Chrysanthemum is an important functional plant that is used for food, medicine and tea. Functional chrysanthemums become infected with viruses all around the world, seriously lowering their quality and yield. Viral infection has become an important limiting factor in chrysanthemum production. Functional chrysanthemum is often propagated asexually by cutting during production, and viral infection of seedlings is becoming increasingly serious. Chrysanthemums can be infected by a variety of viruses causing different symptoms. With the development of biotechnology, virus detection and virus-free technologies for chrysanthemum seedlings are becoming increasingly effective. In this study, the common virus species, virus detection methods and virus-free technology of chrysanthemum infection are reviewed to provide a theoretical basis for virus prevention, treatment and elimination in functional chrysanthemum. Chrysanthemum × morifolium Ramat.), a perennial herb of the genus Chrysanthemum in the family Asteraceae, has a history of 4000 years in Chinese written records [Chrysanthemum  and is the most commonly used and easiest-to-perform method in scientific research experiments. It can detect Peake (pg) or even Feke (fg) grade  and can RT-PCR involves reverse transcribing the target RNA virus into cDNA and then amplifying it, resulting in the exponential amplification of minute viral nucleic acids. DNA viruses can be directly amplified using PCR. This technique achieves detection sensitivity at the Feke (fg) level , featuriSingle RT-PCR refers to the extraction of RNA from tissue samples and reverse transcription into cDNA combined with a viral primer for RCR amplification to detect whether the plant contains a virus. However, this method can only be used to test for a single virus species. This technique was used to identify and detect TAV , TMV 8686 and CVThe operation process and reaction principle of multiple RT-PCR are no different from those of conventional PCR. Multiple viral primers can be added into a reaction system for reverse transcription with two or more target DNA or RNA regions for simultaneous detection of multiple PCR targets . BecauseFluorescence quantitative RT-PCR (qRT) is a method that combines PCR technology with fluorescence detection. The principle is to use probes that can specifically bind PCR products and label fluorescent substances. The probes mainly include molecular beacon probes, hybridization probes and TaqMan fluorescence probes. At present, Taq DNA polymerase 5’ exonuclease activity is often used to degrade it in the reaction process, and the fluorescence groups are far away from the quenched groups to emit fluorescence to realize the monitoring of the whole process. Liu established a real-time PCR system for the detection of CVB, TAV, CMV, TMV, PVY, CChMVd and CSVd . The sevCommon PCR techniques are able to detect very low contents of target genes, but in some cases, the virus content is extremely small, the target genes are unstable, and the product amplified by the primary primer still cannot be detected in gel electrophoresis, so a second round of PCR amplification based on the product of the first round of PCR is needed . Nested LAMP technology uses the chain replacement activity of Bst DNA polymerase to provide the reaction power and completes the efficient, specific and sensitive amplification of target DNA in a short time under constant temperature conditions. Park et al. used LAMP technology to rapidly detect CChMVd at 65 °C . Liu et Multiple RT-LAMP can detect multiple pathogens simultaneously in the same reaction system. Liu established a multielement RT-LAMP method for the simultaneous detection of CVB and CSVd infected chrysanthemum . This meAs shown in To date, the chrysanthemum viruses and viroids reported to have been removed include CVB, CMV, TMV, TAV, CSVd and CChMVd. The main methods of virus elimination include shoot tip elimination, heat treatment combined with shoot tip elimination, antiviral treatment elimination combined with shoot tip elimination, ultralow temperature treatment, etc.Viruses are unevenly distributed in the plant ,113, beiViruses and host plants have different high temperature tolerances, which makes the growth rate of plants at high temperature higher than the virus diffusion rate, and partial virus-free meristems can be obtained . Heat trAt present, antiviral agents are mainly natural compounds and chemical agents. This method exploits the principle that antiviral agents prevent viral RNA from forming a cap structure in the triphosphate state and thus inhibit the virus. Commonly used antiviral agents include 5-dioxyuracil (DHT) and riboside triazolium (ribavirin) . Among tUltralow-temperature treatment is a method for treating virus-infected samples in liquid nitrogen for a short period of time to eradicate the virus, developed based on ultralow-temperature preservation . It explIn summary, the main elimination methods currently used are shoot tips, heat treatment, antiviral treatment and ultralow temperature. The most common is shoot tip elimination, but shoot tip virus elimination does not have a good effect by itself, so it is often combined with heat treatment technology and antiviral agent treatment, which can achieve better elimination effects. Among them, CVB and CMV can be completely removed by combining heat treatment with antiviral agents at shoot tip, and viroid CSVd can also be completely removed by using antiviral agents after low temperature treatment at 4 °C. These effective elimination methods are helpful to study the differences from before to after elimination from chrysanthemum plants, and to clarify the specific effects of CVB, CMV and CChMVd on the disease symptoms, physiological indexes and nutrient content of chrysanthemums. The existing elimination technology cannot effectively remove TAV, TMV and CChMVd, so much more research is still needed. At present, CVB, CMV, TAV, TMV, CSVd and CChMVd viruses have been more extensively studied in chrysanthemums, while PVX, PVY and TSWV have been studied less. The elimination technology of these three viruses still needs further research.The propagation of chrysanthemums is mainly carried out by cutting, which is easy to popularize and inexpensive. However, in long-term production, due to virus accumulation, compound infection and other factors, plant dwarfing, nonflowering, yellow leaves and other problems greatly reduce the quality and value of chrysanthemums, affecting the development of the functional chrysanthemum industry ,131. UndMS has been used as the basic medium for the primary culture of chrysanthemums , and appDeng et al. established a rapid propagation technology system for virus-free ‘Chuzhou’ Chrysanthemum seedlings by using improved heat treatment combined with shoot tip meristem culture and concluded that the best medium for inducing calli from stem segments was MS + 2.0 mg/L 6-BA + 0.1 mg/L NAA. The optimal medium for shoot tip proliferation of ‘Chuzhou’ Chrysanthemum is MS + 1.0 mg/L 6-BA + 0.1 mg/L NAA . Wang etSecondary culture of functional chrysanthemums is an important method to obtain a large number of virus-free seedlings. One is to separate the callus from the bud and then inoculate the separated callus on the medium for culture to differentiate into more buds. The other is to cut the grown rootless test-tube seedlings into small segments with leaves or buds, inoculate them on the medium, and then grow them into seedlings . When HoStable rooting is a necessary condition for transplanting cultivated seedlings. When auxin is present, most of the roots of tissue culture seedlings are stimulated and begin to grow . IAA, IBChrysanthemum virus-free seedlings are always grown in controlled indoor environments from inoculation to root growth. When tissue culture seedlings are removed from the tissue culture bottle, their growth environment changes greatly, and the external natural environment affects the survival rate of the seedlings. Therefore, it is necessary to cultivate seedlings before transplanting in the open field. The commonly used time to cultivate seedlings is when the rooting tissue culture seedlings are approximately 3 cm high, the white roots are exposed and the side roots are grown. Zhao et al. showed that 3 d was the most opportune time for seedling cultivation, when the survival rate of seedlings was the highest, reaching more than 95% . If it w2·s) and culture temperature (25 ± 2) °C [Virus-free seedling propagation technology can effectively solve the problem of chrysanthemum viral infection, promote the growth and development of chrysanthemums from the root, and improve the fine quality of chrysanthemum plants and the content of effective chemical components . Liu et  ± 2) °C . The ste ± 2) °C . After iIn conclusion, different media should be used in different stages of tissue culture to achieve the best efficiency of clonal reproduction. In the culture propagation of rootless seedlings, the selection of explants and disinfection methods are different, and the hormone ratio of the callus-inducing medium is also different. KT and TDZ will also be used in relevant studies , and an At present, nine kinds of chrysanthemum viruses, including CVB, CMV, TMV, TAV, PVX, PVY, TSWV, CSVd and CChMVD, have been reported in the main production areas of chrysanthemum . The traThe current detection technologies are mainly divided into four categories: indicator plant assay, electron microscopy, ELISA and molecular biology, among which the molecular biology has the most different techniques, the highest detection speed and the highest sensitivity . MultiplThe main methods of virus elimination include shoot tip culture, heat treatment, chemical treatment and ultralow temperature treatment. Shoot tip elimination is the most commonly used method of virus elimination, but the existing research shows that the combination of various methods can greatly improve the efficiency of virus elimination. For heat treatment combined with shoot tip elimination, the method of changing the temperature can be used for elimination. Due to the wide selection range of antiviral agents, the elimination effect from different plants is different, and it is necessary to consider the nature of the plant itself and the characteristics of the virus to avoid hindering the growth of the plant itself while improving the elimination rate. Research on ultralow-temperature elimination is still relatively inchoate, and further experimental research is needed.During the propagation of sterile and virus-free seedlings, the selected explants and disinfection methods differ, as do the hormone ratios of the callus-inducing medium. An MS medium with different concentrations of 6-BA and NAA is commonly used, and KT and TDZ are also used in some studies . In the Chrysanthemum virus R (CVR) , zucchin


