The world’s staple food crops, and other food crops that optimize human nutrition, suffer from global virus disease pandemics and epidemics that greatly diminish their yields and/or produce quality. This situation is becoming increasingly serious because of the human population’s growing food requirements and increasing difficulties in managing virus diseases effectively arising from global warming. This review provides historical and recent information about virus disease pandemics and major epidemics that originated within different world regions, spread to other continents, and now have very wide distributions. Because they threaten food security, all are cause for considerable concern for humanity. The pandemic disease examples described are six . The major epidemic disease examples described are seven . Most examples involve long-distance virus dispersal, albeit inadvertent, by international trade in seed or planting material. With every example, the factors responsible for its development, geographical distribution and global importance are explained. Finally, an overall explanation is given of how to manage global virus disease pandemics and epidemics effectively. Virus disease pandemics and major epidemics threaten cultivated plants grown not only to feed humankind and its livestock, but also to produce fiber, ornamental plant or medicinal products. When many plants become systemically virus-infected and this infection causes severe disease symptoms, the magnitude of the resulting losses in overall crop yield or quality of produce can be devastating. Moreover, such losses happen in both annual and perennial cultivated plants ,6,7,8,9.In global agriculture, once the cultivation of annual crops as monocultures became widespread, this practice provided an underlying basic ingredient of instability . This inSeveral factors have contributed to the currently rapidly deteriorating global plant virus disease situation. Firstly, the rapid expansion of international trade in plants and plant produce by multinational companies is introducing damaging virus diseases to parts of the world where they were formerly absent. This is occurring for three main reasons. (i) Trade globalization involving international agreements over free trade or tariff reductions has opened up new pathways for the large-sale transfer of crop produce from one continent or distant country to another. (ii) Lower subsidies for developed country production have enabled developing countries to expand their trade in international crop produce. (iii) More efficient methods of rapid transport by air and sea, combined with the loosening of plant quarantine regulations to meet revised World Trade Organization rules, have facilitated this trade ,19,20,21Reviews and research papers by the late J.M. Thresh  describeThis review focusses mainly on virus disease pandemics or major epidemics that are known to have, or considered likely to have, originated within crop domestication centers in different parts of the world, became distributed from there to other continents, and now have predominantly global distributions. The examples described infect cereals , root and tuber crops (potato and sweet potato), plantation and orchard crops , grain legumes (faba bean) and annual horticultural crops (tomato and cucurbits). Both historical and recent information is provided about seven examples of virus diseases that threaten staple food crops and therefore are of critical importance regarding global food security. Three of these seven staple food crop examples involve diseases caused by virus complexes. The remaining six examples involve virus diseases threatening other food crops that play important roles in achieving balanced human nutrition. Three of the latter examples are of seed-borne virus diseases currently of concern because of their inadvertent dispersal by the international seed trade. The ways each of these 13 examples arose, and then became distributed globally, is described, and information is given about the principal factors favoring their development. Lastly, a brief overall summary is provided concerning how to achieve successful management of global virus disease pandemics and major epidemics that afflict important food crops.In his 1973 review of plant diseases threatening worldwide agriculture, Thurston  categoriWhen he reviewed catastrophic plant diseases in 1970, Klinkowski ,37,45. JTo understand plant virus epidemics and pandemics, it is also necessary to understand what plant virologists mean when they refer to the term ‘plant virus disease epidemiology’. Various definitions have been suggested, including the study of: (i) “the patterns