification and reporting of dengue-related productivity costs. This will be helpful for developing more accurate estimates of the economic burden of dengue as well as helping policymakers and healthcare funders in decision making regarding dengue interventions.We searched for relevant articles using the PubMed and Web of Science electronic databases using variants of the following keywords  on 24th of April 2019, without any date or language restrictions. The search terms were discussed and tested by two reviewers (TMH and HCT). A detail information of search terms and PRISMA checklist were provided in Additional file One key feature of dengue is the variety of types and definitions of dengue episodes. Many studies focus implicitly on episodes managed in the formal health care system, while episodes managed outside the formal healthcare system were commonly excluded . In someWhen possible, we stratified the results according to the severity of the dengue episode  and the source of care . Notably, the World Health Organization (WHO) classification for dengue has been updated from dengue fever or dengue hemorrhagic fever to dengue or severe dengue . We repoThroughout this paper, productivity losses were defined as the estimated amount of productive time (i.e. paid or unpaid work) lost due to a dengue episode. Productivity costs were defined as the monetary value of these productivity losses.As the studies were performed in different years, it was necessary to adjust for inflation to make the estimates more directly comparable. We therefore adjusted the reported productivity costs to 2017 US prices. For the majority of the studies, the reported productivity costs were converted to their country’s currency and adjusted for inflation using the specific country’s Gross Domestic Product (GDP) deflator rates from the International Monetary Fund and then reconverted to US$ using the 2017 exchange rate –11. FurtWe identified 56 potentially relevant articles reporting the cost of dengue illness in Asian countries. Of these, 23 of the studies only reported direct costs and were therefore excluded. The remaining 33 studies were included in the analysis and reviewed in further detail. Details of the identified articles are described below Tables  and 2. GThere was substantial variation regarding how the different studies defined and estimated productivity losses and productivity costs. For instance, some studies only considered the productivity losses of the patients, while others also accounted for the losses experienced by the patients’ informal caregivers  associated with non-fatal dengue episodes and their informal caregivers.The estimated productivity costs associated with non-fatal episodes i.e. the monetary value of the productivity losses.The estimated productivity costs related to fatal dengue episodes.How these estimated productivity costs of dengue episodes influence estimates of dengue’s total economic burden.How these productivity costs can be incorporated into cost-effectiveness analyses of dengue interventions.In the following sections we summarize:A number of studies reported the productivity losses associated with dengue episodes . However, none of the studies appeared to explicitly quantify the productivity losses relating to presenteeism. Presenteeism is likely to be present at every phase of the illness. It is probable that presenteeism is even more significant in low and lower-middle-income countries, as the patients will be less likely to have access to paid “sick days” and many would not be able to afford to take much time off work. Studies on arthritis and mental health have found that presenteeism accounted for the majority of their associated total productivity losses –45. CompThe patients’ productivity losses were found to correspond to the severity of the dengue episode. Three studies differentiated the productive time lost based on disease severity , 22, 41.The majority of studies focused implicitly on episodes managed within the formal health system. Although this is understandable, this focus excludes episodes that cause productivity losses but do not receive a formal diagnosis or management in the formal health care system.Four studies reported the patients’ productivity losses pre, during and post-hospitalization period , 21, 46.Within the literature, there is variation regarding the reported proportion of cases (2–68%) experiencing post-acute consequences of dengue infection , as well as the reported duration of these symptoms . The majMany dengue patients require support from informal caregivers: individuals (often family members) who provide unpaid assistance and support while the patient is ill. Within the studies we identified, the average number of days lost by the patients’ informal caregivers ranged between 1.0–25.5 days for hospitalized episodes and 0.1–2 days for outpatient episodes  discussed previously. The estimated productivity costs from the different studies are summarized in Table The methods for placing a monetary value on productivity losses can vary depending on whether the loss is experienced by an adult patient, a child patient or an informal caregiver. These issues are discussed more in the following subsections.The productivity losses incurred by adult dengue patients are typically valued based on a specified wage source(s), which reflects the average income of the population at risk of disease in the study setting. Many of the identified studies used a minimum wage for this , 25, 27.Currently, there is no standard method for estimating productivity costs. This has resulted in variation across different studies, even when they are reporting to have used the same approach; this is true not just for dengue but for the health economics field in general . For exaA key area of debate in the health economic field is regarding the use of the human capital approach or the friction cost approach Fig.  and the The productivity costs of lost paid work were commonly quantified and reported across the studies. However, those related to lost unpaid work were often ignored. Specifically, some studies did not value the productivity losses of unemployed patients, students or individuals not earning money at the time of the survey (such as farmers). It should be acknowledged that quantifying monetarily unpaid productivity losses is challenging, and the correct methodology is debatable. Though there are some recommendations , there aValuing monetarily the productivity losses of children is challenging, and currently there is no broadly accepted guideline or methodology for this , 66. BecThe time that informal caregivers spend caring for a patient is typically considered to be unpaid work. Therefore, the opportunity cost method or replacement cost method are generally used to quantify the productivity losses of informal caregivers in monetary terms Fig. . The aveThe majority of the studies did not specifically report informal caregivers who were teenagers or retirees. Teenagers and retirees could conceivably spend a lot of time caring for patients, though similar to children, quantifying their productivity costs can be challenging and the correct methodology to use is under debate , 68, 69.More transparency is needed in future studies regarding how the productivity costs related to patients’ informal caregivers are quantified.We have discussed the estimated productivity costs associated with acute non-fatal dengue episodes; however, a small proportion of dengue patients die. Some studies estimated the economic burden associated with these fatal dengue episodes. The average estimated productivity costs related to a fatal episode varied across the different studies between US$15,568-1,453,237 for episodes in children and US$12,035–948,121 for episodes in adults  2017 study estimated that there were 86,183,620 symptomatic dengue episodes in Asia in 2017  . This nuProductivity costs account for a significant proportion of the economic burden of dengue . ConsequThose corresponding to the productivity losses directly associated with accessing the intervention (e.g. someone takes a day off work to go to an outpatient clinic to get a vaccine).Those corresponding to the averted productivity losses that result from prevented morbidity/mortality . These averted productivity losses are often referred to as productivity gains.Within cost-effectiveness analyses, there are effectively two types of productivity costs Fig. :Those cThe productivity costs of dengue episodes discussed in the previous sections of this paper could be used to quantify the productivity costs associated with the productivity gains that result from a dengue intervention within a cost-effectiveness analysis (based on the number of episodes averted by an intervention and their corresponding productivity costs). Within the cost-effectiveness ratio, these productivity costs associated with productivity gains are effectively a negative cost Fig. .The recommendations for which productivity costs should be included in cost-effectiveness analysis vary Fig. . The firThe WHO’s cost-effectiveness analysis guide recommends that the productivity costs associated with both productivity losses and productivity gains should be excluded from a cost-effectiveness ratio . The reaOwing to the debate in this area within the health economic field, and the significance of the dengue related productivity costs, researchers performing cost-effectiveness analyses of dengue interventions should clearly state their assumptions regarding the methodology being applied and highlight the fact that the appropriateness of certain methods is still under debate.A potential source of bias in our search strategy is that it did not capture studies published outside of the searched electronic databases . For example, we found few articles reporting the burden of dengue in China and India, which may be due to studies relating to these countries not being published in journals which are indexed in PubMed or Web of Science. Efforts were made to minimize this bias by searching Google Scholar, WHO’s Dengue Bulletin and the bibliographies of selected studies. It should be noted that the selection process was not performed independently by two researchers (i.e. not in duplicate), which could result in selection bias. To mitigate this, the papers where there was uncertainty regarding their inclusion were discussed between two reviewers. Furthermore, because there is no standard methodology for conducting cost of illness studies, the quality of the identified studies was not assessed. The variation in quality could result in biases in the estimated productivity costs of dengue episodes . However, this observed variation further supports our primary finding regarding the need for greater consistency in future studies.A major impetus for calculating the cost of an illness episode is to have a standard burden measure that can be used for comparisons across countries and time periods, and for use within economic evaluation of interventions. Productivity costs can be a key component of the cost of an illness dengue episode. We found that the estimated productivity costs associated with dengue episodes in Asia are notable. However, owing to the significant variation in methodology and approaches applied, the reported productivity costs of dengue episodes were often not directly comparable across studies.The majority of studies we identified only valued absenteeism from paid work. The losses associated with lost unpaid work and presenteeism have not been adequately addressed or characterized. Further work is also needed to account for the productivity costs incurred by patients’ informal caregivers. Due to such losses not being fully captured, the productivity costs of dengue could be underestimated.In the future, further guidelines and recommendations regarding how to calculate productivity costs would be invaluable and make different studies more consistent and comparable. What is most important moving forward is that the assumptions and methods used to estimate productivity costs are clearly reported.More consistent and transparent methodology regarding the estimation of productivity costs would help the estimates of the economic burden of dengue be more accurate and comparable across studies. Ultimately, accurate estimates of the economic burden of dengue will be crucial to informing policy in both the prevention and treatment of this highly prevalent disease, particularly in settings with limited resources.Additional file 1. Overview of the methods applied to calculate productivity costs.Additional file 2 Description of the adjustment for inflation, Table S1: The estimated productivity losses of patients and their informal caregivers, Figure S1: The average reported productivity costs as a fraction of the study settings per capita GDP, The search terms and PRISMA CHECKLIST.