Although global food consumption costs more in terms of impact on human life than money is spent on it, health costs have not been consistently quantified or included in food prices to date. In this paper, a method to determine the external health costs of nutrition and dietetics is developed by employing the cost-of-illness (COI) and true cost accounting (TCA) approaches. This is done exemplarily for the reference country Germany. The results show that 601.50 € per capita and 50.38 billion € in total external health costs are incurred annually due to nutrition. Overall, most costs are accrued through excessive meat consumption (32.56% of costs), deficient whole grain intake (15.42% of costs), and insufficient uptake of legumes (10.19% of costs). Comparing the external health costs with the external environmental costs in Germany, it can be seen that of the total annual costs of around 153.86 billion €, 67.26% originate from environmental impacts and 32.74% from impacts on human life. In order to achieve the 17 Sustainable Development Goals and to increase family as well as public health, there is a need to internalise these external costs into actual food prices. Intense droughts, water scarcity, severe fires, rising sea levels, melting polar ice, and declining biodiversity are just a few examples of the consequences of global climate change. Since the 1800s, humans have been the main reason for these extreme events . The SDGGHG emissions represent an example of an externality, which is defined as any action that affects the welfare or available opportunities of an individual or group and is neither directly paid for nor compensated   Food Systems Summit shows that food externalities amount to USD 20 trillion per year, which corresponds to more than twice the global food consumption. While USD seven trillion is the result of environmental costs, the bulk of the costs of USD eleven trillion is attributable to impacts on human life . Of this, an average of 57% is due to direct healthcare costs and 43% to indirect costs such as informal care and losses in labour productivity. The high proportion of unpriced health externalities makes sustainable and healthy food less affordable for consumers and less profitable for producers than comparatively unhealthier alternatives; hence, representing one main reason for global malnutrition. For example, 690 million people were undernourished in 2019, and ten million die every year due to unhealthy eating habits. Global nutrition guidelines could save five million lives and 79 million life years per year. Moreover, according to this study, up to USD 735 billion in health costs could be saved if such guidelines were introduced .The COI approach was used to calculate the potential cost savings due to healthier diets. COI studies aim to identify and measure all the costs of a disease  by consiIn the scientific literature, no concrete, calculated health premiums or discounts for certain foods exist. The reason for this absence is both the difficulty to measure food products externalities in general as well as large uncertainties. The latter is due to incompletely captured fields of influence as well as fundamental uncertainties in the primary literature, in trade datasets, in modelling the strands of influence, and in monetising external costs. Specifically, estimates of health costs from food vary due to differing approaches and assessment methods, as well as inconsistent standards and guidelines , as thisFurthermore, data sources and data collection in combination with the high complexity of the underlying value chains represent a key challenge. One of the main reasons why health and social components have not yet been sufficiently taken into account in corresponding approaches such as TCA to date is the main focus of companies on improving the natural capital base in impact assessment to improve shareholder value creation. The center of attention lies in reducing carbon emissions, as this is also a global focus, above all visible in the example of the 2030 Agenda. However, impacts on the oceans or workers’ health, for example, are hardly addressed. Therefore, a transdisciplinary approach is necessary to consider health and social consequences along the entire value chain .The mentioned problem areas have the consequence that currently no uniformly quantified nutrition manual that can be utilised to determine concrete price premiums for the health effects of food has been published .One solution to this problem is the Planetary Health Diet (PHD), which was designed for a global population of about 10 billion people in 2050. The basic ideas are a shift to healthy diets and sustainable food production   , a tax oIn summary, the vast majority of studies deal with the environmental costs of food and only a few focus on calculating the health costs of food. The COI approach is predominantly used for internalising health costs, in which direct and indirect costs of diseases are taken into account. Up to now, however, either the total global health costs have been calculated or only those for a group of countries, subdivided according to income. A specific health premium per kilogram was calculated for red and processed meat only, whereby here again a high level of aggregation was applied at the country level. To date, no methodology has been elaborated that determines the country-specific health costs of individual foods or their ingredients and, thus, creates reference values for potential price increases. Therefore, in this paper, we aim to develop a true pricing approach using the COI and TCA approaches to calculate the specific external health cost premiums of selected dietary risk factors for a reference country. Finally, different implementation options and their advantages and disadvantages will be discussed. Thus, this study answers the following research question: how high are the external health costs of food consumption?In this section, we first describe the methodology to calculate health premiums, which represent external costs to date, for foods by dietary risk factors. In the first step, an approach is developed to quantify and monetise the quantity-standardised health costs of the harmful quantity for individual dietary risk factors. In the second step, the methodology is extended to include an approach for determining the concrete health premiums of individual products. Finally, we explain the calculation and sourcing of the input data.Before starting the method, a reference country is defined for which the health costs are determined in the following by utilising the procedure illustrated in Then, relevant diseases through food are selected based on Afshin et al.  (pp. 195d) and those for specific dietary risk factors , the DALY rate (q) of the individual diseases (d) is calculated, by which the percentage of the respective disease burden caused by the individual dietary risk factors is obtained.From the general DALYs of the diseases (DALYdir) and indirect costs (cind) of the selected diseases are determined for a given year (y) and the values are summed up. Here, one can distinguish between three cases concerning the data situation.Then, using the COI approach already mentioned, the direct  and on population figures (py). In the following calculation, it is assumed as an example that the data on direct costs originate from the year 2010 and the data on indirect costs from the year 2011. Thereby, the costs are standardised to the year 2010.In the second case, data on both types of costs are available for different years. Here, an adjustment is needed with regard to inflation and population size in the summation. This requires information on consumer price indices  and indirect per capita costs  exist, but only the total direct costs (cdir) for one year are available.In the third case, data is available on both cost types, with one cost type being calculated from the ratio of total costs to per capita costs of the other cost type. In the following calculation, for example, it is assumed that both the direct , the inflation-adjusted per capita costs of the selected diseases are first multiplied by the calculated DALY rates, resulting in the per capita health costs of the respective diseases of the individual dietary risk factors .In order to ascertain the per capita health costs of the individual dietary risk factors , one needs the average daily intake of the respective dietary risk factors in the reference country  and their daily recommended intake . The former is retrieved from a global database listing the intakes of all dietary risk factors [In order to be able to calculate the quantity-standardised health costs of the harmful quantity that is consumed deficiently or excessively of the respective dietary risk factors on average in the reference country  by calculating the difference between the values just determined.Now we detect the specific harmful amount of the dietary risk factors that is consumed too little or too much per capita daily , which is estimated by the PHD at 2500 kcal per day [p,cal). From these values, the quantity of the product that would be required if one were to cover the daily requirement exclusively with it is calculated .In the first case, for the individual ingredients, one needs a reference number of kilocalories consumed daily  to obtain the total amount of the dietary risk factor that would arise when the daily requirement is completely covered with the product .This is then multiplied with the amount of the dietary risk factor contained in the product .Subsequently, the daily recommended intake of the respective dietary risk factors already determined is needed, and is subtracted from the total amount of the dietary risk factor when the daily requirement is completely covered with the product. This shows the harmful amount contained in the product when the daily requirement is fully covered by it . This value is now divided by the reference number of kilocalories consumed daily, which yields the average share of the product in daily consumption in the reference country .The average consumption of the product group in the reference country per day is then determined and the kilocalories of the specific product standardised on this per day is calculated  by multiplying the harmful amount of the dietary risk factor when the daily requirement is fully covered by the product with the share just determined. This value indicates how many grams of the dietary risk factor one would consume excessively on average per day in the reference country if one were to rely exclusively on this product within the product group. Although the standardisation is not needed to arrive at the same final result, this intermediate step is included in the method for better and easier understanding.Then, we calculate the average unhealthy amount of the dietary risk factor of the product in the daily consumption in the reference country  by dividing the number of kilocalories of the product by the average consumption of the product group in the reference country per day and multiplying it by the quantity just calculated.This is then adjusted to the quantity of the product (np) by multiplying the amount of the dietary risk factor of the product in daily consumption in the reference country by the quantity-standardised health costs of the harmful amount of the dietary risk factor.The last step is to calculate the specific health premium for the product .In the case of under-consumed product categories, the harmful quantity calculated above is divided by the daily recommended intake of the respective dietary risk factor to obtain the quantity quota, which represents the basis for internalising health costs (lr). Here, the average daily intake is extracted directly from studies by national or supranational authorities or, if this is not possible, from databases. If aggregated values are not available, the average is determined using population statistics. The underlying data is explained in more detail in the following subchapter.In the case of over-consumption, the harmful quantity calculated above is divided by the average daily intake of the respective dietary risk factor in the reference country to obtain the quantity quota, which represents the basis for internalising health costs (hr). This is used to calculate the quantity of the product that causes external benefits or costs .Now we determine the specific amount of the product category or dietary risk factor (mr) is calculated by multiplying the quantity of the product category that causes external benefits or costs by the quantity-standardised health costs of the harmful quantity for the dietary risk factor.In the last step, the specific health premium for the product category  is utilised, which contains the direct as well as the indirect expenses for the year 2015 . Here, dNumerous studies exist for T2DM, all of which include direct costs. A distinction is made between overall, diabetes-specific, and diabetes-associated direct costs. According to Jacobs et al. (2017)  , which includes estimated daily intakes of all dietary risk factors for any country . In GermDifferent sources are also required to determine the daily recommended per capita intake of the respective dietary risk factors.For sodium, the suggestion of the German Nutrition Society (DGE) is utilised. It recommends a daily intake of 6.00 g of salt, which corresponds to 2.30 g to 2.40 g of sodium   is used   that are consumed deficiently or excessively of the dietary risk factors per capita daily and the