of disease within space and time, and within populations” ; (ii) “tPotyvirus, family Potyviridae), potato virus S  and potato virus Y  were collected from plantings in potato’s Andean crop domestication center [Potyvirus, family Potyviridae) [Tungrovirus, family, Caulimoviridae) and rice tungro spherical virus  [Babuvirus, family, Nanoviridae) [Some pandemics or major virus disease epidemics result from new encounter scenarios when a crop first domesticated in one continent ,53 is inn center ,58,59. Uviridae) ; rice tuviridae) ,62; and viridae) ,64. Zea mays) is now the world’s most important staple food crop, and the second most important in the developing world [Zea mays ssp. parviglumis [Overall, maize  was reported initially in 1977 in the USA, where it was called corn lethal necrosis disease . Its dra2 ,72. In E2 ,73. MoreMachlomovirus, family, Tombusviridae) and one or other of several different cereal viruses belonging to the Potyviridae, such as sugarcane mosaic virus , maize dwarf mosaic virus  and wheat streak mosaic virus  [Potyviridae that infect maize, especially SCMV, commonly occur in warmer climates globally, whether MLND occurs in a region depends on the presence of MCMV [MLND is caused by mixed infection between maize chlorotic mottle virus  . The two of MCMV . MCMV wa of MCMV , and in  of MCMV ,72. HoweSaccharum officinarum), sorghum (Sorghum bicolor), millet (Panicum miliaceum), wheat (Triticum aestivum), barley (Hordeum vulgare), and several pasture and weed grasses. Cereal infecting Potyviridae are transmitted by aphids  or eriophyid mites (WSMV). They infect a diverse array of alternative hosts amongst poaceous crops, weeds and pasture plants [Potyviridae to maize plants, sowing MCMV-contaminated maize seed seems responsible for MLND’s recent emergence in SSA, and other continents outside the Americas [MCMV has very stable virions, and is transmitted by contact, contaminated maize seed and soil, and several beetle and thrips species act as its vectors. Alternative hosts for MCMV include sugar cane  but also of SCMV and other cereal potyviruses infecting maize and other poaceous hosts [What is responsible for the devastating MLND pandemic that emerged after MCMV’s arrival in SSA? Factors exacerbating MLND’s appearance include sowing vulnerable maize cultivars, agricultural intensification including sowing multiple maize crops annually, and the widespread occurrence not only of its maize thrips vector  crops. In addition, maize, triticale  and Asian rice (Oryza sativa) crops are sometimes affected [Yellow dwarf disease (YDD) causes the virus disease epidemic of greatest global significance for wheat . It was affected ,76,77,78affected  chose YDLuteovirus, family, Luteoviridae), and cereal yellow dwarf virus . Their infections occur singly or in mixed infection. Both viruses cause the same types of YDD symptoms in infected wheat plants, and both are persistently aphid transmitted. Their most important aphid vectors are Rhopalosiphum padi (bird cherry-oat aphid), R. maidis (corn leaf aphid), Sitobion avenae (grain aphid) and Schizaphis graminium (wheat aphid). CYDV is transmitted by R. padi, whereas the main vectors of BYDV strains PAV, MAV, RMV and SGV are R. padi (PAV), R. maidis (RMV), Sitobion avenae (MAV) and Schizaphis graminium (SGV) [In wheat, YDD is caused by two different viruses, barley yellow dwarf virus  ,79,142. um (SGV) ,77,78,79um (SGV) . How BYDum (SGV) .The development of severe YDD epidemics varies seasonally. It depends upon weather conditions  that promote early build-up of aphid vectors in alternative aphid and BYDV/CYDV hosts , and their large-scale migration to wheat crops at an early growth stage. When this occurs, widespread YDD develops causing significant losses. Mean daily rainfall and temperature data before sowing allow predictions of infection incidence and losses, and provide decision support over insecticide applications to kill aphid vectors ,143,147.2. However, unlike YDD, which is most damaging when wheat crops are gown under cool conditions, WSMD is at its most destructive when they are grown under warm conditions [Wheat streak mosaic disease (WSMD) causes the second most important virus disease epidemic of wheat globally . It was nditions ,83,150. nditions ,79,83. Inditions ,83,150. Aceria tosichella). When viruliferous WCM are blown away from infected plants onto healthy plants by the wind, WSMV is transmitted to them [WSMD is caused by infection with WSMV, which was recently described as “a century old virus with rising importance worldwide” . WSMV is to them ,72,79,83 to them ,151,152. to them ,83. In t to them ,83. Howe to them ,82. Epid to