This review summarizes research on virus diseases of cereals and oilseeds in Australia since the 1950s. All viruses known to infect the diverse range of cereal and oilseed crops grown in the continent’s temperate, Mediterranean, subtropical and tropical cropping regions are included. Viruses that occur commonly and have potential to cause the greatest seed yield and quality losses are described in detail, focusing on their biology, epidemiology and management. These are: barley yellow dwarf virus, cereal yellow dwarf virus and wheat streak mosaic virus in wheat, barley, oats, triticale and rye; Johnsongrass mosaic virus in sorghum, maize, sweet corn and pearl millet; turnip yellows virus and turnip mosaic virus in canola and Indian mustard; tobacco streak virus in sunflower; and cotton bunchy top virus in cotton. The currently less important viruses covered number nine infecting nine cereal crops and 14 infecting eight oilseed crops (none recorded for rice or linseed). Brief background information on the scope of the Australian cereal and oilseed industries, virus epidemiology and management and yield loss quantification is provided. Major future threats to managing virus diseases effectively include damaging viruses and virus vector species spreading from elsewhere, the increasing spectrum of insecticide resistance in insect and mite vectors, resistance-breaking virus strains, changes in epidemiology, virus and vectors impacts arising from climate instability and extreme weather events, and insufficient industry awareness of virus diseases. The pressing need for more resources to focus on addressing these threats is emphasized and recommendations over future research priorities provided. On a global scale, virus disease outbreaks, epidemics and pandemics threaten all kinds of crops and, depending upon individual circumstances, cause damage varying from total crop failure to small-scale losses. They threaten crop plants not only by impairing their growth and vigor thereby diminishing gross yields, but also by damaging produce quality thus decreasing marketable yields. Agricultural globalization, climate change-driven influences and other factors, such as pesticide resistance in virus vectors, are aggravating their spread and making their control more difficult ,9,10,11.Triticum aestivum), barley (Hordeum sativum), oats (Avena sativa), triticale , sorghum (Sorghum bicolor), maize and sweet corn (Zea mays), and rice (Oryza sativa); oilseeds–canola , sunflower (Helianthus annuus), soybean (Glycine max), peanut (Arachis hypogea) and cottonseed (Gossypium hirsutum); and grain legumes–lupin (Lupinus spp.), chickpea (Cicer arietinum), field pea (Pisum sativum), faba bean (Vicia faba), lentil (Lens culinaris) and mungbean (Vigna radiata). In addition, Australia grows sugarcane (Saccharum officinarum) and a wide range of horticultural crops , relies on improved pastures to provide the main the feed-base for its extensive wool, dairy and meat industries, and has a large timber industry. The most important agricultural crop is wheat with c. 20 million tonnes produced annually. Barley, canola, cottonseed and rice are also produced on a very large-scale, with production of other cereal and oilseed crops, and of grain legumes occurring on a smaller scale . Thr. ThrMachviridae)  7,8]. T. TMachlo viruses . Since WP. graminis which has motile zoospores that swim in soil moisture [P. graminis resting spores, as was suggested previously for SBWMV when it reached New Zealand (see Pecluvirus (family Virgaviridae) , and wheat yellow mosaic virus  (Bymovirus (family Potyviridae) viruses barley yellow mosaic virus (BaYMV) and barley mild mosaic virus (BaMMV) , and oat golden stripe virus , and, for triticale and rye, they are SBCMV, SBWMV and WSSMV  . Both ocviridae)  [325,329viridae) C,E, SBCMviridae) , Chineseviridae) . For bar (BaMMV) . For oatnd WSSMV . P. gramst crops ,328,329.Begomovirus, family Geminiviridae), present in the Indian subcontinent, and Central and North America, and cotton leaf curl disease caused by cotton leaf curl viruses , present in the Indian subcontinent, East Asia, Southeast Asia, and North, East and West Africa (A. gossypii) is very common in subtropical and tropical regions of Australia [B. tabaci) in warmer regions of eastern Australia [To date, the Australian cotton industry has avoided the damage to international cottonseed oil production from cotton blue disease caused by infection with CLRV see  above, wt Africa  [103,224ustralia ,293, as ustralia . Therefoustralia , and in ustralia . This hiustralia  so its aP. graminis transmitted viruses PCV and IPCV (see above in this Section) , groundnut rosette assistor virus  and a virus satellite; and groundnut bud necrosis disease which is present in the Indian subcontinent, Central and East Asia and Southeast Asia, and is caused by groundnut bud necrosis virus   and F. schultzei (common blossom thrips). T. palmi has a widespread distribution across northern Australia, and F. schultzei occurs commonly throughout the Australian continent [Major biosecurity virus threats to Australian peanut oil production include damaging diseases caused by the Section) . They alviridae)  [7,103,3ustralia ,107, andontinent . Groundnontinent , so if iontinent ,336,337,Worldwide, PCR, tissue blot immunoassay (TBIA), ELISA and biological indexing were formerly the standard procedures for detection of cereal and oilseed viruses with PEQ. However, biological indexing is both labor-intensive and time consuming, and obtaining supplies of reliable and accurate antisera to support TBIA and ELISA poses a significant limitation to their use. In addition, they are sometimes unable to identify diverse virus variants. Fortunately, the cost of high throughput sequencing has declined rapidly due to advances in sequencing technology, so it is now feasible to use it to overcome such drawbacks and deploy as a routine diagnostic tool for detection of cereal and oilseed viruses in PEQ ,339.Future research efforts need to focus on developing more robust and cost-effective diagnostics and surveillance strategies to help avoid establishment of damaging cereal and oilseed viruses and their vectors within Australia. This includes improving diagnostic standards at the border and providing tools to support cost effective biosecurity measures both there and during area-wide surveillance. In addition, with appropriate modifications, such as the metabarcoding already used for insect identification , high thM. persicae and infection with TuYV, resulting in increased yields  and M. persicae (TuYV vector), the whitefly species B. tabaci (CLCuV and CLCrV vector), the thrips species Frankliniela oxidentalis (TSWV vector) and Thrips palmi (GBNV vector), and the eriophyid mite vector WCM (WSMV vector)  . This makdr-type) conferred by multiple point mutations in voltage-gated Na+ channels effective against pyrethroids and the now banned organochlorines; (ii) modified acetylcholinesterases (MACE) conferred by mutations at the AChE target site effective against carbamates; (iii) insensitive gamma-aminobutyric acid (GABA) receptors effective against cyclodienes and phenylpyrazoles; (iv) over-production of esterases E4 or FE4 effective against organophosphates, carbamates and, to a lesser extent, pyrethroids; and (v) over-production of cytochrome P450 genes reducing sensitivity to neonicotinoids [In addition to behavioral resistance involving insecticide avoidance, e.g., in eriophyid mites , three dotinoids .M. persicae have developed target site resistance to carbamates (S431F mutation) and synthetic pyrethroids , strong metabolic resistance to organophosphates and carbamates (enhanced expression of E4/FE4 esterase), and mild metabolic resistance to neonicotinoids (enhanced expression of cytochrome P450 gene CYP6CY3) [M. persicae in Australia, although the resistance mechanism involved has not yet been elucidated [M. persicae populations [A. gossypii populations have also developed resistance to carbamates and organophosphates , pyrethroids (L1041F mutation) and metabolic resistance to neonicotinoids (enhanced expression of a cytochrome P450 gene) [A. gossypii having developed resistance to sulfoximines or the butenolide insecticide flupyradifurone (group 4D) yet [A. gossypii has also been reported to develop target-site resistance to neonicotinoids (R81T mutation) [B. tabaci MEAM1 (=virus vector bioytpe B) is reported against organophosphates, carbamates, synthetic pyrethroids and the insect growth regulator pyriproxyfen, but not yet against neonicotinoids [B. tabaci MEAM1 populations have developed resistance to neonicotinoids such as imidacloprid [M. persicae and A. gossypii populations with stronger resistance to neonicotinoids and sulfoxamines, and B. tabaci MEAM1 populations with neonicotinoid resistance arriving in imports, presents three serious vector biosecurity threats for Australia. They pose a threat to Australian canola and cotton production because both industries rely heavily on these two insecticide groups to control insect vectors. Uncontrolled populations of virus vectors in these crops would lead to increased virus epidemics and resulting losses from TuYV in canola and CBTV in cotton. Such uncontrolled vector populations would also increase damaging epidemics of CLRDV, CLCuSV and CLCrV in cotton should these viruses become established in Australia. They would also increase the insect vector reservoir for viruses of other crops. As in other parts of the world, in Australia F. occidentalis can no longer be controlled by any of the major groups of insecticides, only by newer groups such as the spinosyn insecticides spinosad and spinetoram [T. palmi also cannot be controlled by applying any of the major insecticide groups, application of insecticidal soaps constituting the only current recommendation for their management using chemicals [Australian populations of CYP6CY3) ,254,346.ulations ,348. Aus50 gene) ,342,349. 