quantity quota which represents the basis for internalising health costs (lr/hr) are calculated there.The just mentioned quantity quota is required when calculating the health premiums for dietary risk factors that are available as product groups. In addition, there is a necessity for a concrete quantity for which external health costs are internalised. In this paper, in the first step, premiums and discounts are calculated for one kilogram of the respective dietary risk factor. In the second step, this is adjusted to the quantities of selected foods. Furthermore, information on the prices of the specific products is required. This study examines some products of Edeka, a large German retailer. Here, apples , minced To calculate the health premiums of dietary risk factors as individual ingredients, the daily consumed reference number of kilocalories already addressed in In this paper, the health premiums due to sodium are calculated for four products as examples. For these products, one needs the selling price, the specific number of kilocalories, and the amount of sodium contained. Here, we determine corresponding health premiums for the types of salami from Edeka already mentioned above and for gouda , as wellFinally, the health costs for certain food products from the Penny-market are determined and applied to a study that already calculated the influence of external environmental costs on corresponding products . The valResults show that EUR 601.48 per capita health costs occur annually in Germany due to dietary risk factors. The six largest dietary risk factors account for 72.48% of the costs, which is why we focus on these factors in this paper. The total annual external health expenses of CVD, T2DM, and neoplasms caused by dietary risk factors are approximately EUR 50.38 billion based on the most recent population status as of 1 January 2022. Over-consumption occurs when the amount that is actually consumed exceeds the recommended amount, while under-consumption refers to the opposite. Of CVD health costs from dietary risk factors, 38.89% are the consequence of over-consumption, while 61.11% are attributable to under-consumption. Legumes with 17.20% and whole grains with 17.07% account for the highest share of the resulting total CVD health expenses. Red meat and sodium turn out to cause the largest shares of health costs due to over-consumption with 11.32% and 9.80%, respectively.T2DM occurs mainly because of excessive consumption of dietary risk factors. Of the health costs, 70.27% are due to over-consumption, of which 84.22% are ascribable to red and processed meat. Excessive consumption of the two types of meat thus poses the greatest risk for dietary T2DM. The remaining 29.73%, which occur due to undersupply, are mainly caused by deficient consumption of whole grains and fruits.Neoplasms result predominantly from an undersupply in the diet. Thus, 67.44% of the health costs are attributable to under-consumption, of which 37.02% are traced back to a too low daily intake of whole grains. The remaining 32.56% largely arise from red and processed meat. Excessive consumption of the two types of meat is responsible for 87.98% of the health costs from neoplasms due to over-consumption.In summary, CVD and neoplasms are predominantly caused by an undersupply of food. In both cases, too little consumption of whole grains is elementary. T2DM is mainly a consequence of over-consumption of food, with red and processed meat being the main contributors. Red and processed meat also contribute largely to the health costs of neoplasms and especially red meat to the health costs of CVD.For dietary risk factors as whole product groups, we calculate health premiums for each kilogram, which are presented in To illustrate the concrete impact of internalised health costs on food, we ascertain the price changes of selected products from Edeka, one of the leading retailers in Germany, in With the information on the quantity-standardised health costs of sodium, the average daily reference number of kilocalories and the data on individual products, their specific health premiums are obtained. The inflation-adjusted external ecological cost premiums of certain products from the above-mentioned Penny-market in some cases differ greatly depending on the production practice. Thus, the organic and conventional variants of the same product entail different environmental cost premiums. Since the production method has no significant influence on the extent of external health expenses, the cost premiums and discounts for the organic and conventional variants of the product are assumed to be identical. In the following, one kilogram is always applied as the reference quantity. The environmental surcharge for conventional gouda is EUR 4.69, while that for organic gouda is EUR 3.49. For both, the health expenses amount to EUR 0.16. For conventional mozzarella, the environmental cost premium is EUR 3.04, and for organic mozzarella EUR 2.29. The health costs of both variants come to EUR 0.01. Conventional mixed minced meat should become more expensive by EUR 10.35, and organic mixed minced meat by EUR 12.39 with the internalisation of external environmental costs. The health cost surcharge is EUR 7.51. Conventional apples should become higher priced by EUR 0.19, and organic apples by EUR 0.13 when internalising environmental costs. For conventional bananas, the environmental surcharge is EUR 0.21, and for organic bananas EUR 0.16. If health costs are internalised, both fruits just mentioned become cheaper by EUR 0.68, which corresponds to overcompensation of ecological costs.This effect can also be observed with price reductions. Here, too, the percentage price decrease is greater for the conventional variants than for the organic variants. Thus, the price of conventional apples would drop from EUR 2.31 to 1.83 per kilogram (79.08% of the current price), while that of the organic variety would decline from EUR 3.47 to 2.92 (84.20% of the current price). The price per kilogram of conventional bananas would diminish from EUR 1.09 to 0.63 (57.67% of the current price), and the price per kilogram of organic bananas would decrease from EUR 1.79 to 1.27 (71.15% of the current price).Now that the external health costs of food in Germany resulting from nutrition have been presented and the impact of internalising these expenses on consumer prices has been evaluated, the results and the uncertainties will be discussed.First, we describe, explain, and discuss the dealing with uncertainties based on the input data breakdown in This paper uses a variety of data from different sources, each of which is subject to uncertainty. In measuring the burden of disease on life, a tool from the University of Washington is employed . This prIn determining the costs of the selected diseases, aggregated costs for the super categories of diseases are used in this study. Concerning CVD, there is no subdivision into individual subcategories such as ischemic heart diseases or stroke. In neoplasms, the different types of cancers are not differentiated. As a result, the costs of illness cannot be calculated in a fully differentiated way for each dietary risk factor. Because of the coarser granulation, the total costs are higher than the sum of the individual costs in a more disaggregated view. This problem is counteracted in this study by using the DALY rate, as this normalises the costs to the over category under consideration.Furthermore, significant uncertainties arising from the heterogeneous sources regarding the age of the data and the data contained exist, which is particularly important when calculating the costs of diabetes. Numerous studies are published containing varying direct costs, most of which are more than ten years old. In this paper, the study with the highest sample size was selected, as it is the most representative. It represents almost all people with T2DM in Germany, and its result is similar to that of the Costs of Diabetes Mellitus (CoDiM) study, which analysed data from the same year from about 1.5 million insured people and came up with a per capita cost of EUR 5239  , assignable to the lack of national and international guidelines   , organicAs already illustrated, the majority of diseases occur due to an undersupply of food. Consequently, there should be an incentive to consume more of it. Based on the calculated results, under-consumed food becomes cheaper when both health costs and environmental costs are internalised, because the impact of health externalities exceeds that of environmental externalities. However, for over-consumed food, both types of costs add up, whereby the share of environmental costs is greater than that of health costs for animal-based and dairy products used in the aforementioned study .The Boston Consulting Group (BCG) estimates the total environmental external costs of agriculture in Germany at around EUR 90.77 billion in 2018 . AdjusteThus, in comparison, the production increases in Germany, which leads to higher environmental costs, while, because the diseases examined here are in general mainly caused by an undersupply of food, there are fewer external health impacts on human life, explaining the dominance of environmental costs over health costs in Germany.The results illustrate that an integrated internalisation of all external costs from food consumption leads to incentives for a healthier diet. Food that is taken in deficiently becomes cheaper for the consumer, and food that is taken in excessively becomes more expensive. In the process, poor production methods and the resulting environmental costs or benefits increase or decrease the calculated health costs. Thus, the side effects of economic activity are monetised and become part of prices. The market is equalised, and a shift to healthier and more sustainable diets is motivated.In this study, a methodology is developed to determine the health costs of individual foods and their ingredients for the reference country to, thus, create reference values for potential price increases or decreases. In our framework, first, a new type of DALY rate is introduced to determine the percentage of the diseases CVD, T2DM, and neoplasms of the most influential dietary risk factors in their total numbers in Germany. This rate is used in the next step to calculate the per capita health costs of the individual dietary risk factors using the COI approach. By determining harmful levels of dietary risk factors, quantity-standardised health costs are calculated, and concrete health premiums or discounts for individual ingredients as well as individual products are determined.We find, that EUR 601.50 per capita in total health costs are incurred in Germany per year due to nutrition. Almost one third of these is caused by an over-consumption of meat, thus, highlighting an excessive meat consumption as a main driver of health costs in Germany. Further results underline the insufficient consumption of grains, legumes, and fruits, as well as the excessive consumption of sodium as significant drivers of health costs.This thoroughly answers the research question formulated in the introduction.Overall, 48.78% of health costs in Germany are due to an oversupply of food, while 51.22% are caused by an undersupply. While both globally and in Germany most DALYs are incurred by an undersupply of food, the proportion in Germany is significantly lower than in international comparison, which is primarily due to Germany’s high consumption of meat. Comparing the calculated health costs of minced meat, apples, bananas, gouda, and mozzarella to the environmental costs calculated by Michalke et al. (2019) , an inteIn summary, the costs of nutrition through impacts on human life, which to date are not included in market prices and thus not compensated, can be quantified and monetised by applying the methodology developed in this paper. By pricing food appropriately, the 17 SDGs could be advanced by improving the health aspect in particular. For this, an international application in addition to the calculation of health costs through this method for Germany is required for a globally healthier diet and an equalisation of the market.