Vitis vinifera cv Nebbiolo and Chardonnay virus-free plantlets regenerated by somatic embryogenesis. Grapevines were then artificially inoculated in the greenhouse with either Plasmopara viticola or Erysiphe necator spores. GFLV-infected plants showed a reduction in severity of the diseases caused by powdery and downy mildews in comparison to virus-free plants. GFLV induced the overexpression of stilbene synthase genes, pathogenesis-related proteins, and influenced the genes involved in carbohydrate metabolism in grapevine. These transcriptional changes suggest improved innate plant immunity, which makes the GFLV-infected grapevines less susceptible to other biotic attacks. This, however, cannot be extrapolated to GRSPaV as it was unable to promote protection against the fungal/oomycete pathogens. In these multiple interactions, the grapevine genotype seemed to have a crucial role: in ‘Nebbiolo’, the virus-induced molecular changes were different from those observed in ‘Chardonnay’, suggesting that different metabolic pathways may be involved in protection against fungal/oomycete pathogens. These results indicate that complex interactions do exist between grapevine and its different pathogens and represent the first study on a topic that still is largely unexplored.Grapevine may be affected simultaneously by several pathogens whose complex interplay is largely unknown. We studied the effects of infection by two grapevine viruses on powdery mildew and downy mildew development and the molecular modifications induced in grapevines by their multiple interactions. Grapevine fanleaf virus (GFLV) and grapevine rupestris stem pitting-associated virus (GRSPaV) were transmitted by in vitro-grafting to  Vitis vinifera L.) is one of the most economically important and widespread fruit crops in the world. In all viticultural regions, it is affected (often simultaneously) by several pathogens, such as fungi, oomycota, phytoplasma, and viruses, which induce serious damage to the plants with consequent high economic losses1. Among these biotic agents, viruses represent a widespread class of pathogens in all grapevine species. Thanks to deep sequencing technologies and the subsequent discovery of new viruses, more than 80 virus species have been identified in grapevine2. Among these, about half are recognized as agents of ‘major diseases’ in terms of economic relevance3, while others may not show clear, typical symptoms possibly also for mixed infections with other viruses in the same plant. Two well known and widespread viruses are grapevine rupestris stem pitting-associated virus (GRSPaV) and grapevine fanleaf virus (GFLV). GRSPaV is a member of the genus Foveavirus, family Betaflexiviridae, generally associated with “Rupestris Stem Pitting”, a disorder of the “Rugose Wood complex”3, and it usually infects V. vinifera cultivars in a latent state. GFLV is a member of the genus Nepovirus in the family Secoviridae4 responsible for the complex of ‘Grapevine infectious degeneration’ and is one of the most harmful and economically impacting grapevine–virus diseases worldwide3. Several studies have analysed the ecophysiological, molecular, and biochemical modifications induced by viruses in grapevine10, which can then be regarded as a model to study plant–virus interactions in fruit plants and other woody species11. In recent years, it has become increasingly evident that plant–virus interactions are not limited to a simple reaction to pathogen infection and that they can shift from antagonist to mutualistic or beneficial responses to environmental changes13. Viruses can increase the tolerance of plants to drought and cold stress14, high soil temperatures15, and bacterial pathogens17. V. vinifera cv ‘Bosco’ infected by GRSPaV showed greater tolerance to water stress than virus-free plants18. The detrimental effects of grapevine leafroll associated virus-3 (GLRaV-3) have been extensively demonstrated in grapevine8 although Repetto et al.19 reported that GLRaV-3-infected grapevines displayed an increased resistance to downy mildew (DM) caused by Plasmopara viticola. All together these studies show that plant–virus interactions are more complex than expected and a deeper understanding of these interactions would be warranted, considering the plant as a holobiont in which micro- and macrobiomes interact with the environment as a unique organism20.Grapevine . DM is caused by the obligate biotrophic oomycete P. viticola, introduced into Europe from North America in the late 19th century21. DM affects the leaves and berries under warm and humid environmental conditions, reducing grape yield and negatively impacting the quality of berries and the aging ability of wine22. PM is caused by the obligate biotrophic fungus Erysiphe necator, a polycyclic fungus infecting all green tissues with grey-white mycelia on the surface, leading to significant losses in yield and quality, as well as fruit and wine characteristics23. Management of these diseases in the vineyard implicates significant economic and environmental costs, to such an extent that grapevines are one of the most frequently sprayed crops26. The European Commission has already expressed its recommendation to reduce the use of several chemical products (Directive 2009/128/EC), including copper salts (CE no. 354/2014). Consequently, in recent years, several studies have been carried out to investigate the effectiveness of more environmentally friendly products, such as several microorganisms, resistance inducers, biostimulants, and biofungicides30. However, so far, no studies have been reported on the induction of resistance to PM by viruses while only a few studies have been carried out on DM19.If viruses are the most numerous and widespread class of pathogens in all The aim of this work was to study the effects of GRSPaV and GFLV on DM and PM incidence and severity and the systemic molecular modifications induced in grapevines by these multiple interactions under controlled greenhouse conditions.In the present study the interactions among viruses, DM and PM were evaluated on ‘Chardonnay’ and ‘Nebbiolo’ grapevine cultivar. Immature anthers of both genotypes were collected in May 2016 and cultured in an inductive medium for embryogenesis to obtain virus-free plants, as confirmed by RT-PCR. A single line per cultivar regenerated from somatic embryos was multiplied in vitro, thus supplying the virus-free controls (NE_CTR and CH_CTR) for subsequent experiments  belongs to the phylogenetic group designed as GRSPaV-SG1  grouped together in the cluster IB with isolates from Canada and South Africa  was amplified by RT-PCR‘Nebbiolo’ plants infected by GRSPaV or GFLV were then used as “green rootstocks” for the transmission of these viruses through in vitro grafting in virus-free lines of ‘Chardonnay’ and ‘Nebbiolo’, used as scions. One month after grafting, viral diagnoses confirmed the transmission of the viruses and the infected scions of ‘Chardonnay’ and ‘Nebbiolo’ were removed from the graft and multiplied in vitro becoming the virus-infected treatment  for subsequent experiments . The percentage of affected leaves and the percentage of affected leaf area, i.e. disease incidence (DI) and severity (DS), respectively, were evaluated. At the final assessment, higher levels of downy mildew DS were observed in ‘Chardonnay’ compared to ‘Nebbiolo’, whereas in plants infected by E. necator, ‘Chardonnay’ appeared to be more tolerant than ‘Nebbiolo’ to PM. Plant genotype, virus, and their interaction significantly (p ≤ 0.05) influenced the DI and the DS caused by both pathogens. GFLV-infected plants showed lower downy mildew DS compared to virus-free plants, while GRSPaV-infected plants showed a DS not significantly different from CTR, with intermediate values between virus-free and GFLV-infected plants (Table E. necator. The genotype and virus interactions (G × V) were significant, resulting in significant lower values of DS in NE_GFLV compared to CTR for both fungal/oomycete pathogens (Table Virus-free and infected plants (eight plants per treatment) were grown in the glasshouse and, after 2 months, artificially inoculated with t June 208. The pet June 208. The peP. viticola or E. necator for each thesis  were more tolerant to PM than virus-free controls resulting in lower DS. A similar trend was observed for DI, where CH_GFLV plants showed the significantly lowest values  in leaf tissue immediately before fungal/oomycete inoculation (T0) and at the end of experiments (Tf), and was calculated as the relative expression ratio to the virus titre in ‘Nebbiolo’ at T0. GRSPaV concentration in ‘Chardonnay’ and ‘Nebbiolo’ was similar at T0 and decreased significantly at Tf  were much higher than those observed after P. viticola (Tf_PV) inoculation    Fig. . NoteworVvCHS) at Tf was influenced by fungal/oomycete inoculation. After inoculation with E. necator, transcription was reduced, whereas in the presence of P. viticola, we observed a significant increase in gene expression  and Beta-1,3-glucanase (VvBgluc), defense-related genes involved in the response to several biotic factors, was significantly affected by all three factors, namely genotype, virus, time, and their respective interactions , one of the genes linked to E. necator susceptibility, was strongly genotype dependent; the higher expression in ‘Nebbiolo’ at T0 confirmed its higher susceptibility to PM compared to ‘Chardonnay’  expression was modulated by the multiple interactions among genotype, time (after P. viticola inoculation), and viruses (after E. necator) (G × V × T). At Tf, expression was higher in ‘Chardonnay’, particularly in the presence of GRSPaV and GFLV  was regulated differentially following fungal/oomycete inoculation. After inoculation with P. viticola, the gene was significantly overexpressed in ‘Chardonnay’ and downregulated at Tf while, after E. necator inoculation, we observed multiple interactions among all components (G × V × T) with a significant increase in CH_GFLV_TF_En  and sucrose synthase (VvSUSY4) showed higher expression levels in ‘Nebbiolo’ at T0, particularly in the absence of viruses (NE_CTR). Infection by viruses and fungus/oomycete tended to downregulate these genes in both cultivars , which is involved in activation of stress response pathways, was upregulated at Tf with a significant G × T interaction , a transcription factor involved in the response to several stressors that improve the efficiency of the enzymatic antioxidant defence system35, was upregulated at Tf in virus-infected plants , a key gene in abscisic acid synthesis, was upregulated only at Tf in ‘Nebbiolo’, particularly after P. viticola inoculation, while in ‘Chardonnay’ was stable under all conditions , a gene linked to jasmonate biosynthesis, had an expression independent on both the genotype