4D) yet . Furtherutation) . Resistaotinoids . Outsideacloprid , but appacloprid ,355. Theinetoram . Similarhemicals .R. padi, the primary vector of BYDV and CYDV in cereals, have developed tolerance to the pyrethroid alpha-cypermethrin, this aphid vector species remains susceptible to most other insecticides used to control it in Australia [S. avenae, a YDV vector not yet found in Australia, has developed a broad insecticide resistance profile [R. padi or S. avenae populations with multiple insecticide resistances represents two further vector biosecurity threats for Australia.Although local clones of some populations of Australian ustralia . Howeverustralia ,360,361. profile ,126 , biopesticides, RNAi and nanoparticles ,65,369. The likelihood that resistance-breaking virus strains will develop constitutes a major hazard when single gene virus resistances are being incorporated into new crop cultivars by plant breeding programs. It also applies when new cultivars are unknowingly bred from parental lines with resistance genes, as with TuMV in canola . In the The subject of likely climate change induced alterations to epidemics of cereal and oilseed virus diseases around Australia constitutes an issue of considerable magnitude. It can only be dealt with briefly here. A review published in 2009 summarised likely effects on plant virus epidemics, including Australian cereal and oilseed examples, as part of an article also covering virus origins, emergence and evolution . Since t2 on insect and mite vectors can include alterations in their population numbers, growth rates, fecundity and feeding, the actual outcome varies greatly between different types of vectors, and even between similar species within the same group of vectors. The same applies to its direct effects on virus multiplication within infected plants, disease severity and plant defense responses to infection as these vary with the virus-host plant pathosystem studied [2) in Australia. Hence, the need to focus instead on how virus epidemics are likely to be influenced by the alterations in other climate parameters that result from elevated levels of greenhouse gasses  in the atmosphere. These altered climate parameters include rainfall, temperature, wind, and increased climate instability, all of which have major influences on virus epidemic development, and the resulting production losses in cereal and oilseed crops grown in Australia’s diverse agricultural regions.Although the effects of elevated CO studied ,9. This a vector before crops are sown and in the early growing period on volunteer cereal and annual grass hosts. This is because: (i) although there is little WCM population increase under cool conditions, its populations explode under warm conditions, and (ii) it spreads this virus from initial infection foci consisting of infected volunteer or sown wheat plants that grow from WSMV-infected wheat seed  or elevated (650 μmol mol−1) CO2, its titer was increased by elevated CO2 [R. padi vector were held under ambient CO2 (385 μmol mol−1) or elevated CO2 (650 μmol mol−1), on healthy plants elevated CO2 caused the aphid vector population to diminish and its feeding damage to increase, whereas in plants infected with BYDV-PAV neither parameter was altered [2 (550 μmol mol−1) in an Australian free-air CO2 enrichment facility [Although there are a small number for TuYV in canola, TuMV in mustard and BYDV in wheat, relatively few Australian studies, have examined the likely effects of different climate change parameters on virus diseases of cereal and oilseed crops. The transmission efficiency of TuYV by at 16 °C , congruoformance . Thus, h25–28 °C  , but a great deal more needs to be done before the country is ready to face what is likely to occur in the future. More climate change scenario modelling is needed to establish when significant viral pathogens or vectors are likely to increase causing damaging virus epidemics in crops or invade regions where conditions were formerly unsuitable for them. Moreover, initial experiments studying the effects of diverse climate change parameters on viral diseases under controlled environment conditions need to be followed up by examining their effects on virus epidemics occurring in field situations, including in FACE facilities, and in crops growing in regions with high temperatures or where flood or drought conditions occur.To prepare for future climate change induced epidemics of virus diseases in cereal and oilseed crops in Australia, a much greater emphasis on research on this subject is required. Such research would need to focus on the effects arising from increased greenhouse gasses, temperatures or wind speeds, insufficient or excessive rainfall, and extreme weather events upon epidemics of Australia’s major current virus disease threats to cereal and oilseed production  increasing difficulties in controlling them due to the escalating levels of climate instability and extreme weather events; (ii) the rise in insecticide resistance in insect vectors, combined with the likelihood of a future ban on several insecticides currently relied upon to manage them; (iii) the development of virus strains that break resistance genes being used in breeding new virus-resistant cultivars; and (iv) the lack of sufficient industry awareness about the importance of virus diseases. We also provide detailed recommendations for future research needed to address these threats and emphasize the major biosecurity risk to Australian cereal and oilseed industries posed by damaging viruses and their virus vector species spreading from other countries.P. graminis pose a threat to wheat and/or barley crops , and oats , and in subtropical or tropical regions to maize, sweet corn, sorghum, pearl millet and/or rice crops . The oilseed viruses of greatest concern include CLRDV, CLCrDV and CLCuVs in cotton; and GBNV, PCV and the virus complex causing the GRD in peanut and/or soybean. However, currently, there do not appear to be any obvious external biosecurity threats to Australian canola and Indian mustard crops from virus diseases present elsewhere.With the sole exception of rice and linseed, virus diseases are known to occur in all of the diverse range of 19 different cereal and oilseed crops currently grown in the Australian continent’s temperate, Mediterranean, subtropical and tropical cropping regions. The cereal virus diseases currently of greatest cause for concern are those caused by BYDV and CYDV in wheat, barley and oats, WSMV in wheat, and JGMV in maize, sweet corn and sorghum. Those of greatest concern for oilseeds are TuYV and TuMV in canola and Indian mustard, TSV in sunflower and CBTV in cotton. In the past, two of these viruses  have been controlled effectively by planting crop cultivars with virus resistance genes, but this situation is now changing due to the appearance of resistance-breaking virus strains or planting new cultivars lacking virus resistance. In addition, there are 23 other viruses infecting one or more crops that are currently of less importance, nine of which infect nine different cereal crops and 14 of which infect eight different oilseed crops. However, several of these have the potential to cause greater losses in the future. The cereal viruses of greatest concern should they become established in Australia include MDMV, MSMV, MRDV and the virus complex causing MLND in maize and sweet corn crops, and RHBV, RYMV and the virus complex causing RTD in rice. In cooler, wetter regions of Australia, several soil-borne viruses transmitted by Despite the considerable knowledge accumulated in the past, recent information available from (i) virus disease surveys, epidemiology research and yield loss studies from across Australia, and (ii) increased understanding of the growing threats to their effective management from climate instability and extreme weather events, insecticide resistance breakdown, and virus resistance breaking strains, reveal that cereal and oilseed virus diseases and their management require a considerable further research effort. Greater emphasis on traditional research involving field experiments and studies on plant virus disease epidemiology and management is required. Also, a vigorous extension effort is needed to ensure farmers understand the threat virus diseases pose to their crops. Moreover, there is a pressing need to understand how rapidly changing farming systems and alterations in global market requirements, are likely to alter future virus disease epidemics, and the extent to which this will require modification of disease management approaches for cereal and oilseed crops under changing circumstances. Such knowledge will enable farmers to maximize their profits in years when yields are high and minimize their losses in years when they are low. In addition, the climate in Australia’s cereal and oilseed growing regions is changing rapidly as maximum temperatures rise, and rainfall within, or between, growing seasons declines or increases, and becomes more erratic. This is causing rising uncertainty over when virus control measures are needed, and when to intervene within growing crops. This uncertainty will increasingly compromise decision making over which control measures to use in order to optimize the desired control outcome. In addition, farmers need reliable early warning systems based on vector and virus loads from both the current and previous growing season. Such early warning systems enable estimates of risk, inform in-crop monitoring decisions, and assist with the timely and targeted implementation of control strategies. Furthermore, the current approach of utilizing historical data and new research approaches to optimize disease modelling and decision support systems is important. The principal focus of future research on the better understood virus diseases of cereals and oilseeds should involve ensuring that epidemiological models and decision support systems already available  are deployed nationally. Additional local research on their epidemiology and management that complements any research findings already available may be required leading to the development of epidemiological models and decision support systems suitable for use in different parts of the continent.Given the magnitude of the threat posed to Australian cereal and oilseed crops should damaging virus diseases, or any of their insect, mite or protist vectors, that occur in one or more other world regions become established in the country, stringent biosecurity measures are required in pre-border, border and post-border situations see  above. AFinally, preparation of a revised version the book ‘Viruses of Plants in Australia’ that brings it up-to-date by summarizing all new plant virus identifications, and recent studies on the biology, epidemiology and management of each virus, and by confirming, or otherwise, previous assumptions about virus identifications in the light of recent molecular studies, would provide an extremely valuable resource for Australia.