Baculoviruses are insect-specific DNA viruses that have been exploited as bioinsecticides for the control of agricultural and forest pests around the world. Mixed infections with two different baculoviruses have been found in nature, infecting the same host. They have been studied to understand the biology of virus interactions, their effects on susceptible insects, and their insecticidal implications. In this work, we summarize and analyze the in vivo baculovirus co-infections reported in the literature, mainly focusing on pest biocontrol applications. We discuss the most common terms used to describe the effects of mixed infections, such as synergism, neutralism, and antagonism, and how to determine them based on host mortality. Frequently, baculovirus co-infections found in nature are caused by a combination of a nucleopolyhedrovirus and a granulovirus. Studies performed with mixed infections indicated that viral dose, larval stage, or the presence of synergistic factors in baculovirus occlusion bodies are important for the type of virus interaction. We also enumerate and discuss technical aspects to take into account in studies on mixed infections, such as statistical procedures, quantification of viral inocula, the selection of instars, and molecular methodologies for an appropriate analysis of baculovirus interaction. Several experimental infections using two different baculoviruses demonstrated increased viral mortality or a synergistic effect on the target larvae compared to single infections. This can be exploited to improve the baculovirus-killing properties of commercial formulations. In this work, we offer a current overview of baculovirus interactions in vivo and discuss their potential applications in pest control strategies. Baculoviridae family  is classified into four genera: Alphabaculovirus and Betabaculovirus are lepidopteran-specific NPV and GV, respectively, Gammabaculovirus are NPVs that infect hymenopterans and Deltabaculovirus comprise dipteran-specific NPVs. Phylogenetic analysis based on amino acid sequences of core genes separates the Alphabaculoviruses into two lineages, Group I and Group II. The Betabaculovirus genus is also separated into two different clades, a and b [Naturally occurring co-infections involving two or more different species of virus are found in nature in all domains of life ,2. This  a and b ,15. In lIn this review, we focus on the mixed infections of baculoviruses in vivo and analyze their effects on the host from the point of view of biocontrol. We review studies carried out on baculovirus interactions in vivo, focusing on the effects of viral interactions in the host and not on the effects produced by one virus due to the presence of another virus. Thus, when applied to an insect, the effect of a viral interaction can be classified as synergistic, additive, or antagonistic . The terResearch on the in vivo co-infection of baculoviruses aims to improve their use as biopesticides. Most interaction studies have focused on mixtures of an NPV and a GV isolated from the same host species. Also, artificial baculovirus combinations have been assayed using an NPV mixed with a GV that is not infective for the host. In this study, we critically review the literature in this field and propose a classification of virus interaction based on its effect on the host. Moreover, we describe the factors that determine the type of interaction and the techniques involved in the mixed infection analysis. A schematic representation of the topics discussed here is presented in Euxoa segetum in 1936 [Pieris rapae, Pseudaletia unipuncta, Nephelodes emmedonia, Mythimna unipuncta, Choristoneura fumiferana, Spodoptera frugiperda, and Spodoptera Ornithogalli [Pseudaletia unipuncta to virus infections and observed a synergistic effect when an NPV and a GV were co-inoculated into larvae. Subsequent studies aimed to characterize other co-infections involving NPVs and GVs in various larval hosts, leading to the identification of additional phenomena, such as interference and additive effects [A total of 91 baculoviral species have been registered by the International Committee on Taxonomy of Viruses . Of thesthogalli ,23,24,25thogalli ,23. He e effects ,27,28. TChoristoneura fumiferana (Clemens) and found that the mortality of larvae infected with both viruses simultaneously was often higher than that of those infected with each virus individually. Interestingly, he also found that one virus interfered with the replication of the other. The author concluded that no synergistic effect was observed [Trichoplusia ni was comparable to the mortality caused by each virus individually, concluding that no synergism or antagonism was present (referred to as interference). Also, the time to death was delayed in the double infection compared to the NPV infection [In 1959, Bird assayed different combinations of CfMNPV and CfGV in the spruce budworm observed . Nonetheobserved ,29,30, tnfection ,29. ConsHeliothis armigera (homotypic synonym: Helicoverpa armigera), when co-infected with both NPV and GV, exhibited an inhibitory effect compared to individual infections. Mortality rates of insects infected with both viruses were lower than those of larvae infected with a single virus [Xestia c-nigrum. The enhancing effect was dependent on instar and dose, with XcenGV displaying a tendency to outcompete NPV infection. XcenGV enhanced XcenNPV infection in the fifth instar but not in the fourth, where the host exhibited higher susceptibility to XcenGV infection. In this instance, the GV acted as a competitor to the NPV, obscuring its enhancing action. The authors concluded that the presence and concentration of synergic factors associated with the OB play a pivotal role in viral mixtures [Whitlock (1977) found that larvae of le virus . Years lle virus . A similmixtures . In thismixtures .Helicoverpa zea. It was found that HzSNPV was outcompeted by HearGV, despite the fact that HzSNPV is a fast-killing virus in contrast to HearGV. On average, higher doses of NPV caused larval death in 5.5 days, whereas in combination with HearGV at varying doses, the average killing time extended to 16.5 days. This led to the proposition that HearGV not only competed for host resources but also had an inhibitory effect on HzSNPV [S. littoralis obtaining similar results and concluding that both viruses had an antagonistic effect. They combined different doses of SpliNPV with a constant dose of SpliGV, and vice versa. This reinforced the concept that the GV interfered with NPV replication, whereas the NPV did not hinder GV infection [Agrotis segetum larvae. They reported that at low NPV concentrations, larvae died of granulosis, while at higher NPV concentrations, larvae mostly died with polyhedra. Based on the mortalities observed in mixed infections, they concluded that AgseGV and AgseNPV-B acted independently within the larval host, resulting in an additive interaction [Spodoptera frugiperda larvae. However, this synergy was observed only when the proportion of SpfrGV in the mixture did not exceed 2.5% [Spodoptera ornithogalli [Based on these previous reports, Hackett (2000) measured the ‘interference’ of HzSNPV due to HearGV by assessing the survival time of n HzSNPV . Similarnfection . More reeraction . Converseed 2.5% . Similarthogalli .The study of synergistic interactions between different baculoviruses has gained much attention due to its implications for the improvement of strategies in pest biocontrol. Following the discovery that PsunGV was responsible for the synergistic effect on PsunNPV infection, with the synergistic factor residing within the PsunGV occlusion body (formerly named ‘capsule’ and later ‘granule’) ,35, sevePseudaletia separata, slightly enhanced the infection by SpltNPV in Spodoptera litura, and had no effect on the infection of BmNPV in Bombyx mori. Therefore, they concluded that the synergistic effect varied greatly depending on the insect host and the virus used [Lymantria dispar. He found that HearGV could reduce LdMNPV LD50 by a factor of 289, while SpfrGV could reduce it 13-fold, concluding that both GVs were able to enhance LdMNPV infection [T. ni [Spodoptera litura. In this case, where XcenGV does not naturally infect S. litura, they reported that the addition of XcenGV OBs significantly reduced SpltNPV LC50, producing an enhancing effect [Anticarsia gemmatalis larvae was enhanced by the addition of Epinotia aporema GV OBs [A. gemmatalis was not susceptible to EpapGV. In the same vein, Jeyarani et al. (2012) assayed five GVs in combination with HearNPV across various instars of the cotton bollworm, H. armigera. They tested GVs from: Spodoptera litura (SpltGV), Agrotis segetum (AgseGV), Plutella xylostella (PlxyGV), Achaea janata (AjGV), and Chilo infuscatellus (CiGV). Among these, only SpltGV synergized with HearNPV infection, while the remaining GVs had a neutral effect [Hukuhara and collaborators were the first to test the enhancing activity found in PsunGV in hosts that were not susceptible to PsunGV. They used it in combination with various NPVs and found that PsunGV greatly enhanced the infection of PsunNPV in rus used . In addinfection . Similarn [T. ni ,39. Guo g effect . Similara GV OBs . Also hel effect   demonstrated that the synergistic factor in PsunGV was a protein present in the occlusion body, often referred to as the “capsule”. On the other hand, heat inactivation treatments with HearGV revealed that when an interferent effect occurred, it was related to the virion