and virus infection; it was significantly overexpressed at Tf  of all RT-qPCR data showed division of the samples into three main groups Fig. . Data atata Fig. .Fig. 4Pr18, suggesting that such species may be a suitable woody plant model for studies on this topic11. In these works, the molecular, metabolic, and physiological responses to virus infection varied considerably according to the viral strains, presence of multiple viral infections, grapevine genotype, and external environment, including climatic conditions. Starting from these considerations, we set up an experimental design aimed to control some of these variants to better understand whether and which kind of interactions existed between some viruses and P. viticola and E. necator, causal agents of DM and PM, respectively, in grapevine. The analyses were focused on GFLV and GRSPaV because both are widespread viruses, well known from the molecular point of view, and represent two extremes of the biological responses induced in grapevine. As a rule, GFLV is quite harmful to grapevine3, while GRSPaV is generally latent in V. vinifera. On the other hand, positive effects of interactions have been demonstrated in GRSPaV-infected plants subjected to drought18.Over the past decade, studies on plant–virus interactions in grapevine have gained increasing importance37. These cultivars were chosen for their biological characteristics, e.g. different colour of the berries, different susceptibility to DM and PM, and for their importance and diffusion at international (‘Chardonnay’) and local levels (‘Nebbiolo’). In addition, as somatic embryogenesis may induce somaclonal variation38, the genetic uniformity of healthy and infected samples was guaranteed by using one single plant regenerated by a somatic embryo for each cultivar multiplied in vitro and infected with the selected viruses. Considering that the transmission of viruses to grapevine by mechanical inoculation is poor or not efficient at all, the grafting technique was adopted to transmit viruses from infected plants to virus-free ‘Chardonnay’ and ‘Nebbiolo’ cultivar.A critical point in our work was the strong influence of the grapevine genotype, so that healthy controls of the two cultivars, ‘Chardonnay’ and ‘Nebbiolo’, had to be produced by somatic embryogenesis6. Notably, in’Nebbiolo’, GFLV titre in leaves was 5–6 times lower than in ‘Chardonnay’ during the whole sampling time of the experiment .The experimental plan highlighted the influence of the grapevine genotype and/or of the time factors on viral titre. The viral titre of GRSPaV was similar in ‘Nebbiolo’ and ‘Chardonnay’ and decreased at Tf, which is in line with the seasonal variation observed in leaves in the vineyardP. viticola19. Interestingly, the main differences were observed in the severity of PM and DM diseases. In both cultivars, GRSPaV did not induce significant variation in the development of DM and PM and providing DS and DI values that were generally intermediate between the virus-free controls and GFLV-infected plants. However, in the second trial, GRSPaV seemed to favour the development of DM with DS and DI slightly higher than virus-free controls, even of differences were not significant. Notably, plant responses induced by GRSPaV stimulate tolerance to water stress18, whereas they had no effect or worsened the presence to fungal/oomycete pathogens, as shown in this study.Under greenhouse conditions, grapevine-mediated interactions between the two viruses and both the DM and PM causal agents  were demonstrated. Although ‘Chardonnay’ and ‘Nebbiolo’ showed different susceptibility to these diseases, both cultivars infected with GFLV showed a greater tolerance to PM and DM. These results agree with previous observations, in which grapevines infected by GLRaV-3 in field conditions, were more tolerant to late infection of 40. Although poorly explored in grapevine, the simultaneous presence of pathogens can lead the plants to develop a state of readiness or priming that enables earlier, faster and stronger defence responses to a subsequent attack as reported in other plant species41. To further explore these aspects, target genes were selected to investigate whether molecular defence priming can be activated in two different grape genotypes.The complex tripartite interactions have not yet been sufficiently explored in terms of molecular and biochemical mechanisms activated to counteract the disease development. Plants evolved several strategies to control the immune system responses and limit the costs of resistanceP. viticola and E. necator were not always in line with the data reported for these pathogens43. This is because we chose to focus on the systemic responses induced by these biotrophic pathogens over a long period by sampling only asymptomatic leaves at Tf and we did not consider the early molecular responses in infected tissues, which were well characterized in DM42 and PM43. An example of this discrepancy was the regulation of VvPR1 and VvBgluc, two genes codifying PR proteins, which are generally upregulated in the first days after the infection of P. viticola44 and E. necator45. These genes were downregulated at Tf in ‘Nebbiolo’ and substantially not modulated in ‘Chardonnay’, because the acute phase of both pathogen infection disappeared at Tf and the systemic activation of VvPR1 and VvBgluc was likely switched off in asymptomatic tissues. This is supported by previous reports in ‘Sultana’ plants infected by E. necator, in which glucanase activity was concentrated in infected leaf areas, while activity in the uninfected areas of the same leaf was similar to those observed in healthy plants45. Stilbene synthases (VvSTSs) showed strong overexpression at Tf in all cases for both pathogens, and at Tf_EN these genes were expressed 3 to 10 times more than at Tf_PV. Stilbenes are the most important class of phytoalexins, which represent a powerful defence system against several pathogens, including P. viticola and E. necator46, and are key molecules of basal immunity in grapevine48. Defense responses involving VvSTSs persisted for a long period after inoculation with both pathogens, with higher levels after E. necator inoculation.The first important factor inducing transcriptional modulation of the genes was the time (T0 and Tf) associated with inoculation of DM and PM causal agents. In some genes at Tf, the responses induced by P. viticola, E. necator and viruses and its effect was evident for some genes at T0. In detail, vacuolar invertase VvGIN2 and VvPR1, and VvBgluc, the genes involved in stress responses, were constitutively expressed more in ‘Nebbiolo’ than in’Chardonnay’, supporting the previous findings on the specific interactions of ‘Nebbiolo’ with biotic stress49. In addition, these genes were strongly overexpressed in ‘Nebbiolo’ infected by GFLV and GRSPaV, which suggests that these viruses may activate some defence responses that primer plants more promptly to the infection by other pathogens.The grapevine genotype represents another relevant factor in the response to VvGIN2 and VvSUSY4 at T0 was downregulated in the presence of viruses, particularly in GFLV-infected plants. The induction of invertases and sucrose synthases can stimulate the change of leaves from source to sink organs50 and the accumulation of sugars can lead the plant tissues to host more easily the development of biotrophic pathogens, such as P. viticola and E. necator. Therefore, at T0, immediately before the fungal/oomycete inoculation, virus-free plants (NE_CTR and CH_CTR) with high expression of VvGIN2 and VvSUSY4 seem to be more suitable for the development of these pathogens compared to virus-infected plants, as confirmed by the DS data. At Tf, after inoculation with P. viticola and E. necator, systemic activation of these genes in asymptomatic leaves was not observed, as conversely reported in symptomatic tissues51, suggesting a specific role(s) in symptomatic tissues to counteract pathogen development. The sucrose transporters VvSTP13 and VvSUC27 were upregulated at Tf after E. necator inoculation and, in ‘Chardonnay’, virus infection significantly increased this upregulation, particularly VvSUC27 in CH_GFLV_Tf_EN. In Arabidopsis, the high constitutive level of STP13 led to an enhanced basal resistance to Botrytis cinerea52, while in grapevine, high transcription levels of VvSTP13 were induced by the rootstocks in scions that were more tolerant to DM53. In addition, the transcriptional increase of VvSUC27 associated with the downregulation of VvGIN2 is one of the responses observed in the resistant species V. amurensis after P. viticola inoculation54. Therefore, the similar regulation observed in GFLV-infected ‘Chardonnay’ could partially explain the increased tolerance to DM and PM.The interactions between viruses and grapevine genotype significantly modified the susceptibility reaction of grapes to PM and DM. In carbohydrate metabolism, the expression of All these findings, at least in our experimental controlled conditions, suggest a virus primed defence state that increases resilience against fungal/oomycete pathogens.E. necator and P. viticola in grapevine. GFLV infection caused the overexpression of stilbene synthase genes and pathogenesis related proteins  and influenced carbohydrate metabolism thus modifying the expression dynamics of sugar transporters , vacuolar invertase, and sucrose synthase (VvGIN2 and VvSUSY4) genes. These transcriptional changes can trigger the synthesis of defence compounds, improving the innate plant immune response in grapevine and making the plant prompter to respond to biotic stress. However, this does not appear to be a generic reaction to all grapevine viruses, as in GRSPaV-infected plants, the transcriptional modulation of candidate genes was not sufficient to promote protection against E. necator and P. viticola. It is likely that this increased tolerance to fungus/oomycete is an indirect effect of plant responses to viruses, such as GFLV and GLRaV-319. Additionally, ‘Nebbiolo’ showed a higher constitutive expression of some genes in response to pathogen compared to ‘Chardonnay’, confirming previous observations by Gambino et al.49. However, the hypothetical application of GFLV-infected plants within integrated pest management strategies is not feasible, because the GFLV is a harmful virus that must be absent in the grapevine propagation material according to the certification protocols. It will be interesting to broaden our understanding of these interactions which can also be investigated under field conditions for several years, and in the presence of multiple virus and fungal/oomycete infections.In this study, infection by GFLV reduced the disease severity caused by V. vinifera cv. Nebbiolo and Chardonnay were regenerated from somatic embryos obtained