Nicotiana benthamiana or Nicotiana glutinosa. Recently, the Brassica yellows virus (BrYV) genotype A P0 protein (P0BrA) was identified as a strong viral suppressor of RNAi. In this study, we compared the features of the P0 proteins encoded by different genotypes of BrYV and revealed their difference in inducing cell death in N. benthamiana. Key residues in P0BrA for inducing cell death were also identified. We also showed that all three BrYV genotypes had synergistic interaction with PEMV 2 in N. benthamiana. This study provides theoretical guidance for controlling the viral disease caused by poleroviruses in the future.Polerovirus P0 proteins are multifunctional proteins. Besides their viral suppressor of RNA silencing (VSR) functions, several P0 proteins can induce a cell death phenotype within the infiltrated region of Polerovirus, which has at least three genotypes  in China. The P0 protein of BrYV-A (P0BrA) has been identified as a viral suppressor of RNA silencing (VSR), which can also induce cell death in infiltrated Nicotiana benthamiana leaves. In this study, we demonstrated that the cell death induced by P0BrA was accompanied by the accumulation of reactive oxygen species (ROS) and increased Pathogenesis-related protein genes-1 (PR1) expression. Meanwhile, this cell death phenotype was delayed by salicylic acid (SA) pretreatment. Biological function comparison of the three P0 proteins showed that transiently expressed P0BrB or P0BrC induced a significantly delayed and milder cell death response compared with P0BrA. However, like P0BrA, they also suppressed local and systemic RNA silencing. Six residues of P0BrA essential for inducing cell death were identified by comparative analysis and amino acid substitution assay. We also show that all three BrYV genotypes have synergistic interactions with pea enation mosaic virus 2 (PEMV 2) in N. benthamiana. This study provides theoretical guidance for controlling the viral disease caused by poleroviruses in the future.Brassica yellows virus (BrYV) is a tentative species of the genus  Turnip yellows virus (TuYV), Cucurbit aphid-borne yellows virus (CABYV), Potato leafroll virus (PLRV), Beet mild yellowing virus (BMYV), Cotton leafroll dwarf virus (CLRDV), Sugarcane yellow leaf virus (ScYLV), Melon aphid-borne yellows virus (MABYV), Maize yellow mosaic virus (MaYMV), Cereal yellow dwarf virus (CYDV), Wheat yellow dwarf virus-GPV isolate (WYDV-GPV), Maize yellow dwarf virus-RMV 2 (MYDV-RMV2), and Pea mild chlorosis virus (PMCV)  dCTP-labeled DNA probe specific for BrYV or PEMV 2 was generated using the Prime-a-Gene labeling system . Hybridization was carried out at 65 °C for 16 h. After washing, the nylon membrane was exposed to a storage phosphor screen .Northern blot was performed as described . RNAs usNbPR1 gene  and FWR motif [(K/R) IYGEDGX3FWR], indispensable to the silencing suppression activity [FWRIA-223) was present in the three P0Br proteins (BrA and P0BrB had a similar F-box-like motif (63-LPL(L/H)LGDH(V/I)HDDVRKSILVP-82). Interestingly, the amino acid residue 82 of P0BrC in the F-box-like motif was an alanine (A) instead of the consensus proline (P), but residue 84 of P0BrC was a P  essential for inducing cell death were identified by comparative analysis of the P0 proteins. Here, we also showed that all three BrYV genotypes had a synergistic interaction with PEMV 2 in N. benthamiana. This study provides theoretical guidance on controlling the viral disease caused by poleroviruses in the future.In this study, we demonstrated that the cell death induced by P0


We are pleased to present this Special Issue on the topic of “Plant virus Epidemiology”. This issue includes a series of articles on important aspects related to plant viruses. Plant diseases in general are in fact both fascinating and abominable. Although the consumer usually perceives the effect of these diseases only in an anecdotal way, like when the fruit and vegetables in the home spoil, the truth is that they have an extraordinary impact on the life and health of humans and livestock. This impact has profoundly marked the history of humanity, since it is only a little more than a century and a half ago that we have begun to unravel the biological bases of diseases, understand their causes, and mitigate their effects. Among the pathogens that cause them, viruses are admirable weapons of mass destruction. On the one hand, they are a paradigm of biological simplicity that places them on the frontier of life. However, they are also the second group of plant pathogens and they currently cover some 1516 species across 26 families ). Plant viruses are also economically important, and, in general, this increases proportionally with the intensification of agriculture . Two of The possibilities of acting directly on viruses are less than for other pathogens that cause losses, and their control is fundamentally based on two strategies: (i) control of inoculum sources , and (ii) the use of resistant cultivars, obtained by conventional genetic improvement or by genetic engineering methods. Therefore, it is essential to have a thorough knowledge of the epidemiology of diseases caused by viruses, which allows the best use of the control tools available. The epidemic is defined as the variation, in time or space, of the amount of diseases in a host population. In general, the definition of epidemic substitutes variation for increase, but variation reflects a greater number of situations that can occur throughout the development of an epidemic . In the C. melo accessions behave different following the inoculation with either one or the other virus strain. These results are important for melon breeders, but help to explain the recent successful spread of the Asian CGGMV strain in Spain and many other countries in the world [Breeding for disease resistance plays a major role in developing strategies for disease management. This is especially the case for plant virus disease. Cucumber green mottle mosaic virus (CGMMV) is a severe threat to melon production worldwide. At present, there are no cultivars available on the market which show an effective resistance or tolerance to CGMMV infection. Different strains of this virus exist in different parts of the world, and recently strains of putative Asian origin have entered Spain, coinfecting and subsequently displacing the European strain. It was found that he world . As withhe world .The epidemiology of plant viruses becomes more complicated in systems where several virus species are involved. Watermelon mosaic virus (WMV) and Moroccan watermelon mosaic virus (MWMV) infect cucurbitaceous crop species like melon, watermelon, and zucchini. However, a monitoring of crops revealed the different presence of WMV and MWME which could be explained on host plant species, but also on the different ranges of plant host species, and of aphid vector species between the two virus species. In addition, experimental infections suggest that mixed infections with both virus species in the same host also determines the viral loads, and may explain impaired distribution of one of the viruses .Mixed infections of different virus species is also studied in another paper in this Special Issue, and this involves two important emerging viruses in tomato . Since tPlant Virus Epidemiology” highlight elaborate efforts in modern plant virus epidemiology and that they support a broader view of the complex nature of how plant diseases caused by viruses occur and offer clues to its control. In closing, we thank all of the authors for their enlightening contributions to this Special Issue.We hope that the articles in this Special Issue on “


Over the past two decades, several deadly viral epidemics have emerged, which have placed humanity in danger. Previous investigations have suggested that viral diseases can spread through contaminants or contaminated surfaces. The transmission of viruses via polluted surfaces relies upon their capacity to maintain their infectivity while they are in the environment. Here, a range of materials that are widely used to manufacture personal protective equipment (PPE) are summarized, as these offer effective disinfection solutions and are the environmental variables that influence virus survival. Infection modes and prevention as well as disinfection and PPE disposal strategies are discussed. A coronavirus-like enveloped virus can live in the environment after being discharged from a host organism until it infects another healthy individual. Transmission of enveloped viruses such as SARS-CoV-2 can occur even without direct contact, although detailed knowledge of airborne routes and other indirect transmission paths is still lacking. Ground transmission of viruses is also possible via wastewater discharges. While enveloped viruses can contaminate potable water and wastewater through human excretions such as feces and droplets, careless PPE disposal can also lead to their transmission into our environment. This paper also highlights the possibility that viruses can be transmitted into the environment from PPE kits used by healthcare and emergency service personnel. A simulation-based approach was developed to understand the transport mechanism for coronavirus and similar enveloped viruses in the environment through porous media, and preliminary results from this model are presented here. Those results indicate that viruses can move through porous soil and eventually contaminate groundwater. This paper therefore underlines the importance of proper PPE disposal by healthcare workers in the Mediterranean region and around the world. During a viral epidemic, personal protective equipment (PPE) plays a major role in protecting doctors, nurses, and other healthcare or emergency medical personnel from viral infections Islam . PPE is To be transported into the environment, pathogens must be able to stay viable outside the host. How long a pathogen remains viable in the environment depends on the effects of numerous biotic and abiotic stresses on the pathogen   of India states that used PPE  from COVID-19 isolation wards at healthcare facilities should be segregated and sent to common facilities for disposal as per biomedical waste management rules (BMWM rules). However, used PPE (such as masks and gloves) from households, commercial establishments, and institutions must be stored separately for a minimum of 72 h, cut and shredded, and then disposed along with solid waste  via the leaching of contaminated water.Personal protective equipment should include eye and face protection, hand protection (disposable gloves), respiratory protection , skin and body protection, head protection, and reusable leather boots  treatment, thermal disinfection (heat) treatment, ultraviolet germicidal irradiation (UVGI), ethylene oxide (EtO), and bleaching (sodium hypochlorite) treatment are generally used to systematically disinfect PPE. Among these, UVGI and EtO treatment effectively decontaminate PPE components without affecting their protective performance or physical appearance. On the other hand, a single cycle of VPHP treatment does not have a significant effect on respirator performance or appearance. While bleaching (0.1% sodium hypochlorite) treatment does not affect the appearance or performance of the treated respirator, the residual odor of