rather than the capsule ,31,42. Min genes . Severala larvae ,45,46,47enhancin genes are more prevalent in GVs compared to NPVs. Interestingly, the majority of GVs with reported enhancement of NPV infections belong to the phylogenetic clade “a” [enhancin genes in their genomes.The term “Synergistic Factor” (SF) was initially coined to describe proteins associated with the OBs of GVs that enhanced the infectivity of other baculoviruses ,49. Theslade “a”  and encoenhancin genes encoded by this virus are expressed and incorporated into the OB structure , there might be a correlation between the GV’s enhancing ability and the number of enhancin genes encoded by each virus. In this context, AgseGV codes for one enhancin in contrast to HearGV or XcenGV, which code for four enhancins each, in agreement with their significant enhancing capabilities. This notion is reinforced by the inspection of their RP values, as when comparing HearGV with SpfrGV (encoding two enhancins) showing RPs of 286 vs. 13, respectively, in L. dispar.ve hosts . InteresP. unipuncta, specifically the Typical NPV (TNPV) and Hypertrophy NPV (HNPV), which showed distinct signs of infection in the cells. They reported “interference or antagonism” between them, as co-inoculation of both viruses resulted in larvae infected solely with the TNPV strain and did not differ from the single TNPV infection. In addition, the number of cells infected with HNPV was significantly less than in the single infection [Few studies report the detection of different NPVs in natural mixed infections . Early snfection . Del Rinnfection . This wanfection . Althougnfection , attemptnfection . The absnfection .Thysanoplusia orichalcea L. When these viruses were separated and evaluated individually using bioassays, ThorMNPV showed a 271-fold higher potency than ThorSNPV against third-instar Pseudoplusia includens. However, when using the original mixture containing the majority of ThorSNPV and low levels of ThorMNPV, the infectivity on the host was low, similar to the infection caused by ThorSNPV alone. Conversely, when using a mixture containing the majority of ThorMNPV, the effect was similar to the one caused by ThorMNPV alone. Both viruses were capable of replicating in the same tissues, but never simultaneously in the same cell. Therefore, the authors cataloged the interaction of ThorMNPV and ThorSNPV as neutralistic [Cheng et al. (2005) reported that a multicapsid nucleopolyhedrovirus (ThorMNPV) and a single nucleopolyhedrovirus (ThorSNPV) were co-isolated from larvae of ralistic . From thChoristoneura fumiferana). The Choristoneura fumiferana nucleopolyhedrovirus (CfMNPV) and the Choristoneura fumiferana defective nucleopolyhedrovirus (CfDEFNPV) are two different viruses that were detected co-infecting the insect host. The CfDEFMNPV isolate is unable to produce larval infection when administered orally, but in mixtures with CfMNPV, it gains the capability to infect the insect gut. Once in the hemocoel, CfDEFNPV synergizes with CfMNPV infection through unknown mechanisms [A natural viral mixture was isolated from the eastern spruce budworm  is one of the few reported NPVs to contain an nditions . These iRachiplusia nu (Guenée) [R. nu larvae. In laboratory bioassays, these authors compared the effects of single and mixed infections and reached the conclusion that co-infection exhibited a slightly increased insecticidal performance when compared to AcMNPV alone. However, this enhancement was not found to be statistically significant [The generalist Autographa californica multiple nucleopolyhedrovirus (AcMNPV) and Rachiplusia nu nucleopolyhedrovirus (RanuNPV) have been documented in natural infections of the soybean caterpillar (Guenée) ,68. In anificant .Spodoptera exigua. Accordingly, CpGV gp37 was shown to enhance the infectivity of AcMNPV on S. exigua larvae. Furthermore, it was confirmed that CpGV gp37 aided ODVs to cross the insect PM and fuse with midgut cells [Enhancin genes are absent in some GVs that have shown synergistic effects on the infectivity of NPVs ,41. Thisut cells . Howeverut cells ,72.b GV, was shown to augment the virulence of AgMNPV in A. gemmatalis. As EpapGV does not encode enhancin genes, gp37 has been proposed to be responsible for this effect [S. ornithogalli, lacks an enhancin gene, implying that other factors are supposed to act as enhancers [T. ni larvae [EpapGV, a clade s effect . Howevers effect . Therefonhancers . Also, ai larvae . ConsideAdditional proteins have been identified for their role in the disruption of the peritrophic membrane, aiding the encounter of ODVs with target epithelial cells. Baculoviral ODV-E66 is an ODV-specific protein that is conserved in lepidopteran baculoviruses . SugiuraTaking into account that the mixture of two viruses can produce very different outcomes, including undesired effects, from the biocontrol point of view, it is critical to have several technical considerations that will be reviewed in this section.P. unipuncta [In early studies, OB quantification was not performed properly, and they talk about mixing “volumes” and dilutions of the viruses. For example, Tanada used equal volumes of “heavy suspension virus” to test mixed NPV + GV in nipuncta . In morenipuncta  because nipuncta . Later, nipuncta , Lowe annipuncta . Nowadaynipuncta . As an anipuncta .H. armigera [Anticarsia gemmatalis larvae were treated with mixtures of AgMNPV and EpapGV, and doses were selected on the basis of the LD50 in their respective hosts. Two mixtures were defined, using two AgMNPV doses (higher and lower than the LD50) in combination with a fixed EpapGV dose chosen based on EpapGV virulence on its native host, specifically a dose resulting in 100% mortality in the last instar of Epinotia aporema [Spodoptera ornithogalli larvae [When choosing doses of each virus to test in combination, the literature lacks explicit criteria on how to select the amount or proportion of virus to use. If the objective is to observe a difference in virulence between the NPV alone and the NPV + GV treatments, the assay should be designed to produce an observable change in mortality, or LD50, at a fixed stage of the target larvae. The combinations used in the reviewed literature are varied. At first, the authors used a single mixture of equal volumes of dense suspensions of both NPV and GV ,27,28,36armigera . In our  aporema . In anot aporema . Cuartas aporema . With a i larvae .T. ni [Combinations of NPVs and GVs were also tested by administering both viruses at separate times. Bird (1959) found that feeding CfGV granules 1 or 2 days before administering NPV polyhedra had an inhibitory effect on the development of polyhedrosis in the spruce budworm. However, by manipulating the times of virus feeding, distinct levels of double infections were observed, comprising blocks of adjacent cells infected with either NPV or GV. Individual cells infected with both viruses seemed to be an extremely rare event . Lowe anT. ni . MoreoveT. ni .P. unipuncta [P. unipuncta, “Fifth-instar larvae were found to be most suitable for tests on synergism because they were more resistant to virus infections than those in the first four instars, and hence demonstrated the synergistic association between the viruses much more distinctly” [H. armigera with different combinations of HearGV and HearNPV. Although they confirmed that the susceptibility of larvae to individual virus infections decreased with increasing instars, they did not observe an enhancement in any of the assayed instars [A. segetum, an additive effect was observed [A. segetum were employed.The choice of larval instar to which the viral mixture is applied appears to affect the outcome of the interaction. However, most of the studies reviewed here focus on a single instar, and in general, the reasons for that selection are not detailed. Only a few studies have tested viral mixtures across two or more instars. Tanada highlighted the effectiveness of a mixed infection of PsunNPV + PsunGV vs. individual infections in the later developmental stages of the armyworm nipuncta . Accorditinctly” ,81. Moretinctly” . Jeyaran instars . In a stobserved . ConsideIt was not until 1968, when Lowe and Pashke introduced the bioassay technique and quantification of OBs , that daNPV + MGV (1 − MNPV), where M corresponds to the mortality caused by each virus alone, while EM is the expected mortality resulting from the combination of viruses. The calculated mortality (CM) in bioassays performed with viral mixtures is related to EM by chi-square value analysis (X2 = (CM − EM)2/EM). The X2 value is compared to the table value (X2 = 3.841) for 1 degree of freedom (dF) and α = 0.05. If the calculated X2 value is below 3.84, the virus interaction is categorized as additive. Conversely, values higher than 3.84 are associated with synergistic or antagonistic interactions. The difference in value (D) between CM and EM determines whether the virus interaction is synergistic  or antagonistic . In summary, the synergistic, additive, or antagonistic effect can be assigned based on quantitative analysis [H. armigera larvae with NPV and GV mixtures produced a viral effect described as “interference” [2 and D values indicate an antagonistic interaction . This concept was initially introduced when the GV was non-infective to the host and was used in combination with an NPV . The RP C. fumiferana larvae showed different outputs: only polyhedrosis, only granulosis, or the presence of both polyhedra and granules in adjacent cells [In