from in vitro culture of immature anthers collected in spring (May 2016), according to the previously described protocol36. The regenerated plantlets were micropropagated by sub-culturing apical cuttings thus giving rise to individual lines55. Virus and viroid infections were assessed by RT-PCR assays as described below. A single virus-free line originated from a single somatic embryo was selected, multiplied, and used for subsequent analyses.Virus-free plantlets of GFLV or GRSPaV were transmitted to virus-free ‘Nebbiolo’ and ‘Chardonnay’ lines by in vitro grafting. Woody cuttings from plants infected only by GFLV or GRSPaV were collected during winter and forced to sprout in water at room temperature. Herbaceous green branches (about 5 cm) emerging from the buds were collected, surface-sterilized for 15 min with sodium hypochlorite (1.5% available chlorine) and rinsed several times with sterile distilled water. Ten apical cuttings (3–4 cm long) for each virus-free line were grafted on virus-infected herbaceous green branches and maintained under in vitro conditions. After 1 month, the virus infection was assessed by RT-PCR and a single apical cutting of ‘Nebbiolo’ and ‘Chardonnay’ infected by GFLV or GRSPaV were micropropagated by repetitively sub-culturing apical cuttings.For each thesis, ‘Nebbiolo’ and ‘Chardonnay’ virus-free , infected by GFLV , and infected by GRSPaV , 16 plantlets were acclimated out of in vitro conditions in 1 litre pots (10 × 10 × 12 cm) using a peat substrate  and placed in the greenhouse  following the protocol of Glasa et al.57. Multiplex RT-PCR assays reported by Hajizadeh et al.58 were adopted for the detection of five viroids: hop stunt viroid (HSVd), grapevine yellow speckle viroid 1 and 2 , australian grapevine viroid (AGVd), and citrus exocortis viroid (CEVd). One accession of ‘Nebbiolo’, originally infected by GFLV and GRSPaV, was previously subjected to sanitation. The in vitro thermotherapy and meristem tip culture sanitation techniques were used to eliminate GFLV from some explants and GRSPaV from other ones, thus obtaining some lines still infected by only one of these two viruses, as confirmed by RT-PCR. The two plants were used for the grafting experiments described above.The major viruses and viroids commonly present in wine grape cultivar grown in north western Italy are GFLV, GVB, grapevine fleck virus (GFkV), grapevine leafroll associated virus-1, -2, -3 , grapevine–virus A (GVA), arabis mosaic virus (ArMV) and GRSPaV. These viruses were detected by multiplex RT-PCR reported in Gambino49. PCR was carried out using Phusion Hot Start II High-Fidelity DNA Polymerase  according to manufacturer’s instructions. For GFLV, primers GFLVpoly5238Fw and GFLVpoly6048Rev reported by Pacifico et al.33 targeting 829 bp of the putative RNA-dependent RNA polymerase (RdRp) (1EPol) gene localized in RNA1 of the virus were used (Supplementary Table 31 targeting 476 bp of the genomic region encoding the RdRp domain were used (Supplementary Table https://www.ebi.ac.uk/Tools/msa/muscle/). Phylogenetic analysis based on the neighbour-joining (NJ) method  was carried out using MEGAX software version 10.1.759.To characterize the isolates of the two viruses, RT-PCR amplification products were purified and sequenced by Sanger sequencing, as reported previouslyP. viticola and E. necator), four replicates (two plant/replicate) with a randomized block design were used for each infected or virus-free plant for both genotypes. Three months after growing in greenhouse conditions, the plants were artificially inoculated with P. viticola and E. necator collected in Piedmont . The infected leaves with P. viticola from ‘Chardonnay’ plants and of E. necator from ‘Moscato’ plants were shaken in 100 ml of sterile deionized water, and each suspension obtained was adjusted, with the aid of a haemocytometer, to 5 × 103 sporangia/ml and 1 × 104 conidia/ml, respectively. The artificial inoculation of the pathogens was carried out for each trial throughout nebulisation with a laboratory spray bottle (10 ml of capacity) of one ml of suspensions/plant. All plants artificially inoculated with P. viticola or E. necator were maintained in two different greenhouse compartments in order to avoid cross contamination, at temperatures ranging from 20 and 25 °C and relative humidity maintained close to 85–90% for P. viticola and 25–27 °C for E. necator without overhead irrigation. The dates of the different operations carried out are reported under Supplementary Table Two independent trials were carried out in the greenhouse at the University of Turin, Agroinnova Competence Center, located in Grugliasco, in the Northwest of Italy  in summer 2018 and 2019. For each trial and pathogen (P. viticola (PP 1.31) or E. necator (PP1.4) by visually check 10 to 25 leaves / plant for each plant. DS was calculated using the formula: ∑ / .About 10–25 leaves/plant were visually estimated by rating the percentage of affected leaves  for both pathogens. The leaf area affected by the pathogen  was estimated using the severity scale 1–7 corresponding to 1= no symptoms; 2 < 5%; 3 = 5–10% 4 = 10–25%; 5 = 25–50%; 6 = 50–75% and 7 > 75% as reported by the EPPO / OEPP protocols for P. viticola and E. necator), leaves were collected before the fungal inoculation (T0) and on the day of the final assessment of DS and DI of fungal/oomycete diseases (Tf) from three biological replicates for a total of 72 samples  . DNase treatment, first-strand cDNA synthesis, and real-time PCR were carried out as reported by Chitarra et al.53. The results were calculated as expression ratios  relative to NE_CTR at T0.Fifteen genes representative of the most important molecular pathways involved in the response to biotic agents in grapevine were analysed , and their interactions on DI and DS. When the effects of the tested factors were significant (p ≤ 0.05) and interactions were observed among the considered factors, the means were separated by Tukey’s honestly significant difference test (HSD) at p ≤ 0.05.DI and DS were analyzed using p ≤ 0.05. The standard deviation (SD) of all means were calculated.For RT-qPCR data, when ANOVA indicated that either genotype , virus , and time  factors or their interaction was significant, mean separation was performed using the Tukey’s HSD test at a probability level of Supplementary materialSupplementary Table S1Supplementary Table S2


Flaviviruses such as Dengue (DENV) or Zika virus (ZIKV) assemble into an immature form within the endoplasmatic reticulum (ER), and are then processed by furin protease in the trans-Golgi. To better grasp maturation, we carry out cryo-EM reconstructions of immature Spondweni virus (SPOV), a human flavivirus of the same serogroup as ZIKV. By employing asymmetric localised reconstruction we push the resolution to 3.8 Å, enabling us to refine an atomic model which includes the crucial furin protease recognition site and a conserved Histidine pH-sensor. For direct comparison, we also solve structures of the mature forms of SPONV and DENV to 2.6 Å and 3.1 Å, respectively. We identify an ordered lipid that is present in only the mature forms of ZIKV, SPOV, and DENV and can bind as a consequence of rearranging amphipathic stem-helices of E during maturation. We propose a structural role for the pocket and suggest it stabilizes mature E. Here, the authors provide cryo-EM structures of mature and immature Spondweni virus, defining the furin recognition site at high resolution, and identifying a lipid that binds E upon capsid maturation and is also present in Zika and Dengue virions. Flavivirus contains many important mosquito-borne human pathogens, such as dengue virus (DENV) and Zika virus (ZIKV). Flaviviruses represent a significant economic and health-care burden to affected countries, particularly in Southeast Asia and South America1. The surface of mature, infectious flaviviruses is composed of an icosahedral shell of 90 flat-lying envelope (E) protein dimers4, the main target of humoral immunity7. E contains the fusion peptide, which is responsible for insertion into host membranes during infection8. Within infected host cells, progeny immature viruses bud into the lumen of the endoplasmatic reticulum (ER) and follow the secretory pathway through the Golgi apparatus3. In contrast to their mature counterparts, immature virions possess 60 protruding spike-like trimers of precursor membrane (prM)-E complexes (prM3E3)10. The pr domain of prM binds to E, obscuring the fusion peptide and thereby preventing unproductive fusion within the already infected cell11. The M domain of prM is embedded in the membrane and connected to pr via a flexible linker. When immature virions reach the low-pH environment of the trans-Golgi network (TGN), the trimeric prM3E3 spikes rearrange into the flat-lying conformation, characteristic of the mature virus13. The TGN-resident protease furin14 then cleaves a recognition site between pr and M. pr remains associated with E, inhibiting its fusion function, until the virus is transported outside of the cell. There, the neutral pH environment triggers the dissociation of E and pr, rendering the virus infectious12.The genus 16, hampering our mechanistic understanding of the maturation process. Here we investigated flavivirus maturation by high-resolution cryo-EM. We chose to carry out this study using Spondweni virus (SPOV)17, a human flavivirus that is closely related to ZIKV (~75% amino-acid identity) and has remained structurally uncharacterized until now. Owing to their similarity, SPOV was initially misclassified as a strain of ZIKV in neutralization tests19. SPOV was identified in Sub-Saharan Africa, but has recently been detected in Culex quinquefasciatus mosquitoes in Haiti, indicating that the virus may be adapting to vectors that prefer human hosts and that the epidemiology may therefore be changing21. In addition, mutual infection enhancement between DENV, ZIKV, and SPOV by immune sera has been demonstrated in cell culture22. We have used localized reconstruction23 and focused refinement to obtain a 3.8 Å resolution cryo-EM structure. In contrast to previous cryo-EM studies, the improved resolution enabled the building of a refined atomic model for (prM)-E, including the crucial furin protease recognition site. For direct comparison, we have also carried out cryo-EM reconstructions of mature SPOV (2.6 Å resolution), and DENV (3.1 Å resolution). Our data resolve how the furin recognition site on pr interacts with neighboring E and thus is sequestered in the trimeric spike and how prM is anchored to E via a strictly conserved and mutation-sensitive histidine. Comparison with mature virus reveals that rearrangement of amphipathic helices in E during maturation leads to the formation of a binding pocket for a lipid headgroup, which