bleach following the treatment is a potential health risk. In fact, even the presence of a low concentration of this chemical led researchers to discourage the use of bleach for decontamination. The effectiveness of common decontamination techniques has been nicely reviewed by Kharbat et al. .There are several factors that affect the ability of enveloped virus (SARS-CoV-2) to retain its infectivity for a certain period of time on a surface. Environmental factors influence this ability in different ways, with each having some favorable and unfavorable effects. Physical factors include the ambient humidity, temperature, and sunlight . Regardless of the current situation, the emergency procedures recommended by the Centers for Disease Control and Prevention (CDC) should be followed  is subject to many environmental stresses but retains its infectivity . Generally, enveloped viruses prefer low-pH environments. An alkaline environment is harmful to enveloped virus (SARS-CoV-2)  can infect wastewater and groundwater are via patient feces, open-air landfills, and contaminated materials that are left in the environment during rainfall; these contamination routes are crucial to understanding COVID-19 diffusion Islam .To predict the likelihood of SARS-CoV-2 dispersion into soil through virus-contaminated water, a model of virus diffusion through a layered soil sample was implemented. In this model, water is ponded by a ring on the soil surface. Enveloped virus (SARS-CoV-2) is present in the water puddle, and is transported by the water through the dry soil Fig. . The soiC denotes the specific moisture capacity (m−1); Se is the effective saturation of the soil (dimensionless); S is a storage coefficient (m−1), which is expressed in terms of θs and θr: the volume fractions of fluid at saturation and after drainage, respectively; Hp is the pressure head (m), which is proportional to the dependent variable p (Pa); t is the elapsed time; K is the hydraulic conductivity (m/s); and D (m) is the vertical diffusion depth typically, represent in the z-direction and "r" is water paddle radius.The Richards equation governs the saturated–unsaturated flow of water in soil. The soil pores are connected to the atmosphere, so it can be assumed that pressure changes in the air do not affect the flow, meaning that the Richards equation is applicable (Oran and Topol c is enveloped virus (SARS-CoV-2) concentration (PFU/mL); t is the elapsed time; and cp is the mass of contaminant adsorbed per dry unit weight of solid (mg/kg). In addition, ρb is the bulk density (kg/m3) and θ is the volume fraction (porosity) of fluid. Thus, the term ρbcp is the mass of contaminant attached to the soil. It is worth noting that reaction rate terms were omitted from this preliminary model.The most general form of the governing equation for virus transport, which considers the convection and diffusion of a sorbing species in variably saturated soil, can be written as follows:−3 m2/day. Other physical and transport properties are not reported here for the sake of brevity. Different upper soil layer porosities were also analyzed.All of the physical and transport proprieties of soil were fixed in accordance with their literature values (Oran and Topol The finite element (FEM) method was used when performing the modeling, which was implemented in COMSOL Multiphysics 5.5. The three soil layers were discretized into a total of 7098 mapped mesh elements with an average element quality of about 0.9993, leading to 35,999 degrees of freedom. The mesh used provided adequate spatial resolution for the system under study. The solution was independent of the grid size, even with further refinements. On an i7-10750 hexa-core processor computer (16 GB DDR4 RAM) running under Windows 10, a typical parametric sweep simulation was completed in about 36 min.To aid understanding of the model simulation, Fig. Plots showing the time evolution of the distribution of enveloped virus (SARS-CoV-2) considered in 3 soil sections at 0, 2, 5, 10, 15, and 20 days are reported in Fig. r = 0.4 m) is shown in Fig. The evolution of the coronavirus contamination of the soil and the evolution of enveloped virus (SARS-CoV-2) transport streamlines were examined. The third layer (section) only became contaminated after a long period. Additionally, the influence of the upper layer porosity was investigated by performing a parametric sweep analysis using a coefficient ranging from 10% to 190% of the original porosity. The temporal variation in enveloped virus (SARS-CoV-2) concentration at a probe point in the middle layer was derived for various upper layer porosities. The position of this probe point , detailed diffusion data, and other physical quantities will be updated and included in the model as soon as more information on the SARS-CoV-2 virus is available in the literature. Also, we considered that enveloped virus (SARS-CoV-2)-contaminated water in our model was tap water or fresh water, not wastewater. Wastewater contains residual wastes that could affect the diffusion of enveloped virus (SARS-CoV-2) into the soil. To account for the effects of waterwater components on virus diffusion through the soil in our model, we require more detailed information on the physical and/or chemical interaction mechanisms between enveloped virus (SARS-CoV-2) and other water contaminants to be published in the literature.In this brief review and investigation, we have highlighted the risks of uncontrolled personal protective equipment disposal and the mechanism of virus transmission from contaminated surfaces to humans and groundwater. Considering the risk factors involved, PPE should not be reused without proper disinfection. PPE should be worn and removed according to the recommendations of the CDC and WHO, as this should reduce the risk of cross-contamination. PPE should be changed and disinfected at regular intervals (after about 8 h of continuous utilization). Due to the shortage of PPE caused by the current COVID-19 pandemic, there is a strong drive around the world to reuse PPE. However, if the reused PPE is not properly disinfected, enveloped viruses such as SARS-CoV-2 can spread across its surface. All medical wastes, including defective PPE components, should be discarded in designated appropriate containers and disposed of in landfills. It is also known that viruses can diffuse through porous soil, eventually contaminating groundwater. Proper treatment of enveloped viruses is absolutely necessary to reduce virus transmission and contamination of groundwater sources, even in arid areas. The agencies responsible for the proper disposal of used PPE must ensure that this waste material does not pollute the environment. Enveloped virus (SARS-CoV-2) transport model presented in this paper can easily be applied to other enveloped viruses if relevant chemical and physical transport properties are known. We have presented only the main results of our model and simulations here. More analytical research is necessary to determine all of the causes of infections from enveloped viruses and their transmission pathways, as this should enable the creation of adequate guidelines and safety protocols that will prevent their spread and effectively control infections.


Argonaute (AGO) family. Host RNA dependent-RNA polymerases, particularly RDR6, strengthen antiviral silencing by generating more dsRNA templates from RISC-cleavage products that, in turn, are converted into secondary vsRNAs by DCLs. Previous work showed that Pelargonium line pattern virus (PLPV) is a very efficient inducer and target of RNA silencing as PLPV-infected Nicotiana benthamiana plants accumulate extraordinarily high amounts of vsRNAs that, strikingly, are independent of RDR6 activity. Several scenarios may explain these observations including a major contribution of dicing versus slicing for defence against PLPV, as the dicing step would not be affected by the RNA silencing suppressor encoded by the virus, a protein that acts via vsRNA sequestration. Taking advantage of the availability of lines of N. benthamiana with DCL or AGO2 functions impaired, here we have tried to get further insights into the components of the silencing machinery that are involved in anti-PLPV-silencing. Results have shown that DCL4 and, to lesser extent, DCL2 contribute to restrict viral infection. Interestingly, AGO2 apparently makes even a higher contribution in the defence against PLPV, extending the number of viruses that are affected by this particular slicer. The data support that both dicing and slicing activities participate in the host race against PLPV.In plants, RNA silencing functions as a potent antiviral mechanism. Virus-derived double-stranded RNAs (dsRNAs) trigger this mechanism, being cleaved by Dicer-like (DCL) enzymes into virus small RNAs (vsRNAs). These vsRNAs guide sequence-specific RNA degradation upon their incorporation into an RNA-induced silencing complex (RISC) that contains a slicer of the  Viruses must evade the defence mechanisms of the host in order to establish a productive infection. In plants, one of the main defence responses against both DNA and RNA viruses is that based on RNA silencing . This reArabidopsis thaliana have shed light on the particular silencing components that participate in antiviral defence. Among the four DCLs encoded by this plant species (DCL1-4), three of them, DCL4, DCL2 and DCL3, seem to function in a hierarchical fashion to yield viral sRNAs (vsRNAs) of 21, 22 and 24 nt, respectively [A. thaliana encodes ten distinct AGOs and six different RDRs, with AGO1, AGO2 and RDR6 being particularly important in the fight against viruses [Studies mainly performed with the model organism ectively . Concern viruses . The ide viruses .In order to survive, viruses have developed strategies to counteract antiviral silencing, prominent among which is the production of proteins, called viral suppressors of RNA silencing (VSRs), that interfere at different stages of the silencing pathway . For insFrom a theoretical point of view, dicing would be sufficient to clear or diminish virus accumulation in infected cells. Upon onset of an active infection, vsRNAs are both generated and used to target viral RNAs leading, as mentioned above, to their degradation. However, if the infection can be adequately controlled, the necessity to sort viral vsRNAs into RISC might conceivably become less critical. Indeed, dicing, rather than slicing, has been reported to play a main role in the latency of some animal viruses in persistently infected cells . In the Pelargonium line pattern virus (PLPV) is the type member of the new genus Pelarspovirus in the family Tombusviridae [Nicotiana benthamiana plants infected by PLPV accumulate the highest percentage of vsRNAs reported so far for a plant virus , which illustrates the strong defence that the host deploys against the virus. Such strong antiviral silencing was proposed to be a key factor in determining the low viral titers that characterize PLPV infections that, moreover, use to be asymptomatic [N. benthamiana was independent from RDR6 activity, which could be influenced by the absence of AGO-cleaved RNAs that serve as RDR substrates. Here we have pursued to assess the relative contribution of DCL and AGO proteins in anti-PLPV silencing. To this aim, we have used