order to investigate whether larvae that had been inoculated with two viruses succumbed to NPV, GV, or a combination of both, early works diagnosed infected cadavers through light microscopy . In casent cells . Similarnt cells .Rachiplusia nu [A. segetum larvae with mixed infections [Lara-Reyna et al. (2003) analyzed cadavers under the microscope after treatments with AcMNPV + TnSNPV and AcMNPV + TnGV, observing the dominance of AcMNPV OBs . Similarlusia nu . Nowadayfections .Knowledge of in vivo baculovirus interactions offers the possibility of improving pest management strategies. A detailed review of studies on mixed baculovirus infections has enabled us to recognize different factors involved in the final effect on the host. Our extensive review revealed that mixed infections of NPV + GV are more intensively studied in comparison to NPV + NPV interaction studies. Regarding GV + GV mixtures, no study has been published describing natural co-infection produced by different GVs. However, several studies have described natural or experimental co-infections with mixtures of GV genotypic variants ,85,86 orOur analysis of published studies reveals that the terminology used to describe viral interactions is associated with different classification criteria. For example, terms such as ‘interference’ or ‘neutralism’ refer to the consequences of the infection and/or replication of one virus due to the presence of another virus within the same host ,28,42. HSpodoptera frugiperda larvae. Also, it was found that virulence is reduced when the proportion of GV increases [The effect of baculovirus mixtures on the host depends on different factors such as virus inoculum amounts, host instar, host susceptibility, and the presence of infection-enhancing proteins associated with OBs. When both NPV and GV infect the same host, it appears that a low proportion of GV is associated with the synergistic effect, whereas when more GV is included, a neutral or antagonistic effect is promoted, as observed with XcenGV . In the ncreases . A recenncreases . On the ncreases ,80. Therncreases .Pseudaletia unipuncta [Helicoverpa armigera, in which an antagonistic interaction was registered [L. dispar is lost in older instars [Several reports indicate that the effects of mixed baculovirus infections can vary across different larval instars of the host. Initial studies with PsunNPV and PsunGV mixtures performed by Tanada showed a synergistic effect on the third to sixth instars of nipuncta ,23,53. Dgistered . Goto etgistered . Thus, t instars . Taken tenhancin genes. In cases where an enhancin-containing GV elicits interference, approaches such as virus inactivation or granule extract utilization could be viable alternatives to retain the enhancing effect while eliminating the interfering outcome. Furthermore, other proteins encoded in baculovirus genomes, such as ODV-E66 and chitinases, have shown potential for enhancing activities. These proteins were detected in the ODV and OB, respectively. However, their roles in baculovirus interactions remain to be studied. In this sense, proteomic approaches can shed light on which OB proteins are involved in the enhancing effect of some viruses. In line with this, EpapGV and SpfrGV proteomics added important information to the possible enhancer proteins involved in the synergistic effects observed for these viruses  obtained by cell culture selection resulted in an increased OB relative potency and a faster speed of kill. Similar results were observed with co-occluded mixtures of HearNPV in laboratory and field bioassays ,96. AddiFrom an insecticide perspective, synergism emerges as an ideal interaction between viruses included in a baculoviral formulation. Also, additive effects can be tolerated if the combination broadens the spectrum of pest targets that can be controlled under field conditions. However, in cases where antagonism is possible, the selection of viruses to combine needs to be considered cautiously, and proper assays should be performed to determine the final formulation. The use of mixed formulations with GV or NPV that harbor enhancin-like proteins and infect only their respective hosts is certainly of value if target insects appear simultaneously on the same crop . On the In this review, we analyze in vivo baculovirus interactions and their potential implications for pest control strategies. Combining baculovirus species from a biological control perspective requires conducting laboratory and field assays to determine the final effect on target insects. We conclude that the use of baculovirus-based formulations with NPV-GV or NPV-NPV compositions is an attractive approach to enhancing the insecticidal properties of one of the viruses in the mixture and achieving simultaneous pest control.


Leopardus pardalis (ocelot) and a partial genome were assembled and annotated through high-throughput sequencing protocols followed by Sanger sequencing validation. The full-length virus obtained comprises 2,003 bp, while the partial genome comprises 1,224 bp. Phylogenetic analysis grouped these two sequences in two distinct clusters related to previously described Felidae anelloviruses. The ORF1 of the partial genome was identified as a new species provisionally called Torque teno ocelot virus, with 53.6% identity with its sister lineage. The complete genome was inferred as a new representative of the Torque teno felid virus 3 species, with 73.28% identity to the closest reference. This study expands known virus diversity and the host span of anelloviruses.A complete genome of the first anellovirus infecting the wild felid  Anelloviridae family represents non-enveloped single-stranded viruses with circular negative-sense DNA, whose genomes range from 1.6 to 3.9 kb. The group is classified in 30 genera and 155 species by the International Committee of Virus Taxonomy (Puma concolor), bobcats (Lynx rufus), Canada lynx (Lynx canadensis) and caracals  . In the present study, we characterize two novel anelloviruses in a Brazilian felid, the ocelot . The Taxonomy . AnellovTaxonomy . They arTaxonomy  and theiTaxonomy . In wildLeopardus pardalis). This project was performed with the authorization number 64515 from the Brazilian Institute for the Environment and Renewable Natural Resources (IBAMA). Plasma was filtered, centrifuged and the supernatant was discarded, leaving the bottom 0.2 mL to digestion with several nucleases. Viral nucleic acid was then extracted using QIAamp Mini Elute Virus Spin Kit (QIAGEN) and subjected to complementary DNA (cDNA) synthesis using Superscript III First-Strand Synthesis Supermix Kit (ThermoFisher). The second strand cDNA synthesis was performed with Klenow Fragment 3’-->5’ exo- (NEB). To get a highly efficient purification with superior quality DNA without salt carryover, the Agencourt AMPure XP Kit (Sinapse Biotecnologia) was used. To quantify double-strand DNA (dsDNA), the QuBit dsDNA High Sensitivity Assay Kit (ThermoFisher) was used, and the library was prepared using the Nextera XT DNA Library Preparation Kit (Illumina). Next-generation sequencing was conducted using the Illumina MiSeq V2 500-cycle kit. FASTq reads were processed with an in-house pipeline that included quality trimming, host genome filtering  and two rounds of BLASTx against RefSeq virus and nr (non-redundant) NCBI databases, as described by  - Brazil, a blood sample was collected from a nine-year-old female ocelot . Annotation was performed by aligning each ORF of the Torque teno felis virus 2 (accession number: NC_038349) against the novel genome, which allowed the inference of homologous genome regions. Three open reading frames (ORF) encoding ORF 1 , ORF 2 (321-bp) and ORF 3 (468-bp) were inferred. One partial genome was also assembled, comprising an incomplete ORF1 sequence with 1,173 nucleotides and was provisionally named WF10 strain 2 (accession number: OL449682). Felidae anelloviruses can be found in two major lineages and phylogenetic analysis of ORF1 gene grouped both new strains within feline anellovirus Group 1, in distinct lineages (Torque teno felis virus (MT538137) (SH-aLRT = 100 / Bootstrap = 100). Sequences shared 73.8% of pairwise identity, confirming they belong to the Torque teno felid virus 3 species in accordance with findings in Torque teno felid virus 3, indicating that more than one viral species might be found in the present lineage upon further sampling  with robust node support (SH-aLRT = 98 / Bootstrap = 100) (Torque teno viverrid virus 3 (LC387548) was recovered with high support (SH-aLRT = 98.3 / Bootstrap = 100), indicating that the present monophyletic lineage likely descends from an ancient virus that co-diverged along the Suborder Feliformia. Based on ICTV species delimitation criteria (identity below 69%) (Torque teno ocelot virus - with 53.72% identity to the closest reference (accession number: EF538878). The ORF1 of the WF10 strain 2 (OL449682) was retrieved as sister to the p = 100) . As an olow 69%) , the parL. pardalis explored by HTS protocols was coinfected by two different species of Anelloviruses, one new strain of Torque teno felid virus 3 (accession number: MK069470) and a putative novel species provisionally named Torque teno ocelot virus (accession number: OL449682). Anelloviridae co-infection by multiple unique lineages is common in healthy humans (Anelloviridae host span and may assist further studies aiming to characterize the complex evolutionary history of anelloviruses (The captive y humans , and thiy humans . The chaoviruses .