is present in ZIKV, SPOV, and DENV. We propose the lipid stabilizes the conformation of membrane-associated helices of E and the binding site may be a potential target for the development of antiviral compounds. Based on our structures, we suggest a function for the immature trimeric state of flavivirus glycoproteins, and a pathway for glycoprotein rearrangement during maturation.Structural studies of immature flaviviruses via cryogenic electron microscopy (cryo-EM) have thus far been limited to low to medium resolutions10.To facilitate the collection of high-quality cryo-EM data outside of biosafety containment, we developed an optimized UV-exposure protocol that completely inactivates virus, while fully maintaining sample integrity , in which the three pr domains sitting at the tips of E come together and interact  and two transmembrane (TM) helices  of an E protein residue, which in our structure is responsible for sequestering the furin site , allowing us to examine which changes occur during maturation. We selected mature particles and reconstructed a map to 2.6 Å resolution Fig. . The virers Fig. , formingers Fig. . The den30. We also find that a β-hairpin (residues 244–257) of E, located at the interface to the pr domain, shifts its position in the immature virus to optimally bind the pr domain  and a histidine (His447) is found packing against the lipid, in close vicinity to the phosphate group of PE . Although the map is lower resolution than that of SPOV, we can observe prominent density for the lipid in the identical position in ZIKV  in the trans-Golgi leads to release of E, allowing relaxation into the flat-lying conformation. A topologically feasible, tentative model of the immature-mature reorganization is presented in Supplementary Movie A remarkable reorganization of the flavivirus protein shell occurs during maturation and, with no structures of intermediates between the trimeric and dimeric arrangements of E available, the trajectory of this process remains undetermined. Previously, it has been speculated that large-scale rotations of the E ectodomains might be involved in reorienting immature glycoproteins into their mature positions. It has been argued that in the course of this process the prM linker acts as a “drawstring” and pulls on the ectodomains, thereby rotating them. However, simultaneous pivoting of multiple ectodomains in neighboring asymmetric units of the virion is, in our view, sterically problematic and the energetic driver of the pulling motion is unclear. Instead, we anticipate that simple glycoprotein translations in the fluid lipid bilayer impose a much lower energy barrier and are sterically feasible. We propose that translations of glycoproteins in the immature-mature transition make use of the available space in the membrane at the five- and threefold vertices Fig.  to repos11 (as in the ER or early Golgi), when the virus is in the spiky conformation. This led to the suggestion that the protease recognition site is not exposed. Later modeling based on low resolution data inferred that the furin recognition motif is indeed sterically blocked in the immature conformation31. In the current study, our map resolution allows us to elucidate the molecular basis of furin site occlusion within the prM3E3 trimer. However, furin protease is inactivated by binding its prosegment before reaching the low pH of the TGN, where the inhibitory prosegment is released14. If furin is inactive in the ER and early Golgi this begs the question why it is necessary for the virus to obscure the furin site in these early compartments. However, previous studies have shown that furin is occasionally responsible for some proteolytic processing in the ER or early Golgi, for instance, if the substrate outcompetes the inhibitory prosegment38. We suggest that occlusion of the furin site within immature trimers is a critical adaptation for the virus because otherwise high levels of virus expression within an infected cell, combined with high local concentrations of furin recognition motifs on a viral surface could lead to some cleavage events in the ER or early Golgi. As the pH is neutral in these compartments, the affinity of cleaved pr to E would be low and some pr could dissociate. In turn, this would lead to partially matured virus (which is fusion competent3) reaching the low-pH TGN, where unproductive fusion would be triggered prematurely.Early work on flavivirus maturation has indicated that furin cleavage of prM does not occur at neutral pH35, DENV2, ZIKV, and SPOV all possess a lipid-like density clenched between the membrane-associated helices of their glycoproteins. The high-resolution map for SPOV allows us to assign the density to a PE type lipid. Previous computational modeling studies, that took lipidomics into account29, have estimated that there are around 2400 PE lipids in the dengue virion. Tight binding of 180 PE molecules by all copies of E would constitute the immobilization of 7.5% of total PE in the virion, a substantial amount. This lipid site may be involved in the stabilization of the mature virus, preventing the space between membrane-associated helices from collapsing and ensuring their correct orientations. Given the observation of a structurally equivalent lipid in alphaviruses, it is tempting to speculate that this is a strategy common among enveloped viruses. Furthermore, a highly conserved and mutation-sensitive histidine  is located adjacent to the lipid, suggesting a pH-dependent role during the viral lifecycle. Just as small molecules mimicking a lipid pocket factor in picornaviruses have similarly been successfully employed as viral inhibitors41, we suggest that the pocket may be a potential target for the development of antivirals.We have shown that, in addition to Sindbis alphavirusg for 90 min at 4 °C. Pelleted viral particles were resuspended in cold NTE buffer , cleared by centrifugation at 3200 × g, 5 min, 4 °C, layered over a 22% sucrose cushion and centrifuged at 175,000 × g (Beckman Coulter Sw41) for 2 h at 4 °C. The pellet was resuspended in cold NTE buffer and kept overnight at 4 °C and was subsequently applied to a 10–35% potassium tartrate step gradient. The virus-containing fraction was collected and buffer exchanged into NTE buffer by multiple rounds of dilution and centrifugation using a 100 kD cutoff centrifugal filter device (Amicon Ultra).SPOV strain SM6 V-1s and DENV2 strain 16681 were propagated in C6/36 cells  maintained in L15 (Thermo Fisher Scientific) supplemented with 1.5% heat-inactivated fetal calf serum, 1 mM glutamine, and penicillin/streptomycin. To prepare virus for cryo-EM analysis, cell-free virus supernatants were concentrated by polyethylene glycol (PEG) precipitation using PEG-8000, to a final concentration of 8%. The supernatant/PEG mixture was stored overnight at 4 °C before centrifugation at 3200 × To identify optimal UV-exposure conditions for inactivation, virus samples were placed into a UV crosslinker (Uvitec) for different periods of time (from 15 s to 5 min). UV-inactivated virus samples were added to a Vero cell monolayer and incubated for 2 h. Non-UV-inactivated virus was used as a control. A plaque assay was performed by topping up the cells with 1.5% carboxymethyl cellulose and incubation at 37 °C for 3 days to allow virus infection. Finally, viral foci were visualized via the cross-reactive mouse monoclonal antibody 4G2 , followed by anti-mouse immunoglobulin conjugated to horseradish peroxidase and development by DAB  substrate. Although we saw complete inactivation after the minimal exposure of 1 min, we doubled this inactivation time to 2 min for electron microscopy samples. Following inactivation, a volume of 3.5 µl of purified SPOV was pipetted on glow-discharged Quantifoil holey carbon grids  and was blotted for 3.5 s before flash-freezing in liquid ethane using a Vitrobot mark IV (FEI). For DENV, Protochips carbon-coated copper C-flat grids  were used. A sample of the quality of UV-inactivated virus is shown in Supplementary Fig. Cryo-EM movies for SPOV were collected at the Electron Bio-Imaging Centre (eBIC) at Diamond Light Source, UK. A 300 kV Titan Krios instrument (Thermo Fisher Scientific), equipped with a K3 (Gatan) direct detector and a GIF Quantum energy filter (Gatan) with 20 eV slit width was utilized. Data were collected automatically using SerialEM 3.7 in super-resolution mode (0.4225 Å per pixel). DENV2 data were collected at the Division of Structural Biology, Oxford, UK, on a 300 kV F30 Polara (Thermo Fisher Scientific) equipped with a K2 Summit (Gatan) direct detector  and a GIF Quantum energy filter (Gatan) with 20 eV slit width. Data collection parameters are summarized in Table 42, and 2× binning was applied for super-resolution data. Contrast transfer function (CTF) parameters were estimated using Gctf-v.1.0643 (SPOV) and CTFFIND444 (DENV2). Particles were picked using cryoSPARC v.2.12.045 (SPOV) and Ethan1.246 (DENV2). Subsequent classification and refinement steps were performed in RELION3.147. Icosahedral reconstruction (I1 symmetry) of immature SPOV and mature SPOV/DENV2 was carried out using standard methods. In brief, the initial particle sets were cleaned via 2D/3D classifications runs, followed by masked 3D refinement and post-processing. CTF refinement and Bayesian polishing in RELION3.1 were used to further improve the density and resolution of the reconstructions. The numbers of initial and final particles are summarized for all samples in Table Motion correction of cryo-EM movies was carried out using MotionCor2-1.1.023 within Scipion v.2.049 to obtain coordinates, CTF parameters, and orientations of individual spike subparticles and carried out processing of subparticles in RELION. An overview of the workflow is shown in Supplementary Fig. 6, and mature patches on such hybrid particles may also contribute to heterogeneity. We selected two classes with a total of 371,915 subparticles for further processing. A second round of classification, followed by masked refinement and post-processing, yielded a 4.2 Å resolution map of the prM3E3 trimeric spike reconstructed from 305,017 subparticles. Although the quality of the density had dramatically improved by localized reconstruction, we attempted to improve the resolution further to aid in model building and refinement. We performed focused classification and refinement on prM1E1 monomers and finally obtained a map at 3.8 Å resolution, reconstructed from 281,619 subparticles. This map served as the basis for initial building of an atomic model, while the 4.2 Å resolution map of prM3E3 was used to refine an atomic model of the full trimer. Local resolution values of the immature localized reconstructions and map vs model FSC plots are shown in Supplementary Fig. To acquire high-resolution