N. benthamiana plants with DCL or AGO functions impaired either by downregulation of expression through an RNA silencing  approach (DCL1-4) or by protein inactivation through a CRISPR/Cas9 strategy (AGO2) [sviridae . PLPV posviridae . We haveptomatic . It was ptomatic , which wy (AGO2) . ResultsN. benthamiana plants were used for PLPV inoculation: (i) wild-type (wt) N. benthamiana, (ii) N. benthamiana transgenic plants in which the different DCL genes were downregulated, either individually or in double or triple combinations, by expressing hairpin constructs  [N. benthamiana transformant line with a CRISPR/Cas9 modified AGO2 allele leading to dysfunctional AGO2 protein [Different lines of  a male)  and (iii protein . All plaN. benthamiana plants were agroinoculated with binary plasmids containing either a wt PLPV cDNA or a mutated PLPV cDNA (hereinafter PLPV-mutp37) carrying three nucleotide replacements in the p37 gene leading to an aminoacid (aa) substitution (W28A). As described previously [Cauliflower mosaic virus 35S promoter and the terminator sequence of the Solanum tuberosum proteinase inhibitor II gene. Binary construct for expression of tombusvirus p19 (used for VSR function in some experiments) has been described elsewhere [Agrobacterium tumefaciens strain C58C1 by freeze/thaw shock method. Cultures of A. tumefaciens harbouring the corresponding plasmid were infiltrated at an OD660 of 0.5 on the abaxial side of N. benthamiana leaves (two leaves per plant) using a 20 ml needleless syringe. Usually the whole leave was infiltrated unless stated otherwise. Mock inoculated plants were used as controls. All inoculated plants were kept under greenhouse conditions. Local leaves were collected at 7 days post-inoculation (days p.i.) and systemic leaves at either 34 (for wt virus) or 42 (for mutant virus) days p.i. Plant batches for each experiment included 4–6 plants and experiments were repeated at least three times.eviously , such aaeviously , the PLPlsewhere . The plaN. benthamiana leaves were obtained by phenol extraction and lithium precipitation [32P-radioactive RNA probe for detection of PLPV RNAs. Such a probe was generated by in vitro transcription of a pBluescript KS(+)-based construct containing the PLPV p37 gene (nt 2621 to 3637 of PLPV genome). Hybridization signals were visualized by autoradiography or with a PhosphorImager (Fujifilm FLA-5100).Total RNA preparations from pitation . For NorA. thaliana, N. benthamiana encodes four DCL-type RNases [N. benthamiana plants showed that those of 21 and 22 nt are the most abundant ones in the vsRNA population with percentages of 41.65 and 32.90 %, respectively, while the fraction of vsRNAs of 24 nt is much lower (0.82 %) as well as those of other size classes [N. benthamiana transgenic lines in which the different DCL genes were downregulated, either individually or in combinations, through an RNAi approach [in trans with another viral suppressor  (N. benthamiana (N. benthamiana plants (In contrast with the results obtained in wt nes 4–5) . The auga plants .N. benthamiana plants, all inoculated DCL RNAi lines remain asymptomatic during their lifespan. Analysis of local and systemic leaves collected at 7 days p.i. and 34 days p.i., respectively, did not reveal significant differences in viral titers between DCL RNAi plants or when compared with wt N. benthamiana plants  at the right place at the right time, by the accessibility of viral substrates [N. benthamiana struggle against PLPV may be critically assessed as plant lines with other inactivated slicers become available. Interestingly, CRISPR/Cas9-mediated inactivation of the two AGO1 homologues identified in N. benthamiana has been recently reported [The finding of the participation of AGO2 in anti-PLPV silencing extends over the number of viruses that are affected by this particular slicer. For some time, studies performed in  defence . However defence . Informathamiana . In the thamiana . In addi viruses , suggestbstrates  and, alsbstrates . Interesbstrates ). Nevertbstrates  and, on bstrates . In any reported , openingN. benthamiana AGO2-Cr plants infected by the wt virus in contrast with that observed with the distinct DCL RNAi lines. Though these differences may be due to several reasons, a likely possibility is that, whereas inactivation of AGO2 through CRISPR/Cas9 is complete, downregulation of DCL factors through RNAi is only partial and, thus, the remaining DCL amounts may still contribute to limit infection. In addition, RNAi may be weakened by the PLPV VSR, raising DCL levels. The paradox of studying components of RNA silencing by using RNAi techniques has been already pointed out by some authors [In agreement with that observed in other plant–virus interactions (e.g. ), the ef authors , as this authors .The lack of systemic infection by the PLPV-mutp37 in double/triple DCL RNAi or AGO2-Cr lines is puzzling as this virus mutant accumulates at considerably high levels in local leaves of the modified plants. The ultimate reasons of this observation are unclear but they could be related with the need to reach a defined threshold level for effective virus exit from the local leaves to the vascular system and/or a defined concentration of ‘circulating’ virus in the vascular system to efficiently enter systemic leaves . In thesN. benthamiana lines infected by wt PLPV, despite the considerable increase in wt virus titers observed in systemic leaves of AGO2-Cr plants (Finally, there are some examples reported in which the impairment of silencing components led to an increment in wt virus accumulation that, even when being small and/or transient, had drastic consequences on viral symptoms . Conversr plants . This ob


Microencapsulation is a rapidly evolving technology that allows preservation of various high-value, but unstable, compounds, such as polyphenols and volatiles. These components of chokeberry juice are reported to have various health-promoting properties. In the present study, hydrogel beads with alginate or alginate and pectin as wall materials and chokeberry juice as active agent were prepared using Encapsulator B-390. The effects of different compositions of wall material as well as the duration of complexation (30 or 90 min) with hardening solution on microencapsulation of chokeberry polyphenols and volatiles were investigated. Spectrophotometric and HPLC analyses showed that beads with pectin addition contained higher concentrations of polyphenols and anthocyanins compared to those prepared with alginate. Antioxidant activities evaluated with FRAP, CUPRAC, DPPH, and ABTS assays followed the same trend. Encapsulation of volatiles which were determined using GC-MS analysis also depended on the composition of hydrogel beads and in some cases on the time of complexation. Results of this study showed that the selection of the wall material is a relevant factor determining the preservation of polyphenols and volatiles. The incorporation of bioactive compounds in hydrogel beads opens up a wide range of possibilities for the development of functional and innovative foods. Presence of cations determines the gelling ability of alginate, similar to pectin. Alginate chains in the presence of divalent cations interact with the ions and cross-linking with the nearby chains occurs. Formation of hydrogel structures enables the controlled release of bioactives in the food and pharmaceutical industries [Microencapsulation is a process that has developed rapidly and is widely used in the food, cosmetics, and medical fields . It enhadustries . In the dustries .We applied the microencapsulation technique using vibration technology to produce alginate (ALG) and alginate/pectin (ALG/PEC) beads from chokeberry juice. Effects of different wall materials and duration of complexation (30 or 90 min) with a hardening solution on total polyphenols and anthocyanins , antioxidant activity  and volatile profiles of hydrogel beads prepared from chokeberry juice were investigated.Chokeberry juice used for the preparation of hydrogel beads was evaluated. Total polyphenols, monomeric anthocyanins, antioxidant activity  and concentrations of individual polyphenols were determined using the HPLC method and are presented in For the purpose of this study, four different samples were prepared. ALG and ALG/PEC abbreviations denote whether only alginate or alginate and pectin combined were used as the wall material(s). The number next to the abbreviation denotes the time of complexation with hardening solution (30 or 90 min).Results of total polyphenols and anthocyanins encapsulated in hydrogel beads are presented in It was observed that the addition of pectin to the wall material of hydrogel beads was a useful approach for the encapsulation of polyphenols since ALG/PEC samples had a higher concentration of total polyphenols than ALG samples. In the study of Bušić et al.  it was r2+. However, if alginate was used as the only constituent of the bead wall material, its higher concentration caused better preservation and encapsulation of polyphenols [Possible interactions between components that ensured the chemical entrapment of bioactives in the bead’s matrix is another explanation. Additionally, reinforcement of pectin and alginate beads could be achieved by using a combination of other polysaccharides or proteins with alginate and pectin because ionically cross-linked pectin and alginate hydrogels are not homogeneous as a result of the cluster formation of ionically associated domains . Aguirreyphenols ,13.In our study, extension of complexation from 30 to 90 min did not affect the encapsulation of total polyphenols. The effect of the complexation time of polysaccharides with calcium ions has been investigated in other studies as well. Zam et al.  claimed In the study of Guo et al. , the majClitoria ternatea petal flower in the alginate beads. In the study by Li et al. [Encapsulation efficiency was also determined by dividing the concentration of polyphenols in hydrogel beads with the initial concentration of polyphenols and multiplying by 100. Encapsulation efficiency of total polyphenols in ALG hydrogel beads was around 39%, while in the ALG/PEC beads was higher, around 50%. Values were higher for anthocyanins encapsulation for which we observed that ALG-30 hydrogel beads had 72% and ALG-90 69% of encapsulation efficiency, while for ALG/PEC encapsulation efficiency was 76%. Lower values of encapsulation efficiency for total polyphenols could be ascribed to very low encapsulation of neochlorogenic acid. In the literature, there are different results for this parameter, depending on wall materials, cross-linking solution, as well as the polyphenol profile of the encapsulation material and all studies are pointing out that optimization of encapsulation parameters was of main importance. The broad range of the encapsulation efficiencies of polyphenols of chokeberry extract in alginate beads was determined by Tzatsi and Goula  and theii et al. , the autFrom the results of antioxidant activity , it can Due to the importance of the antioxidant defense, various methods for the determination of antioxidant activity were developed. Different methods are based on different principles and thus antioxidant activity should be tested based on several methods and one method cannot be compared to