Water pollution is regarded as a dangerous problem that needs to be resolved right away. This is largely due to the positive correlation between the increase in global population and waste production, especially food waste. Hydrogel beads based on sodium alginate (Alg) and pomegranate fruit peels (PP) were developed for the adsorption of Safranin O dye (SO) in aqueous solutions. The obtained Alg−PP beads were widely characterized. The effects of the contact time (0–180 min), initial concentration (10–300 mg/L), initial pH (2–10), adsorbent dosage (1–40 g/L) and the temperature (293–333 K) were investigated through batch tests. The data proved that the adsorption kinetics of SO reached equilibrium within 30 min and up to 180 min. The dye adsorption is concentration dependent while a slight effect of pH was observed. The adsorption data of SO onto synthesized beads follow the pseudo second-order model. The experimental data fitted very well to Langmuir model with correlation factor of 0.92 which demonstrated the favourable nature of adsorption. The maximum adsorption capacity of Alg−PP could reach 30.769 mg/g at 293 K. Calculation of Gibbs free energy and enthalpy indicated that adsorption of SO onto Alg−PP is spontaneous (negative ΔG) and endothermic (ΔH = 9.30 kJ/mol). Analysis of diffusion and mass transport phenomena were presented. The removal efficiency was found to be 88% at the first cycle and decreased to 71% at the end of the seventh cycle. The reported results revealed that the Alg−PP beads could be used as a novel natural adsorbent for the removal of high concentrated solutions of Safranin O which is a cationic dye from liquid affluents and as future perspective, it can be used to remove various pollutants from wastewater. Punicaceae family, is widely cultivated worldwide. It is a byproduct of the pomegranate juice industry, wine industries, and tanneries :(24The water content of the Alg−PP hydrogel was determined by drying beads to a constant weight at 50 °C (Equation (25)):ImageJ software (Version 1.53t) was used to digitize photographs and determine the mean diameters and distribution of bead sizes of the hydrogel beads. 80 hydrogel beads were measured. Results were reported as mean ± standard deviation.The surface’s functional groups of the beads were investigated using Fourier transform infrared (FTIR) spectroscopy JASCO FT/IR 4600.pzc) [To investigate the surface charge of the hydrogel beads, the pH-drift method was applied to evaluate the point of zero charge (pHpzc) .Thermogravimetric analysis (TGA) was carried out in air atmosphere with a Mettler TC-10 apparatus from 30 to 700 °C, at a heating rate of 10 °C/min.q) and the removal efficiency (R%) of SO adsorbed onto hydrogel beads were evaluated through the following Equations (26) and (27):The dye adsorption experiments were carried out via a batch adsorption process by fixing a stirring rate of 400 rpm. 500 mg of hydrogel beads into 50 mL of different concentrations of dye solutions at room temperature (20 + 2 °C). The adsorbed dye amount onto the hydrogel beads was analyzed by using a UV/VIS spectrophotometer , Taejon, South Korea) at the wavelength λ= 522 nm. The pH effect on SO adsorption was investigated in the range of 2–10 while the initial dye concentration was fixed at 50 mg/L. The dosage effect of Alg−PP beads was varied from 1 to 40 g/L with a set initial dye concentration of 50 mg/L. The temperature was varied from 30 to 60 °C with a set dye concentration of 50 mg/L. The isothermal adsorption experiments were carried out with 50 mL of dye solutions with different initial concentrations (10–300 mg/L). The adsorption capacity .SSE) and residual root mean square error (RMSE) were used to evaluate the fitness of the kinetic and isotherm data with the regression coefficient R2, determined from the linearized model. The best fit of the curves is indicated by smaller error analysis values, and vice versa. The equations are expressed as follows:Squares of the errors (


Many experts in aerobiology have openly opposed that stance, forcing a vigorous debate on the topic. In this review, we discuss the various proposed modes of viral transmission, stressing the interdependencies between droplet, aerosol, and fomite spread. Relative humidity and temperature prevailing determine the rates at which respiratory aerosols and droplets emitted from an expiratory event  evaporate to form smaller droplets or aerosols, or experience hygroscopic growth. Gravitational settling of droplets may result in contamination of environmental surfaces (fomites). Depending upon human, animal and mechanical activities in the occupied space indoors, viruses deposited on environmental surfaces may be re-aerosolized (re-suspended) to contribute to aerosols, and can be conveyed on aerial particulate matter such as dust and allergens. The transmission of respiratory viruses may then best be viewed as resulting from dynamic virus spread from infected individuals to susceptible individuals by various physical states of active respiratory emissions, instead of the current paradigm that emphasizes separate dissemination by respiratory droplets, aerosols or by contaminated fomites. To achieve the optimum outcome in terms of risk mitigation and infection prevention and control (IPAC) during seasonal infection peaks, outbreaks, and pandemics, this holistic view emphasizes the importance of dealing with all interdependent transmission modalities, rather than focusing on one modality.During the recent pandemic of COVID-19 (SARS-CoV-2), influential public health agencies such as the World Health Organization (WHO) and the U.S. Centers for Disease Control and Prevention (CDC) have favored the view that SARS CoV-2 spreads predominantly  Viruses are expelled via respiratory droplets, which are typically categorized according to size. Droplet size (diameter) plays an important role in infectious droplet dissemination, deposition onto fomites in the environment, and access to and therefore viral infectivity in the human respiratory system  pandemic has resulted in a re-examination of pathways for respiratory virus dissemination and transmission. The transmission of respiratory viruses, such as severe acute respiratory coronavirus 2 (SARS-CoV-2) or influenza virus, from an infected to an uninfected person has been characterized as occurring mainly through “close contact” with an infected person . This raRS-CoV-2 . Exposury system . Direct y system . Droplety system . This favia respiratory aerosols (size: <5 µm) also has been advocated as an important transmission mechanism  has been proposed , as will be discussed later in this review.Long-range  dissemination of viruses echanism . Indirecechanism . The posechanism . Finallyproposed . The late.g., walking, opening doors, vacuuming, etc.), contributing to the infectious droplet/aerosol transmission pathway. Aerosol transmission involves virus from evaporated respiratory droplets that form droplet nuclei, aerosols generated through talking, sneezing, coughing, etc., virus re-aerosolized from surfaces, and virus conveyed on aerial particulate matter .Our goal in this review is to take a neutral position in this debate and to define the potential dissemination/transmission pathways and the major arguments for and against the relevance of these pathways. Part of the confusion and the difficulty in resolving the ongoing debate on transmission mechanisms is semantic . That isetc. are not to be found in the literature. Typically, however, the term “short range” denotes 6 ft or less than 1.5 m  risk to help clarify the semantic problems associated with various transmission modalities for respiratory viruses . Clear dan 1.5 m . Short tan 1.5 m . The uppan 1.5 m . We and an 1.5 m  make thei.e., since late 2019). The target audience for this review includes the infection prevention and control community, regional public health authorities worldwide, and the aerobiology research and epidemiology communities.This survey is intended to provide a sampling of the literature emphasizing the different pathways of transmission of respiratory viruses, including the relative importance of each proposed mode, and the interdependencies between the different modes. As such, our methods for ascertaining relevant literature were not intended to access all of the relevant literature, and exclusion criteria were not applied when accessing the literature. Instead, we started with the World Health Organization’s position on transmission modes, articulated in June 2020 , then see.g., coughing or sneezing) or who is talking or singing; in these circumstances, respiratory droplets that include virus can reach the mouth, nose or eyes of a susceptible person and can result in infection.” In the categorization of i.e., size > 100 µm) (The WHO update  on SARS- 100 µm)  is likel 100 µm) .Coronaviridae) and swine influenza virus and avian influenza virus (Orthomyxoviridae) being enhanced at larger particle sizes , relative to survival at particle sizes closer to that of the virions themselves   . It shou–200 nm) ). The sim) e.g., . Infectim) e.g., , hence tm) e.g., .Respiratory droplets emitted from infected individuals can be viewed as one of the primary sources for virus disseminated through other modes of viral transmission. For instance, the evaporation of infectious respiratory droplets to form droplet nuclei contributes to the generation of viral aerosols . As mentvia respiratory aerosols has also been the subject of scientific debate, which in fact predates the SARS-CoV-2 pandemic  transmission pandemic . The WHOpandemic .etc., or from other activities, including but certainly not limited to, flushing or cleaning toilets ) are required, as reported for volunteers without previous SARS-CoV-2 infection or vaccination , although long-range, long-time transmission is also possible    Fig. 2)via proxialation)  and viraalation) . Viral palation) , as wellalation) . In addialation) . Studiesalation) . Additioalation) .via aerosols include the fact that aerosols may remain dispersed and airborne over longer distances and for longer durations than do droplets, albeit with expected dilution with increased distance from the source  in air samples. It is likely that this reflects the relatively high dilution of virus with time and distance from the contaminating source, the relative insensitivities of the viral infectivity assays, compared with the nucleic acid testing approaches more typically employed for detecting virus in air samples, and the actual air sampling technologies that have been used . Unfortue.g., Clinical evidence for the relevance of the aerosol route of transmission of SARS-CoV-2 has been reviewed extensively e.g., .i.e., people who have close contact with the infectious person also may come into contact with potentially infectious surfaces, making the distinction between fomite and respiratory droplet transmission difficult to discern! This same type of argument is commonly used to dismiss the importance of indirect transmission, as “In the few cases where direct contact or fomite transmission is presumed, respiratory transmission has not been completely excluded.”  