maps of immature spikes, we used Localized Reconstruction50 and manual building in Coot v.0.8.9.251. Model geometry was validated using MolProbity v.4.252. Geometry statistics, model B factors, and map vs model cross-correlation values are shown in Table All atomic structures in this study were refined by alternating cycles of real-space refinement in PHENIX v.1.17.153 was performed using Pfx DNA polymerase (Invitrogen) according to the manufacturer’s protocol. Primer sequences are listed in Supplementary Table To introduce amino-acid exchanges at the lipid pocket, site-directed mutagenesis of prM/E in a pHLsec vectorTo produce virus from DNA, five PCR products for assembly of viral constructs were amplified with Phusion® High-Fidelity DNA polymerase (NEB) according to the manufacturer’s protocol. PCR product of the expression vector was amplified with pcDNA-hCMV-R and DV1-2-3-3UTR-HDVAr-F. PCR products of cDNAs of DENV2-16681 genomes were carried out by three pairs of primers: hCMV-DV2-16681-5UTR-F and DV2-16681prM-start-R (F1), DV2-16681-Eend-F and DV2-16681-6685-6709-R (F3), and DV2-16681-6695-6724-F and DV1-2-3-3UTR-R (F4). PCR product of the pHLsec-prM/E wild-type and mutants were amplified by DV2-16681prM-start-F and DV2-16681-Eend-R (F2). All PCR products were purified using the QIAquick Gel Extraction Kit (QIAGEN).54. PCR products of the expression vector (0.04 pmol) were assembled with 0.04 pmol of each PCR product of the viral genome in the enzyme mix consisting of 5× isothermal buffer , T5 exonuclease, Taq DNA ligase and Phusion DNA polymerase (New England Biolabs). The reaction was incubated at 50 °C for 3 h.PCR products of the expression vector and viral PCR products were then assembled using the Gibson assembly technique55. In brief, to determine DENV production, virus supernatants were captured onto plates coated with antibody 4G2 and then incubated with antibody 749B12 , followed by alkaline phosphotase-conjugated anti-human IgG . Reactions were developed by the addition of para-nitrophenylphosphate substrate and were stopped by addition of NaOH. The absorbance was measured at 405 nm.Twenty microliters of the Gibson assembly reactions were diluted in Opti-Minimal Essential Media I (Thermo Fisher Scientific), mixed with Lipofectamin 2000 (Invitrogen), before transfecting human embryonic kidney 293 T cells  for 4 h at 37 °C. The medium was then exchanged to Dulbecco’s Modified Eagle Medium (Thermo Fisher Scientific), supplemented with 10% heat-inactivated fetal calf serum. After overnight incubation, the medium was removed and replaced by Ultradoma Protein-Free (LONZA). The cultured medium was harvested and replenished on the second, third, and fourth days after transfection. Cell-free supernatants were collected and stored at −80 °C. Wild-type and mutant viruses from transfected HEK-293T cells were further propagated in C6/36 cells. Medium was changed on the fourth day and then every 3 days. Viral production and titers were determined by enzyme-linked immunosorbent assay and focus-forming assay, as described previouslyFurther information on research design is available in the Supplementary InformationPeer Review FileDescription of Additional Supplementary FilesSupplementary Movie 1Supplementary Movie 2Reporting Summary


Mitigating the transmission of SARS-CoV-2 in clinical settings and public spaces is critically important to reduce the number of COVID-19 cases while effective vaccines and therapeutics are under development. SARS-CoV-2 transmission is thought to primarily occur through direct person-to-person transfer of infectious respiratory droplets or through aerosol-generating medical procedures. However, contact with contaminated surfaces may also play a significant role. In this context, understanding the factors contributing to SARS-CoV-2 persistence on surfaces will enable a more accurate estimation of the risk of contact transmission and inform mitigation strategies. To this end, we have developed a simple mathematical model that can be used to estimate virus decay on nonporous surfaces under a range of conditions and which may be utilized operationally to identify indoor environments in which the virus is most persistent. Coronavirus disease 2019 (COVID-19) was first identified in China in late 2019 and is caused by newly identified severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Previous studies had reported the stability of SARS-CoV-2 in cell culture media and deposited onto surfaces under a limited set of environmental conditions. Here, we broadly investigated the effects of relative humidity, temperature, and droplet size on the stability of SARS-CoV-2 in a simulated clinically relevant matrix dried on nonporous surfaces. The results show that SARS-CoV-2 decayed more rapidly when either humidity or temperature was increased but that droplet volume (1 to 50 μl) and surface type  did not significantly impact decay rate. At room temperature (24°C), virus half-life ranged from 6.3 to 18.6 h depending on the relative humidity but was reduced to 1.0 to 8.9 h when the temperature was increased to 35°C. These findings suggest that a potential for fomite transmission may persist for hours to days in indoor environments and have implications for assessment of the risk posed by surface contamination in indoor environments.IMPORTANCE Mitigating the transmission of SARS-CoV-2 in clinical settings and public spaces is critically important to reduce the number of COVID-19 cases while effective vaccines and therapeutics are under development. SARS-CoV-2 transmission is thought to primarily occur through direct person-to-person transfer of infectious respiratory droplets or through aerosol-generating medical procedures. However, contact with contaminated surfaces may also play a significant role. In this context, understanding the factors contributing to SARS-CoV-2 persistence on surfaces will enable a more accurate estimation of the risk of contact transmission and inform mitigation strategies. To this end, we have developed a simple mathematical model that can be used to estimate virus decay on nonporous surfaces under a range of conditions and which may be utilized operationally to identify indoor environments in which the virus is most persistent. Several studies have reported the presence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genetic material on surfaces in coronavirus disease 2019 (COVID-19) patient rooms and throughout hospital wards, indicating environmental contamination. Commonly contaminated items included office equipment  and metal furnishings  15. HoweveTwo previous laboratory studies demonstrated persistence of infectious SARS-CoV-2 on numerous surfaces over extended periods; however, each of these studies evaluated only a single set of indoor environmental conditions and used virus suspended in cell culture media. Using 50 μl of virus-containing droplets deposited onto nonporous  and porous (cardboard) surfaces at 21 to 23°C and 40% relative humidity (RH), van Doremalen et al. were able to show persistence of virus on plastic and stainless steel for up to 72 h, whereas the durations of persistence on cardboard (24 h) and copper (4 h) were much shorter . Chin et8–t1/2) for each set of experimental conditions. A single coupon loaded with 0.1-μm-diameter fluorescently labeled polystyrene latex microspheres was also collected at each time point, resuspended in buffer, and then evaluated for fluorescence. These controls showed that there was not a significant decrease in fluorescence over time (n = 6), we also quantitated viral RNA using reverse transcription quantitative real-time PCR (RT-qPCR) as a second measure of physical losses but observed that its decay rate was dependent on both temperature and humidity conditions and thus concluded that viral RNA could not be used as a physical tracer (data not shown). Although this observation requires further experimental validation, these data suggest that the utility of viral RNA for contamination surveillance may be confounded by environmental conditions.To determine the stability of SARS-CoV-2 on surfaces, virus was diluted 1:10 in simulated saliva and droplets were deposited onto stainless steel, acrylonitrile butadiene styrene (ABS) plastic, or nitrile rubber glove coupons. These surfaces were chosen to represent two common sources of fomite transmission (door knobs/handles and office electronics) and also to address the risks associated with contaminated PPE. Virus stability was measured using multiple RH and temperature combinations ranging from approximately 20 to 80% RH and 24 to 35°C. Coupons were placed in an environmentally controlled testing plenum, which maintained RH and temperature set points with low variability over a total of 32 trials. During each trial, virus was recovered from three randomized coupons into culture medium at predetermined time points over a period of 48 h. The amount of infectious virus remaining was determined using a quantitative cell-based infection assay. Virus infectivity data collected over time were fitted to statistical models to estimate the infectivity decay rate and half-life ; as a result, we did not further evaluate droplet size on ABS plastic or nitrile rubber. Comparison of levels of virus decay determined from all 32 trials, which used a variety of volumes, temperatures , and RH conditions, showed there was not a significant difference in half-life estimates between virus deposited on stainless steel, ABS plastic, or nitrile glove coupons  (Using virus deposited onto stainless steel coupons at ambient indoor temperature (24°C) and maintained using various levels of RH , we found that droplet size was not a significant factor influencing the half-life of SARS-CoV-2  . Based ot1/2 = 15.33 ± 2.75), 40% (t1/2 = 11.52 ± 1.72), 60% (t1/2 = 9.15 ± 3.39), and 80% (t1/2 = 8.33 ± 1.80) at 24°C, with the results indicating that virus at ambient indoor temperature is most stable at relatively low RH . We also evaluated the effect of temperature on the surface stability of SARS-CoV-2 (t1/2 = 7.33 ± 1.33), 40% RH (t1/2 = 7.52 ± 1.22), and 60% RH (t1/2 = 2.26 ± 1.42). Pairwise comparisons of mean half-life results between 24 and 35°C under each set of RH conditions (Student’s t test) demonstrated that these differences were significant . At 40% RH, virus decay was also evaluated at 28°C (t1/2 = 6.11 ± 3.02) and found to be significantly faster . The combination of 35°C and 80% RH could not be evaluated due to test system limitations.We estimated the mean half-life values in hours (± standard deviations [SD]) for RH of 20% (y low RH . PairwisRS-CoV-2 . When teP < 0.0001 for both). Furthermore, we were able to fit a linear regression equation (adjusted R2 = 0.71) that models the half-life of SARS-CoV-2 in simulated saliva on nonporous surfaces  (n = 4) were not included in the model, as we were able to evaluate 80% RH only at 24°C. Although our data indicate that the virus half-life was inversely proportional to increasing temperature and RH, extrapolated estimates for conditions beyond those empirically tested may not be valid.Analysis of estimated half-life data from 28 trials by two-way ANOVA demonstrated that both temperature and RH were significant factors influencing virus decay ( rubber) . This mo rubber) . Trials These data provide the first evidence that increasing temperature and/or RH decreased how long SARS-CoV-2 remained infectious on two hard, nonporous surfaces representative of commonly contaminated objects, suggesting that persistence and, ultimately, exposure risk may vary significantly depending on environmental conditions. In addition, our data on virus stability on nitrile gloves serve as a reminder that PPE, when used inappropriately, has the potential to be a source of contamination.Although indoor temperatures can be effectively controlled by HVAC  systems, in the absence of active humidity control, indoor RH is influenced by outdoor weather conditions. The data shown here are most relevant for indoor conditions as they do not address the significant effect of solar radiation upon virus decay . However1/2t) under a wider range of environmental conditions than was previously possible.While we have demonstrated the impact of RH and temperature on the stability of SARS-CoV-2 on stainless steel and ABS plastic as representative nonporous surfaces, the applicability of these results to porous surfaces  is currently unknown and bears further investigation. In addition, data regarding the amount of infectious virus present on surfaces in patient rooms are required in order to determine how long such surfaces would remain a source of virus transmission. Finally, the infectious dose of SARS-CoV-2 is currently unknown and thus it is difficult to evaluate the risk of infection following contact with a contaminated fomite. Despite these caveats, these data can be used to determine how long it would take to reduce levels of infectious virus on nonporous surfaces by a specific factor  as previously described (Vero (ATCC CCL-81) cells were used to propagate SARS-CoV-2 and in virus microtitration assays. Cells were cultured at 37°C and 5% COescribed . A ViaFi6 Vero cells suspended in 30 ml of gMEM was combined with SARS-CoV-2 at a multiplicity of infection equal to 0.01 and was then seeded into a T-150 flask and incubated at 37°C and 5% CO2. After 24 h, a suspension of 1.2 × 106 uninfected Vero cells mixed in 0.5 ml gMEM was added to the infected cells. At 72 h postinfection, the flasks were frozen at –80°C for at least 1 h and then thawed at 37°C, and a cell scraper was used to remove any remaining cells from the flask. The combination of the infected cell lysate and culture supernatant was clarified by centrifugation at 1,000 × g for 10 min at 4°C. Virus was aliquoted and stored at –80°C prior to use in experiments. The viral stocks were sequenced and found to match the consensus sequence previously described (MN985325.1). All work with SARS-CoV-2 was performed under conditions of biosafety level 3 containment.SARS-CoV-2 isolate USA-WA1/2020 (NR-52281) was deposited by the Centers for Disease Control and Prevention and obtained through BEI Resources, NIAID, NIH. This virus (passage 4) was propagated twice in Vero cells to yield the working stocks (passage 6) that were used for this work. Briefly, a volume of 2.4 × 102PO4 and K2HPO4, which were present at 15.4 mM and 24.6 mM, respectively. The simulated saliva was characterized for its pH, surface tension, viscosity, percent solids, and protein content  of SARS-CoV-2 on stainless steel or plastic, virus-laden wet droplets were deposited onto coupons, which were then incubated under various RH and temperature conditions, followed by recovery of virus from the coupons for microtitration infectivity assays. For each test performed on either grade 304 stainless steel  or ABS plastic, sterilized 19-mm-diameter circular coupons were loaded with 1, 5, or 50 μl of SARS-CoV-2 diluted 1:10 in simulated saliva. For each test performed on nitrile gloves, a 0.25-in. square cut from nitrile gloves was placed on a stainless steel coupon and loaded with 5 μl of SARS-CoV-2 diluted 1:10 in simulated saliva. The coupons were immediately placed into a custom 20-port temperature- and relative humidity-controlled plenum supplied with constant airflow at 5 liters/min. Humidity control was performed by mixing streams of HEPA-filtered dry and humidified air with mass flow controllers (Alicat Scientific). Humid air was generated by circulating ultrapure water through Nafion gas humidification bundles (Perma Pure). For temperatures in the range of 24°C, the plenum was equilibrated to room temperature. For temperatures outside this range, the plenum temperature was controlled by circulating a sealed loop of chilled or heated water around the plenum. Temperature and RH were monitored continuously via temperature/RH probes inserted directly into the plenum , and data were collected with either LabVIEW  or Vaisala Insight PC software. During the experiments, temperature fluctuated ±2°C and RH  was controlled at ±10%. In each experiment, the initial virus titer was determined by immediately placing virus-laden coupons into 4 ml gMEM, vortex mixing for 30 s at 2,400 rpm, and then quantifying infectivity via microtitration assay as described below. To control for physical efficiency and sample extraction, 0.1-μm-diameter green fluorescent polystyrene latex microspheres  diluted 1:1 in gMEM were spotted onto coupons , incubated in the test plenum with the virus-laden coupons, and recovered as described for the virus, and fluorescence (expressed in relative fluorescence units [RFU]) was measured on a GloMax luminometer (Promega). Viral RNA was quantified (as described below) in a subset of the samples. All coupons were processed identically at 0.75-, 3-, 9-, 24-, and 48-h time points.−1 through 10−4) in 96-well, clear-bottom plates containing confluent monolayers of Vero cells. For each dilution, a total of 10 replicate wells were infected. The infected plates were incubated at 37°C and 5% CO2 for 4 days, and then individual wells were visually inspected using a Nikon TS100 microscope for the presence of virus-induced cytopathic effects (CPE) at each dilution compared to a negative (medium only) control. The median 50% tissue culture infectious dose (TCID50) was estimated for each of the samples using the Spearman-Karber method  gene. Briefly, viral RNA from test samples was isolated and purified using the Qiagen Viral RNA Mini Kit centrifugation protocol per the manufacturer’s instructions. The PCR master mix was composed of sterile, molecular biology-grade water, 1× SuperScript reaction mix, 1× SuperScript reverse transcriptase, 0.2 μM forward and reverse primers, and 0.1 μM 6-carboxyfluorescein (FAM)-labeled fluorescent probe. The primers and probe were based on primer and probe sequences published previously by Corman et al. (TC]). Data representing numbers of viral RNA copies present in test samples per milliliter were interpolated from the standard curves.SARS-CoV-2 RNA was quantified by a reverse transcription quantitative real-time PCR (RT-qPCR) assay using an Applied Biosystems 7500 Fast real-time PCR instrument and SuperScript III one-step RT-PCR MasterMix with Platinum n et al.  but wereTrials performed on both stainless steel and ABS plastic encompassed a complete design space that included 5-μl droplets incubated at 24°C , 28°C (40% RH), and 35°C . On stainless steel, additional trials were performed using 1-μl and 50-μl droplets at 24°C . For analyses using nitrile glove rubber, a reduced design space was utilized that permitted comparison between surface types, and trials were performed at 24°C (20 and 60% RH) and 35°C (20 and 60% RH).ANOVA was used to compare the effects of droplet size and surface on the decay half-life. Bartlett’s test was used to show that there was no statistical difference in half-life variance across the droplet size or surface type data. Analysis of the data indicated that virus decay was biphasic and was related to the drying time of virus-containing droplets. Thus, each data set was divided into a wet phase and a dry phase. For 20, 40, and 60% RH, the wet phase was defined from time point 0 to 0.75 h and the dry phase was from time point 0.75 h to 48 h. For 80% RH, the wet phase was defined from time point 0 to 3 h and the dry phase was from time point 3 h to 48 h. These estimates were based on observations of droplet drying during experiments as well as on RH data collected from the surface test decay system (data not shown).50 data, and a linear model was fitted using MATLAB’s FITGLM with an identify link function and a normal distribution. Points that were below the assay’s limit of detection (0.2 log10 TCID50/ml) were removed from the analysis. The half-life (t1/2) was also determined as follows:A logarithm (base 10) transformation was applied to the TCIDTx represents temperature in degrees Celsius, xRH represents percent relative humidity, and the β terms represent coefficients:Regression analysis was applied to determine the dependence of the virus half-life on temperature and relative humidity. The following model was initially assumed where F-test on the ratio of the SSE with and without that predictor; the most statistically insignificant predictor (if there is one) is then removed, and the process is repeated until no further predictors can be removed without a statistically significant loss in the SSE. After determining the model, an F-test was performed to confirm that the model provided a better fit than an intercept-only model. The fitted residuals were then analyzed to verify the absence of any apparent structure, and the Shapiro-Wilke test was used to verify residual normality. The final model for the decay of SARS-CoV-2 on stainless steel, ABS plastic, and nitrile rubber as a function of temperature in degrees Celsius and percent relative humidity is as follows:Stepwise regression (MATLAB STEPWISELM.M) was then used to identify and remove predictors that were insignificant using a backward elimination approach. The stepwise procedure starts with the full model and then measures the contribution of each predictor to the residual sum of squared errors (SSE) using an It should be noted that this model is valid for the range of conditions tested; extrapolation outside the range of temperature and relative humidity values tested is not advised and should be done with caution.GraphPad Prism version 8.4 was used to generate graphs. JMP Genomics version 9.1 was used to prepare a contour plot of half-life versus temperature and RH.