another. The information related to the hydrogen atom transfer or donating capacity of the electrons provides important information about the antioxidant potential of the samples with minimal environmental interference . Another2 values represent the highest correlation of polyphenols to antioxidant activities. Regardless of the method used to determine antioxidant activity, total anthocyanins were the most appropriate predictor of antioxidant activity , followed by correlation coefficient for total phenolic acids, which was satisfactory (R2 > 0.6554). It was observed that total flavonols showed a lack of correlation with antioxidant activity. Total anthocyanins as a good predictor of antioxidant activity were also reported in other studies [ studies ,37.Volatile compounds in chokeberry juice and hydrogel beads were divided into several groups; alcohols, acids, carbonyl compounds, esters, and terpenes. Their properties and flavor notes are presented in Amounts of volatiles in hydrogel beads  dependedFive esters were determined in chokeberry juice, while in hydrogel beads two of them (ethyl hexanoate and hexyl acetate) were not detected. The higher encapsulation efficiency was evaluated for methyl dihydrojasmonate for ALG beads, while for phenethyl acetate there was no significant difference between hydrogels. Nine terpenes were detected in samples. Linalool, phellandral, β-damascenone and α-ionone were determined in equal amounts in all hydrogels, thus hydrogel composition and complexation time had no effect on these volatiles during the encapsulation process. The amount of D-limonene was the highest in ALG-30, while there was no difference between all the other hydrogels. Linalool oxide had the highest amount in ALG/PEC hydrogel beads when complexed for 90 min, while for ALG beads there was no difference between complexation times. γ-ionone and β-ionone were evaluated in higher amounts in ALG, while α-cedrol had the highest levels in the ALG/PEC hydrogel beads.It was reported that the volatile profiles of fruits are affected by the addition of hydrocolloids by two possible mechanisms. One is due to the increased viscosity of the system which consequently lowers the diffusion rate of volatiles to the environment and the other is the binding of volatile compounds to the hydrocolloids . ChemicaEach volatile compound has its characteristic flavor note, therefore based on this property, the contribution of each flavor note to the overall flavor of samples was determined . Green, In the present study, microencapsulation of polyphenols and volatiles from chokeberry juice was applied, and the effects of the wall material composition and complexation time with the hardening solution on the polyphenol and volatile profile were investigated. Results revealed that alginate hydrogel beads combined with the pectin addition contained higher concentrations of polyphenols compared to those prepared with only alginate, except for some of the individual polyphenols determined by the HPLC method, which showed a slightly different trend. Encapsulation of volatiles also depended on the composition of hydrogel beads, and, in some cases, on the time of complexation. This investigation demonstrated that hydrogel beads can serve as a potential matrix for the microencapsulation of bioactive compounds and thus ensure preservation of their positive properties, such as antioxidant activity. However, the proper formulation of wall material is important because its interactions with polyphenols or volatiles have an impact on the retention of these valuable compounds. After preparation, hydrogel beads may be used as an ingredient of functional foods since components used for their production are nutritionally rich and safe for consumption.Chokeberry fruits were bought at the local market. Alginic acid sodium salt (very low viscosity) was obtained from Alfa Aesar , while low-methoxyl pectin (Genu pectin type LM-5-CS) was from CP Kelco . Calcium chloride, ethanol, sodium acetate, potassium chloride and ammonium acetate were from Gram-mol . Sodium carbonate was purchased from T.T.T. . Potassium persulfate and Folin-Ciocalteu reagent were procured from Kemika . Cupric chloride, neocuproine and 2,4,6-tri(2-pyridyl)-s-triazine (TPTZ) were bought from Acros Organic . Methanol (HPLC grade) was from J.T. Baker  and orthophosphoric acid  was from Fisher Scientific . Hydrochloric acid (37%) and methanol were bought from Carlo Erba Reagents  and acetic acid (>99.5%) was from Alkaloid . Trolox, 2,2-diphenyl-1-picrylhydrazil, 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt, 4-dimethyl-amino-cinnamaldehyd and most of the standards used for HPLC analysis  were from Sigma-Aldrich , while the standards of cyanidin-3-galactoside, neochlorogenic acid and hyperoside were products of Extrasynthese .2 in chokeberry juice  was used. Hydrogel beads were formed using Encapsulator B-390  under fixed conditions: 1000 µm vibrating nozzle, pressure 200 mbar, frequency 200 Hz and electrode 1000 V. After the formation of beads, they were left in a hardening solution for 30 or 90 min to investigate whether the time of complexation of hydrogel beads with CaCl2 had an impact on the preservation of bioactives from chokeberry juice. Produced hydrogel beads were then filtered and used for further analyses.Chokeberry juice was prepared by pressing the fruits and filtering the obtained mass. The juice  was then used for the preparation of encapsulation mixture in which 2% of alginate was dissolved by stirring using a stick blender . Another set of samples was prepared by adding 1% of pectin to the alginate-chokeberry juice mixture . After all components in the encapsulation mixture have been dissolved, they were complexed for 30 min before performing the microencapsulation process. As a hardening solution, 7% CaClThe method of Kopjar et al.  for extrTotal polyphenols were determined using the method by Singleton and Rossi  with sli515 represents absorbance read at 515, while A700 is the absorbance read at 700 nm. The concentration of monomeric anthocyanins was calculated as follows:W represents the molecular weight of cyanidin-3-glucoside (449.2), DF is the dilution factor, ε is the molar absorptivity  and l is the cuvette length (1 cm). Since the concentration of monomeric anthocyanins was calculated using data for cyanidin-3-glucoside, their contents were expressed as mg of cyanidin-3-glucoside per gram of beads (mg cyanidin-3-glucoside/g).Determination of monomeric anthocyanins content was carried out using the pH differential method . BrieflyFirstly, to determine ferric reducing ability extract (0.2 mL) was added to FRAP reagent (3 mL). The mixture was left in the dark for 30 min and absorbance was read at 593 nm. This method was from Benzie and Strain . SecondlFor the determination of antioxidant activity, each sample was analyzed in triplicate and the calibration curve was created for Trolox. Results were expressed as µmol of Trolox equivalents per 100 g of hydrogel beads (µmol TE/100 g).2 = 0.9969 for cyanidin-3-galactoside, R2 = 0.999 for chlorogenic acid, R2 = 0.9986 for neochlorogenic acid, R2 = 0.9998 for quercetin, R2 = 0.9993 for hyperoside and R2 = 0.9993 for rutin. Cyanidin-3-arabinoside and cyanidin-3-xyloside were detected tentatively with the help of literature data [Agilent HPLC system 1260 Infinity II  was used to evaluate the individual polyphenols in extracts and chokeberry juice. The system consisted of a quaternary pump, a vial sampler, Poroshell 120 EC C-18 column  and a diode array detector (DAD). Before injecting into the system, extracts were filtered through PTFE filters with 0.20 µm pores. The method used was previously described by Buljeta et al. . The injure data ,54. Theim/z between 45 and 450. Ion source temperature was 230 °C and the temperature of the quadrupole was maintained at 150 °C. Confirmation of the compounds was performed by matching their mass spectra with the NIST  and through retention time (RT) and retention index (RI) (Extraction of volatile compounds from hydrogel beads was conducted using solid-phase microextraction (SPME) and fiber coated with divinylbenzene/carboxen/polydimethylsiloxane (DVB/CAR/PDMS) sorbent . Firstly, 0.3 g of sample, 4.7 g of water and 1 g of NaCl and were weighed in glass vials, which were then conditioned in a temperature-controlled heating module under the following conditions: 40 °C, 45 min and 350 rpm. After extraction, fiber was removed from samples and volatiles were thermally desorbed in the injector port of the GC 7890B gas chromatograph  which was used to perform the analysis. It was equipped with a 5977A mass spectrometer . Volatiles were desorbed into the GC injector port at 250 °C for 7 min in a splitless mode. The gas chromatograph was fitted with an HP5 capillary column (60 m × 0.25 mm × 0.25 µm). Helium was used as the carrier gas, and its flow rate was set at 1 mL/min and 40 °C. The initial temperature of the oven was 40 °C and it was held for 2 min and then the temperature was set to rise at 6 °C/min up to 230 °C. For the identification of volatiles, mass selective detector was used and operated in the range of dex (RI) . For thep < 0.05 were used for data analysis.All results were expressed as the mean values ± standard deviation. Statistical analysis was performed using software STATISTICA 13.1 . Analysis of the variance (ANOVA) and Fisher’s least significant difference (LSD) with the significance defined at


Aedes (Ae.) genus is a vector of several viruses such as Dengue, Chikungunya, and Zika viruses that can cause serious pathologies in humans. Since 2004, Ae. albopictus has been encountered in the South of France, and autochthonous cases of Dengue, Chikungunya, and Zika diseases have recently been reported, further highlighting the need for a comprehensive survey of the mosquitoes and their associated viruses in this area. Using high throughput sequencing (HTS) techniques, we report an analysis of the DNA and RNA viral communities of three mosquito species Ae. albopictus, Culex (Cx.) pipiens, and Culiseta (Cs.) longiareolata vectors of human infectious diseases in a small sub-urban city in the South of France. Results revealed the presence of a significant diversity of viruses known to infect bacteria, plants, insects, and mammals. Several novel viruses were detected, including novel members of the Rhabdoviridae, Totiviridae, Iflaviviridae, Circoviridae, and Sobemoviridae families. No sequence related to major zoonotic viruses transmitted by mosquitoes was detected. The use of HTS on arthropod vector populations is a promising strategy for monitoring the emergence and circulation of zoonoses and epizooties. This study is a contribution to the knowledge of the mosquito microbiome.Some mosquito species have significant public health importance given their ability to transmit major diseases to humans and animals, making them the deadliest animals in the world. Among these, the  Culicidae) are an insect family comprising more than 3600 species [Mosquitoes  and, accidentally, to dead-end hosts such as humans and horses [The south of France is now known as a potential risk area for mosquito-borne viral infections. After its introduction in the French Riviera in 2004, the Asian tiger mosquito bopictus ,9 has prbopictus ,11,12,13d horses . Very red horses ,16. Thesd horses ,16.In recent years, HTS and the “vector enabled metagenomics approach” have emerged as promising tools for virus surveillance using arthropods as a template. Viral metagenomics has proved to be an interesting tool for zoonotic disease control ,18. SeveIn this context, the present study explores the DNA and RNA viromes of mosquito populations in a sub-urban area of the large Marseille city, South of France. Mosquitoes were first submitted to MALDI-TOF MS for precise identification and classification prior to performing viral metagenomics analysis.This study was conducted from April to November 2016 in the village of Cuges-les-Pins (5000 habitants), in the south of France, located 30 km east of Marseille. This area was chosen based on complaints of mosquito nuisances provided by the citizens and registered by the Marseille city healthcare department and its large horse population (>5000 horses). Mosquitos were sampled using BG-sentinel traps , placed in six sampling sites . Once a Mosquitos were separated from the other arthropod families collected in the trap. They were sorted per genus, sex and were counted prior to perform morphological identification under Binocular loupe , using dichotomous keys . All samEach week, the legs from six specimens morphologically identified per species and per collection site were submitted to MALDI-TOF MS analysis for identification, as previously reported . ProteinAe. albopictus); pool B , and pool C . For each pool, mosquitoes collected at distinct time points per site were selected  by ultracentrifugation at 130,000 g for one hour at 4 °C, as previously described [The viral fractions of the pools A, B, and C mosquito samples were concentrated for TEM. The viral fraction was supplemented with PBS and loaded sucrose fraction on Amicon Ultra 4 mL Centrifugal filters . The viral fraction was then centrifuged 2 min at 3000× escribed . Grids wRaw reads were imported into the CLC Genomics Workbench 6.0.1 program  and trimmed according to their quality score, the presence of ambiguities, and their length (reads shorter than 50 nt were discarded). Trimmed reads were compared to the NCBI non-redundant (nr) protein database using the BlastX algorithm using DIAMOND software . The resThe open reading frames (ORFs) were aligned with other amino-acid sequences retrieved from the GenBank database using MUSCLE aligner (v3.8.31)  implemenAe. albopictus) using the viral sucrose fraction of the pool A containing Ae. albopictus. The cultures were maintained at 28 °C and 37 °C for C6/36 and Vero cells, respectively. One hundred and twenty microliters of HBSS were added to 100 µL of the viral sucrose fraction, then two dilutions 10−1 and 10−2 were realized, and 1 mL was inoculated into 12.5 cm2 flasks with confluent monolayer cultures of both Vero and C6/36 cells and adsorbed for 2 h. They were maintained for 9–10 days and were examined daily for evidence of viral cytopathic effect (CPE). Cultures showing CPE were harvested for transmission electron microscopy.Virus isolation was carried out using Vero cells (African green monkey kidney cells) and C6/36 , followed by Cx. pipiens with 25% (1054/4265), and Cs. longiareolata with 18% (790/4265). The nature of the site influenced the composition of the collection. Ae. albopictus was the most abundant species collected in urban areas and, to a lesser extent, in peri-urban areas, whereas Cx. pipiens dominated in rural environments  mosquito legs MS spectra queried against the database conducted to relevant identification (LSVs > 1.8). Of these 542 specimens, 82.65% (n = 448/542) were identified with elevate log score values (LSVs) ranging between 2.001 to 2.593. MS spectra from solely 42 specimens did not succeed in reaching the LSV threshold value of 1.8 for confident identifications. This identification failing was mainly attributed to the lower quality of their MS spectra. The reduction in MS spectra intensity and diversity was attributed for the majority of them to the loss of several of their legs. These damaged specimens occurred during mosquito collection and transport.A total of 584 specimens were blindly submitted to MALDI-TOF MS to confirm the morphological identification. More than 92% , Cx. pipiens  and Cs. longiareolata  by MALDI -TOF MS  were also identified in the DNA viromes of pools A, B, and C, at 14.07%, 2.81%, and 4.15%, respectively. The unclassified “Ixodes scapularis associated virus 2” and viruses within the Iflaviridae family represented the majority of viral reads of RNA viromes for pool A and pool B, at 71.78% and 91.74%, respectively. In contrast, in the RNA virome of pool C, we observed that 40.21% of total viral reads were assigned to the Rhabdoviridae family and 26.11% to the Totiviridae family, while insect viruses represented only 1.1%. Plant viruses  comprised 7.41%, 1.38%, and 3.00% of the total viral reads from pool A, pool B, and pool C RNA viromes, respectively, as well as 0.17% and 0.41% of total viral reads for pool A and pool B DNA viromes, respectively  represented 0.18% and 0.06% of DNA viromes of the pool B and C, respectively. Few DNA viruses were also identified in the RNA viromes , possibly due to residual contamination of the RNA fraction by viral DNA  were identified in the Cs. longiareolata RNA virome. These reads assembled into six contigs ranging from 431 to 4126 bp in size and presenting a nucleotide identity of 33% to 96% with Arboretum virus within the proposed Almendravirus genus [Rhabdovirus close to Arboretum virus and Almendras virus isolated from Psorophora albigenu and Ochlerotattus fulvus mosquitoes in Peru [Arboretum virus CLP-1 (mAV-CLP-1). Analysis of the mAV-CLP-1 sequences indicated that it shared a similar genome organization with Arboretum virus and Puerto Almendras virus [Rhabdovirus structural protein genes (3′-N-P-M-G-L-5′) but lacked the M gene.Sequences (reads) assembled from the mosquito viromes had similarities to four families of animal viruses . Among t in Peru   were replaced in a phylogenetic tree of the Circoviridae family among the proposed Cyclovirus (n = 1), Krikovirus (n = 3), and Smacovirus (n = 1) genera. These were named mosquito Cyclovirus CLP-1, mosquito Krikovirus CLP , and mosquito Smacovirus CLP-1. Except for mosquito Krikovirus CLP-3, which was found in the Cx. pipiens virome, all other identified viruses were detected from the Ae. albopictus virome . The ORFs matched with a wide range of poxviruses, belonging to both Chordopoxvirinae and Entomopoxvirinae sub-families. The Chordopoxvirinae sub-family was represented mostly by the Avipoxvirus genus, including the Turkeypox virus, Fowlpox virus, Canarypox virus, and Pigeonpox virus, with identities ranging from 42% to 75%. While the Entomopoxvirinae sub-family was mainly represented by the Alpha entomopoxvirus and Beta entomopoxvirus, including the three species Mythimna separata entomopoxvirus, Anomalacuprea entomopoxvirus, and Melanoplussanguinipes entomopoxvirus, with an amino-acid identity ranging from 27% to 69%.Finally, within the DNA viromes obtained from Totiviridae and Genomoviridae . All ORFs matched with the viral RNA polymerase RdRp protein segment of Anopheles totivirus, with an amino-acid identity of 38% to 46%. Phylogenetic tree based on RdRp protein (>300 aa) revealed that the majority were grouped with an uncharacterized clade close to an Anopheles totivirus isolated from Anopheles mosquitoes in Western Africa [Sequences related to protozoan and fungus viral families were detected in the mosquito viromes, namely oviridae . Virusesn Africa   assembled from the mosquito viromes had similarities to five families of arthropod viruses, namely  virus 2 .Ixodes scapularis associated virus 2” were particularly abundant within the RNA virome of pool A , with a total of 95,536 reads. They were assembled in three contigs ranging from 679 to 2529 bp in size, from which three ORFs were predicted (104 aa to 266 aa). Two of the ORFs matched with the hypothetical protein 2 of Wenzhou sobemo-like virus 4, with 100% identity, and one matched with the hypothetical protein 1 of Wenzhou sobemo-like virus 4, with 95% identity. Phylogenetic tree based on the hypothetical protein revealed that our viruses named mosquito sobemo-like virus CLP-1 and CLP-2 were close to other sobemo-like viruses (unclassified RNA viruses) isolated from mosquitoes in China [Sequences matching with “Cx. pipiens and Cs. longiareolata pools assigned to the Iflaviridae family were detected in the RNA viromes. Sequence assemblies allowed the reconstruction of five contigs ranging from 481 to 9767 bp, matching with a nucleotide identity of 38% to 61% to the Hubei picorna-like virus 35 and 41, and the Nasonia vitripennis virus. Gene prediction identified six ORFs of 101 to 3126 aa in size that matched mainly with hypothetical protein from the Hubei picorna-like virus 35 with 31% to 61% identity.A total of 71,471 reads from the Parvoviridae within the DNA virome of the pools A and C . They were assembled in 17 contigs ranging from 305 to 3827 bp in size, from which 19 ORFs were predicted (97 aa to 618 aa). The majority of the ORFs had similarities to insect densoviruses with an identity of 26% to 100% . They were assembled in 40 contigs ranging from 357 to 9000 bp in size, from which 47 ORFs were predicted (55 aa to 544 aa). The ORFs matched with different species with a range of 31% to 69% identity . The ORFs matched with Musca domestica salivary gland hypertrophy virus and Glossina pallidipes salivary gland hypertrophy virus.Finally, 2997 reads were assigned to Luteoviridae, Sobemovirus, Bromoviridae, Tomato matilda virus and Geminiviridae. However, the majority of these groups were represented just by a few reads detected in the RNA viromes such as Luteoviridae, Sobemovirus, and Tomato matilda virus. A total of 4037 reads were assigned to Bromoviridae family within the RNA virome of the Cs. longiareolata sample. They were assembled in one contig of 1481 bp leading to the prediction of one ORF sized 441 aa. This ORF matched with 86% identity with Blackberry chlorotic ringspot virus.Sequences with similarities to plant virus families were notably identified in the mosquito viromes . Namely,Geminiviridae family, two ORFs, coding for 185 and 186 aa putative proteins, were predicted. Both sequences matched with putative replication initiation protein (Lytechinus variegatus variable sea urchin associated circular virus) with 36% and 37% identity.For the Mimiviridae family were detected in the DNA virome of Ae. albopictus. A contig of 665 bp matched with Moumouvirus monve with 38% nucleotide identity. ORF prediction led to a sequence of 221 aa with 38% similarity with an integrase zinc-binding and helicase domain-containing protein of Moumouvirus monve.Interestingly, sequences related to the Ae. albopictus pool into flask cultures of Vero cells, a cytopathic effect (CPE) was observed after 9–10 days. The infected Vero cells were dead and formed holes in the cellular mat (After inoculation of the sucrose fraction (dilution 1/10) containing purified viral particles from the ular mat . HoweverAe. albopictus, Cx. pipiens and Cs. longiareolata collected from the French Riviera region. Since the introduction of the invasive Asian mosquito Ae. albopictus in 2004 on this area [In this study, we performed