or indirectly   . There id hands) .Reports of the disruption of the chain of infection through targeted surface hygiene represent an indication of the possible relevance of the indirect transmission pathway. An example is a description of the reduction of norovirus contamination following use of targeted hygiene . Literate.g., The ability of bacteriophage deposited on hands to be transferred to environmental surfaces has been demonstrated previously e.g., . Transfeetc.) taking place on or around such surfaces once contaminated, there is a possibility of re-aerosolizing the virus. We have depicted some of the possibilities for pathogen re-aerosolization in Re-aerosolization  of viruses from environmental surfaces is not viewed as an additional transmission mode, but rather is a contributing factor for aerosol virus burden. Re-aerosolization, therefore, represents an interdependency between droplet transmission, environmental surface contamination, and aerosol transmission of infectious respiratory viruses. The topic of virus re-aerosolization from environmental surfaces is relatively novel, and not surprisingly, the WHO has not—to our knowledge—acknowledged the possibility or possible relevance of re-aerosolization of virus from environmental surfaces in its website addressing transmission pathways . What doIronically, as depicted in etc. from patient beds  and removal of protective equipment itself may represent a source of viral aerosols and, therefore, a route for dissemination of such viruses from infected individuals to healthcare workers in patient care settings.There is also the possibility of re-aerosolization of virus from contaminated personal protective garments when removing these, and from face mask materials that have been contaminated with virus . In addie.g., dust, allergens, PM) to be discussed in the next section of this review. Therefore, it needs to be emphasized that decontamination of environmental surfaces also reduces infectious droplets/aerosols by reducing fomites available for re-aerosolization through various activities as noted above  the various types of aerial particulate matter , dust, and allergens  . That isllergens . As of tllergens .2.5). In fact, infectious respiratory droplets, droplet nuclei, and aerosols might be thought of as relatively transitory states, with conveyance on aerial particulates representing a condition leading to greater airborne distribution, and possibly stability  in animal studies  has been difficult to resolve in the face of knowledge gaps that have yet to be closed. What data do we need in order to resolve the debate over the relative significance of transmission via infectious respiratory droplets vs. aerosols? Is this more a debate over short- vs. long-range transmission? What roles do indirect transmission and re-aerosolization of viruses play, and does viral conveyance on aerial particulate matter really play a significant role in aerosol transmission and acquisition or severity of infection?The debate over the relative importance of aerosol i.e., the upper respiratory mucosa) . Specifically, how many infectious virus particles (not RNA!) are released from the infected person as a result of breathing, talking, coughing, sneezing, or mechanical activities, e.g., vacuuming floor/carpet, toilet flushing, etc.? The extent to which the released infectious viruses end up in respiratory droplets or aerosols could be inferred from the data on size distribution of droplets expelled during different expiratory activities ) are required, as reported for volunteers without previous SARS-CoV-2 infection or vaccination  . In any  mucosa) . For instivities , but thicination . The relcination . More clsmission . However50) may depend upon the infection initiation site  may determine the clinical outcome post-infection. Certain aerial particulates can act synergistically to increase likelihood of acquiring an infection , or influencing the clinical severity of the acquired infection. These represent topical areas deserving of further study. Resolving these knowledge gaps may help investigators to determine the relative relevance of the various transmission pathways discussed in this article.The WHO has made the point  that it vs. ∼18 for measles virus) has been used as an argument against an important contribution of long-range aerosol transmission . This heterogeneity in the case of SARS-CoV and SARS-CoV-2 might relate to the possible synergistic effects of modifiers of host cell receptor or protease expression, rather than to mode of transmission. Until this is worked out, a reasonable position to take is that the transmission of SARS-CoV-2 is multimodal  per liter of air sampled. This level of virus was sufficient to cause infection in the Syrian hamster animal model  and non-pharmaceutical interventions (NPI) to mitigate the risks associated with the illness and risk of transmitting the virus to others (etc. (During the SARS-CoV-2 (COVID-19) pandemic, health authorities worldwide responded by implementing a variety of pharmaceutical (o others . The NPIo others . That thrs (etc. .etc.) should also be acknowledged. In this section, we discuss available means of mitigating these transmission risks.Going forward, we advocate taking the most conservative approach informed by the results of the present review, that is, in a confined indoor space occupied both by infected individual(s) and susceptible individual(s) including those immunocompromised, the risk of exposure to infectious respiratory viruses may include infectious respiratory droplets and infectious aerosols of varying size, indirect transmission from environmental surfaces, and virus re-aerosolized from previously contaminated environmental surfaces . Under cThe most commonly employed NPI for mitigating risk of acquiring a respiratory viral infection COVID-19 pandemic worldwide, has been universal use of face masks. Countries which responded to the pandemic with universal mask wearing mandates (a good example being Taiwan) have been most effective in reducing virus spread . A propeetc.) settings, healthcare settings, and in the presence of an individual known to be infected with a respiratory virus, etc.  to the airborne route . One might argue that prevention of re-aerosolization from environmental surfaces might be adequately prevented through stringent surface hygiene practices, although the possibility exists that at least some re-aerosolization may occur because of cleaning practices (e.g., Although we now apparently are in the final stages of the ongoing SARS-CoV-2 pandemic, and the past three years have seen an explosion in research on all aspects of the pandemic, it is clear that we still have much to learn about transmission of SARS-CoV-2 and other respiratory viruses. New variants of SARS-CoV-2 will likely continue to emerge e.g., . It woul


Dear Editor:The recent spike of a rare zoonotic infection caused by the monkeypox virus is gaining global attention due to its high transmissibility and fear of stigmatization. Without proper measures to prevent the spread of virus, monkeypox could potentially set the stage for another widespread outbreak leading to worldwide health calamity as witness during the coronavirus disease 2019 pandemic. As a lesson learnt, it is now evident that rapid screening and diagnostic testing is the most efficient approach toward containment of any disease outbreakOrthopoxvirus. The Laboratory Response Network was set up with the goal of forming a stable network across the nation to effectively and rapidly share diagnostic tools, interpretation of results, training of laboratory technicians, and reporting of any errors through critical evaluation and communication networks during regular as well as emergency health crisisThe Centres for Disease Control and Prevention recommend the testing of all patients who present with any noticeable rash resembling monkeypox virus or patients with other symptoms of the disease in order to prevent the recent monkeypox surge from emerging as another pandemic of global concern. Currently, the confirmatory test performed monkeypox virus is the Nucleic Acid Amplification Test that uses the PCR to detect the viral DNAIn India, the central government has established a network consisting of a total of 15 Virus Research and Diagnostic Laboratories across the country in 13 states to track the prevalence of monkeypox and promote rapid testing facilitiesThroughout the world, monkeypox disease is raising concern, and rapid screening and testing is the most efficient step toward curbing the disease’s outbreak. Not only symptomatic but also asymptomatic patients and close contacts should also be rapidly screened for the monkeypox virus. This mandates a well-organized and rigorous laboratory network system across the world to collect, transport, test, and report the result of the sample. Laboratory networking functions beyond the scope of testing. This complex and vigilant network system serves as a platform for providing emergency response, training of laboratory personnel, communication across different testing centres, surveillance of laboratory data, and diligent management of patient informationNot applicable.Not funded by any funding agency.V.P.V., S.P., J.N.B, L.P., U.M.: conceptualization, data curation, writing – original draft preparation. S.K.M.: conceptualization, study design, data curation, writing – original draft preparation, reviewing and editing, visualization and supervision.No conflict of interest.Not ApplicableDr Surapaneni K. Mohan, corresponding author.This correspondence is based exclusively on resources that are publicly available on the internet and duly cited in the ‘References’ section. No primary data was generated and reported in this manuscript. Therefore, data has not become available to any academic repository.