This study aimed to evaluate and quantify the different factors affecting the costs of mastitis in cattle, to quantify the annual and monthly financial losses attributed to mastitis, and to estimate production losses using average linear scores found on The Dairy Herd Improvement Association somatic cell count (SCC) sheets and bulk tank SCC per lactation number.All data (bovine mastitis-associated costs and expenditures in Egyptian dairy herds) were analyzed using SPSS/PCT, 2001. A partial budget technique using spreadsheet software and the general linear model procedure was used to analyze the productive and financial measures.3. The annual subclinical mastitis (SCM)-related economic loss was 21,933,258.6 LE, and the two most important cost components were the subsequent decrease in milk production and quality premium losses . The quality premium loss was 1,369,602.1 LE. On the other hand, the annual economic loss due to decreased milk production as a result of SCM was 20,563,656.5 LE. The total cost of 80 CM cases, including the failure and preventive costs, was 1,196,871.4 LE, including 1,169,150.4 LE failure costs  and 27,721.0 LE preventive costs. The average cost per CM case was 28,760.9 LE, including veterinary time and consultation fees of 250.0 LE (1%), labor 562.5 LE (2%), premature culling 736,000.0 LE (77%), decreased milk production 4085.18 LE (13.7%), discarded milk 185.3 LE (1%), and drugs and treatments 328.9 LE (1%). The total costs of CM (expenditures) extra control and preventive measures, including the diagnosis of CM for 80 cows annually in 20 Egyptian dairy farms were 27,721.0 LE, representing 346.5 LE or 1% of the total cost of CM cases. The cost of monitoring and diagnostic measures was 8635.2 LE, representing 107.9 LE or 1% of the total cost of a case of CM.Mastitis was present in 57.1% of cows (240/420), while clinical mastitis (CM) was present in 19% of them (80/420). The geometric mean of SCC/ml in bulk tank milk samples of 150 cattle dairy farms was 556.3×10The method used for cost estimation, in this study, is highly adaptable to individual cattle farms and had a major role in assessing specific control and management measures. The concepts described in this paper help to improve our understanding of the full economic impact of clinical and subclinical mastitis in cattle in Egypt. Assessing the economic losses from mastitis to determine the economic costs and losses occurring in Egyptian dairy farms is critical for encouraging farmers to acknowledge the scale of the problem and implement effective management practices aimed at improving mastitis control and reducing the associated costs. Bovine mastitis is regarded as one of the most economically damaging diseases in the dairy industry globally -3. ThereSCM has been shown to be responsible for most of the economic losses due to mastitis, with a reduction in milk production being the main factor. It has been reported  that theAgainst this background, the main objectives of this study are to quantify and assess the annual herd economic losses caused by bovine clinical and subclinical mastitis within large Egyptian dairy herds. These objectives are often achieved using the following approaches: Quantification, determination, and evaluation of the incidence and prevalence of clinical and subclinical mastitis in the populace, which is essential for estimating its actual cost to the dairy industry. In addition, identification of the preventive and therapeutic measures undertaken is required. It is usually easy to calculate the expenditure on mastitis control, through collection and analysis of various sources of data, substances and records, the scientific literature, regulations, reviews, reports, suggestions, and recommendations, as well as websites and the internet. Evaluation and quantification of the different factors affecting CM costs are also important, including estimation and evaluation of selected factors associated with current expenditures for mastitis treatment and control and mastitis-associated output losses, including factors related to drugs, discarded milk, veterinary services, labor, product quality, diagnostics, reduced milk yield, and culling. Moreover, there is a need for quantifying annual and monthly financial losses attributable to SCM and estimating production losses using average linear scores found on The Dairy Herd Improvement Association (DHIA) SCC sheets and BTSCC per lactation number.The approval from the Institutional Animal Ethics Committee to conduct this study was not required as no invasive procedure on the animals was performed. However, this study was conducted in accordance with the standards of Institutional Animal regulations and Ethics of Animal Reproduction Research Institute, Haram, Giza, Agricultural Research Center.A cross-sectional study was conducted from 2017 to 2019 on field data collected during field Ph.D. study entitled “Epidemiological studies on bovine mastitis in Egypt in Delta Region, Alexandria Road and the upper and lower Egypt Districts.” Samples were collected from a total number of 170 private, dairy cattle farms with total population number 9810, lactating cows belonging to Cairo, Giza, Qalubia, Sharkia, Monofia, Alexandria, Behera, Dakahlia, Bensulf, Fayoum, Sohag, and Aswan and Asuit governorates.The study animals were dairy cattle originating from intensive dairy herds. The intensive dairy farms contained cattle from small, medium, and large dairy herds. The farms were selected based on the availability of lactating cows within the farm and the owners’ willingness to participate. Economic data were obtained from relevant records stored at those farms. Cow parity was defined as follows: Primiparous (first-lactation) and multiparous (second lactation or later). Cows were machine-milked 3 times a day in milking parlors. Teats of cows were sanitized by dipping them in a 0.5% iodine teat dip before and after milking. The age of the cows was between 2.5 and 6 years in different stages of lactations. A total of 420 composite milk samples and 1680 quarter milk samples were collected from 420 lactating animals at 20 cattle dairy farms, having 1178 dairy cows, among which 80 showed clinical signs of mastitis. Overall, 150 bulk tank milk samples were collected from 150 cattle dairy farms, derived from a total of 8667 dairy cows. The udder was examined clinically in accordance with methods described previously -20. The All variables needed for the economic model were determined in line with previously described methods . The maiThe mastitis economic framework was carried out in accordance with previously described methods ,23-26. AVeterinary services, time, and consultation fees were quantified in accordance with previously described methods ,24,26. TAssessment of drug and treatment costs was carried out in accordance with the methods described previously ,26,27. TThe economic losses from discarded milk were calculated in accordance with the methods described previously ,23,24,26Labor costs were quantified in accordance with the methods described previously ,23,24,26Milk production yield losses were quantified in accordance with the methods described previously . SubclinQuality premium loss and penalties were quantified in accordance with the methods described previously ,23,26. APremature culling costs were calculated in accordance with previously described methods ,24,26,28The costs of diagnostic and monitoring measures could be calculated in accordance with previously described methods ,26. ThisThe costs of control measures were quantified in accordance with previously described methods ,26,27. TAll data (bovine mastitis-associated costs and expenditures in Egyptian dairy herds) were analyzed using SPSS/PCT, 2001, in accordance with previously described methods . A parti3.Clinical and subclinical CM was found in 57.1% of cows (240/420). CM was found in 19% of cows (80/420). The geometric mean of SCCs/ml in BTM samples of 150 cattle dairy farms was 565.3 × 10Analyzing the economic losses due to SCM revealed that the annual SCM economic loss was 21,933,258.60 LE . It alsoThe quantification of the economic losses due to CM for 80 CM cases from 20 dairy cattle farms revealed that the total cost of these cases, including the failure and preventive costs, was 1,196,871.4 LE, including 1,169,150.4 LE for failure costs  and 27,721 LE for preventive costs.The quantification of the veterinary time and consultation fees for CM revealed that the mean cost of veterinary time and consultation fees was 250 LE per case. The cost of veterinary time and consultation fees represented 1% of the total costs per CM case. The mean cost of labor was 562.5 LE per CM case  extra control and preventive measures, including the diagnosis of CM for 80 cows annually in 20 Egyptian dairy farms, was 27,721 LE, as shown in The mean drug and treatment cost of 80 CM cases were 328.85 LE per case, as shown in Tables-The main goals of this study were to evaluate the economic costs of cattle CM and SCM in Egyptian dairy farms and to determine the distribution among the various cost components. Mastitis is regarded as the most common disease of dairy cows and causes great economic loss to the dairy industry ,30,31. TThe indirect losses of SCM do not seem to be well known and visible among many farmers. It is mostly accepted that SCM is responsible for the majority of economic losses due to mastitis . The twoThe annual economic loss due to SCM was 21,933,258.6 LE. The two most important cost components were the subsequent decrease in milk production and quality premium losses .The results of assessing quality premium losses for 150 cattle dairy herds with a total of 8867 cows gave a value of 1,369,602.12 LE. As shown in the tables, these quality premiums are mostly based on SCC because SCC reflects the inflammatory process and thus the changes in milk composition. Most milk purchasers select milk with low SCC and offer financial incentives to farmers for high-quality milk. In the dairy industry, all over the world, penalty and premium programs have been designed to produce incentives and motivations for dairy producers to boost milk quality . Most ofOn the other hand, the annual economic loss due to decreased milk production as a result of SCM was 20,563,656.5 LE, which is close to a previously reported value of $800,000 . A decreThe results of quantifying the economic losses of CM for 80 CM cases from 20 dairy cattle farms revealed that the economic impact per case of CM could be classified into two main categories: Failure costs and preventive costs. Failure costs were, in turn, classified into two main categories: Direct and indirect costs. Direct costs include diagnostic testing, therapeutics, discarded milk, veterinary services, and labor. Indirect costs include future milk production loss and costs related to premature culling. Preventive costs are related to preventive measures in terms of equipment, consumables , and the use of other resources to prevent diseases (increased labor) . The totThe mean cost of veterinary time and consultation fees of CM was 250 LE per case. This result is discordant with previously reported values of $4  and €54 The mean cost of labor related to CM was 562.5 LE per CM case, which matches previously reported values of €24  and $21 Indirect CM costs such as premature culling costs are complicated to determine . The cosst month after CM was assumed to be reduced by 40% in primiparous and 50% in multiparous cattle. The second parity multiparous cows are at a higher risk of developing CM  and suffer more severe milk yield loss than primiparous cows , which agrees with the previous findings  and BTSCC per lactation number. It gives insight into current market conditions and management practices in Egypt. The prevalence of cattle mastitis (CM and SCM) in individual cows at the examined dairy farms was 57.1% (240/420). CM was found in 19% of cows (80/420). The analysis of BTM samples for SCC revealed that the geometric mean of SCCs/ml in BTM samples at 150 cattle dairy farms was 565.3×10MFA and HMY designed the concept of the review article, and MFA and HMY designed and performed study design and the economic framework. SAE drafted and revised the manuscript. All authors read and approved the final version.