viral metagenomics on pooled his area ,9, severhis area  in diffehis area ,47. In 2his area . Cases ahis area ,33 collehis area . Using tAe. albopictus species was observed especially close to schools and individual houses situated in the center of the village. This could be explained by the fact that the area is endemic to the tiger mosquito, which has invaded it since 2011 and by the anthropophilic character of Ae. albopictus. Indeed, Ae. albopictus originated in Asian forests and commonly found in rural and peri-urban settings is currently occurring in highly dense urban areas and rapidly extended from one city to another [The monitoring of mosquito populations during these five months showed variations in density and species from one site to another. High prevalence of  another .Cx. pipiens is the second most abundant species collected. Cx. pipiens were found in all prospected sites, but higher abundance was found in traps close to ranch housing horses. Cx. pipiens is a species with two forms with an intermediate (hybrid) form feeding on both mammals (humans and equids) and birds making it a bridge for zoonotic agent transmission. Finally, Cs. Longiareolata, a bird-seeking mosquito was found abundant in the most rural area. Both Ae. albopictus and Cx. pipiens mosquitoes are incriminated as competent vectors of several human arboviruses.In this study, we reported an extensive description of the DNA and RNA viral communities of three mosquito species collected in Cuges-les-Pins. Analysis of the taxonomic assignment of reads revealed a high proportion of sequences related to viruses which can reflect the database increment with RNA and DNA viral sequence these last years. Although the three mosquito species were collected at the same location sites during the same week using the same traps, the six RNA and DNA metagenomes have highly divergent profiles in terms of relative abundance and composition of viral communities ,34,51.Ae. albopictus, Cx. pipiens, and Cs. longiareolata were largely dominated by sequences related to bacteriophages from the Myo viridae, Podo viridae, Sipho viridae, and Microviridae families and unclassified phages. These sequences may derive from phages infecting the bacterial microflora of the mosquitoes or that of the plant/animal hosts on which they fed upon. In that sense, sequences related to mammal viruses belonging to the Rhabdoviridae, Circoviridae, Anelloviridae, and Poxviridae families were also detected in the DNA and RNA viromes and may reflect the blood meal of the mosquitoes. These families were already identified in mosquito viromes [The DNA viromes of  viromes .Rhabdoviridae-related sequences consisted mainly of the RNA virome of Cs. longiareolata. Rhabdoviridae is a family of negative-sense RNA viruses, which includes 19 genera according to the International Committee on Taxonomy of Viruses (ICTV). Among them, the rabies virus, which causes fatal disease in humans and continues to kill an estimated 40,000–70,000 people yearly [Arboretum virus CPL-1 strain described here is closely related to Rhabdoviridae viruses isolated from different mosquito species in the Americas [Circoviridae is a single-stranded circular DNA (ssDNA) genomes viral family composed of two genera Circovirus and Cyclovirus. Porcine circovirus 1 and 2 belonging to the Circovirus genus have been extensively documented to replicate in a mammal, cause a variety of clinical symptoms with significant economic impact [Circoviridae were identified belonging to the proposed Cyclovirus, Krikovirus and Smacovirus genera. So far, the Krikovirus genus was only composed of viruses identified in mosquitoes and bat feces [Krikoviruses identified in the Ae. albopictus and Cx. pipiens mosquito pools of this study form a distinct clade in the proposed Krikovirus genus.The e yearly . Rabies e yearly . Indeed,e yearly ,54,55. TAmericas ,44. Thesc impact . In thisat feces . Based oAnelloviridae is a family of negative sense ssDNA viruses. In the present study, the sequences had low similarities with Torque tenocanis virus (genus Thetatorquevirus) isolated from the sera of domestic pigs and dogs [Nayun tick torquevirus isolated from Rhipicephalus ticks in China [and dogs  and Nayuin China , suggestin China ,59.Poxviridae is a family of large dsDNA viruses divided into two sub-families Chordopoxvirinae and Entomopoxvirinae composed of 11 and 3 genera, respectively. It is a group that can infect a large number of animals causing virulent pathologies like variola in humans [Ae. aegypti from infected to susceptible cattle [Anopheles maculipennis complex [n humans . A few yn humans ,62,63,64e cattle . More re complex . Likewis complex . These s complex .Totiviridae was the second most abundant family composing the RNA virome of Cs. longiareolata. To date, according to ICTV, the Totiviridae family comprises five genera formally recognized: Giardiavirus, Leishmaniavirus, Totivirus, Trichomonasvirus, and Victorivirus. These genera include viruses infecting fungi and protozoa. Here, the sequences clustered with Anopheles totivirus, a virus recently isolated in Anopheles mosquitoes in Africa [Totiviruses have been identified in different mosquito species [n Africa . In rece species ,70,71.Genomoviridae is a new family of ssDNA viruses; it contains one genus Gemycircularvirus with one recognized virus species, Sclerotinia gemycircularvirus 1, which was isolated from Sclerotinia sclerotiorum a plant-pathogenic fungus [Genomoviridae family were detected in four mosquito viromes collected in China [Ae. albopictus was dominated by sequences matching almost a unique virus identified as Ixodes scapularis virus 2 after annotation of the reads with DIAMOND/MEGAN. However, further blast P analysis of the ORFs against the whole NCBI revealed a similarity of 95%–100% with the recently described Wenzhou sobemo-like virus 4 [Ae. albopictus individuals collected in China [Cx. pipiens was also largely dominated by sequences assigned to the unique Iflaviridae family. Blast P analysis of the ORFs against the NCBI database showed a best-blast hit with Hubei picorna-like virus 35, a virus identified in an odonata mix from the Hubei region in China [Cx. pipiens represent novel picorna-like virus. Parvoviridae is an ssDNA viruses family composed of two sub-families Parvovirinae and Densovirinae, regrouping eight and six genera, respectively, according to the ICTV. Parvoviruses were already detected in wild-caught mosquitoes worldwide [Ae. albopictus and Cs. longiareolata mosquito species. However, the presence of Parus major densovirus in mosquitoes probably attributed to the blood-sucking of the mosquitoes from viremic birds.c fungus . Sequencin China . Althougin China , the facin China ,73 stronin China . The RNA virus 4 . This viin China . The verin China , but theorldwide ,50,58. IBaculoviridae is a family of dsDNA viruses including four genera according to ICTV Alphabaculovirus, Betabaculovirus, Deltabaculovirus, and Gammabaculovirus. Members of this family infect larvae of the insect orders Lepidoptera, Hymenoptera, and Diptera [Baculoviridae related sequences were already detected in Culex quinquefasciatus and Culex tritaeniorhynchus collected from Kenya and China [Hytrosaviridae is a new family of insect dsDNA viruses [Glossinavirus and Muscavirus [Glossina pallidipes salivary gland hypertrophy virus (SGHV) and Musca domestica SGHV. To our knowledge, it is the first time that Hytrosaviridae-related sequences were detected in mosquito viromes. Interestingly, Mimiviridae related sequences were detected in this study. The sequences had low similarities (38%) with Moumouvirus Monve. Moumouvirus represents the sublineage B of the Mimiviridae family [ Diptera  exclusivnd China . Hytrosa viruses , groupinscavirus . Actualle family . As gianin natura.Isolation is mandatory to take a step further in the characterization of these newly described viruses. Indeed, additional in vivo and in vitro infection experiments would help to fully understand their biology. In this study, isolation of viral particles on C6/36 and Vero cells was unsuccessful, although a CPE was observed in the last one. Such attempts should be conducted again on these and other relevant cell lines to demonstrate the ability of these viruses to replicate in mammalian cells/mosquito cell lines or both, as the first step to decipher the role of these viruses This study contributes to the knowledge of the repertoire of viruses associated with mosquitoes collected in France and highlights the potential pool of unknown viruses yet to be characterized, which could either represent emerging human pathogens. Using viral metagenomics, we detected one novel rhabdovirus, five novel circoviruses, eight novel totiviruses, and two sobemo-like viruses. Further in-depth studies are essential to characterize these newly described mosquito viruses and to distinguish mosquito-specific viruses from those that may present a serious risk of human infection. It would also be very interesting to carry out seasonal monitoring to determine if viral population dynamics of each mosquito species are modified according to the season and consequently by the influence of the environmental factors.


Dysregulated formation of neutrophil extracellular traps (NETs) is observed in acute viral infections. Moreover, NETs contribute to the pathogenesis of acute viral infections, including those caused by the dengue virus (DV) and severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). Furthermore, excessive NET formation (NETosis) is associated with disease severity in patients suffering from SARS-CoV-2-induced multiple organ injuries. Dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN) and other members of C-type lectin family  have been reported to interact with viral glycans to facilitate virus spreading and exacerbates inflammatory reactions. Moreover, spleen tyrosine kinase (Syk)-coupled C-type lectin member 5A (CLEC5A) has been shown as the pattern recognition receptor for members of flaviviruses, and is responsible for DV-induced cytokine storm and Japanese encephalomyelitis virus (JEV)-induced neuronal inflammation. Moreover, DV activates platelets via CLEC2 to release extracellular vesicles (EVs), including microvesicles (MVs) and exosomes (EXOs). The DV-activated EXOs (DV-EXOs) and MVs (DV-MVs) stimulate CLEC5A and Toll-like receptor 2 (TLR2), respectively, to enhance NET formation and inflammatory reactions. Thus, EVs from virus-activated platelets (PLT-EVs) are potent endogenous danger signals, and blockade of C-type lectins is a promising strategy to attenuate virus-induced NETosis and intravascular coagulopathy. Listeria (spleen tyrosine kinase (Syk)-coupled C-type lectin member 5A, CLEC5A) . Ev. Ev58]. It is interesting to note that EV serum level increased in COVID-19 patients, and correlates with clinical symptoms and lethality , 110. ItIt is also interesting to note that severe pulmonary inflammation in COVID-19 patients is associated with thrombotic complications, such as microangiopathy and pulmonary embolism , 115. AsEVs have been implicated in regulation of infectious and autoimmune diseases, but the underlying molecular mechanisms are still unclear. We have demonstrated that platelets secrete EVs after incubation with DV, LPS, and thrombin. Interestingly, all the