them . The morAn ancestor of WSMV most likely spread from infected wild wheat or other grasses to infect wheat when this crop was first domesticated. Due to the high temperature requirement 24–27 °C) for optimum activity of its WCM vector  °C for o, its iniOryza sativa) is ranked first and third in importance in developing countries and the world, respectively [As a staple food crop, Asian rice  constitutes the most devastating virus disease of rice in tropical regions of Southeast Asia, southern China and the Indian subcontinent, where it causes a devastating pandemic ,38,84,85Nephotettix virescens [RTD is caused by mixed infection with RTBV and RTSV. Although plants infected with RTD develop severe foliage symptoms, plants infected solely with RTSV only show mild stunting, whereas those infected with RTBV develop mild ‘tungro’ symptoms. RTSV and RTBV are transmitted semi-persistently by several leafhopper vector species, the most efficient of which is irescens ,84,85. Iirescens . HoweverThe viral ancestors of RTBV and RTSV most likely emerged from infected wild rice or grasses to infect rice soon after this crop was domesticated. Phylogenetic analysis of RTBV isolates from India and Southeast Asian countries revealed two major phylogroups, one containing South Asian (=Indian sub-continent) and the other Southeast Asian isolates ,62,84. SSolanum tuberosum) crop was domesticated from wild potato species native to the Lake Titicaca sector of the Altiplano in the Andean region of South America [Approximately 9000 years ago, the potato . However, PVY also formed complexes when present in mixed infection with two other common viruses, PVA and potato virus X , the resulting synergistic interactions resulting in the severe foliage disorders ‘crinkle and ‘rugose mosaic’, respectively [After the potato was introduced to Europe in the second half of the 16th century, propagation of its tubers by replanting them continually year after year led to a devastating foliage degeneration disorder that diminished its tuber yields drastically. Until potato breeding from crosses between selected parental lines commenced in the 19th century , addressectively ,164,166.ectively . PVY infectively ,168. Myzus persicae (green peach aphid), by planting PVY-infected potato seed tubers, and, occasionally, by plant-to-plant contact [Solanaceae species [Ny and Nc, elicited by its main biological strain groups PVYO and PVYC, respectively [N, spread from South America to Europe. It caused obvious veinal necrosis symptoms in tobacco plants, but its subtle symptoms in potato foliage often went unnoticed, such that infected plants were missed during seed potato field inspections. It also overcame hypersensitive resistance genes Ny, Nc and additional PVY resistance gene Nz. These characteristics resulted in its widespread distribution through large-scale inadvertent planting of PVYN-infected seed potato stocks [O and PVYN eventually led to recombination between these two stain groups [Ny and Nc, [Nz [N, they too were often missed in seed potato field inspections. In addition, their aphid transmission was more efficient. These properties favored their inadvertent widespread dissemination on a global scale within infected seed potato stocks [PVY is transmitted from plant to plant non-persistently by several aphid species, e.g.,  contact ,168,169. species ,168. It  species ,170,171. species  combinedectively ,172, greo stocks ,166,167.n groups ,89,91. T and Nc, , but R2  Nc, [Nz . These Ro stocks ,89,91. Mo stocks ,89,91,92O, PVYC and PVYN in most potato-growing regions of the world [PVY’s R2 phylogroup was found first in the early 1980s in Europe, but its R1 phylogroup apparently arose earlier . Apart fhe world ,173,174.he world ,89,90. TIpomoea batatas) was domesticated approximately 10,000 years ago. Its domestication occurred twice, once each in Central and South America, the two gene pools subsequently becoming mixed. In the pre-Colombian era, it was taken from the Americas and dispersed widely around Polynesia and from there to Melanesia [Sweet potato  and a member of the Potyviridae family. The latter virus is usually sweet potato feathery mottle virus  or sweet potato mild mottle virus . A synergistic interaction between SPCSV and SPFMV or SPMMV causes a very damaging disease consisting of severe leaf mosaic and malformation and plant stunting, and a tuberous root yield reduction of >85% (Sweet potato virus disease (SPVD) causes the most devastating virus disease of the sweet potato crop. It is caused by mixed infection with the sweet potato chlorotic stunt virus  and Trialeurodes abutilonea (glasshouse whitefly) and SPMMV by B. tabaci. SPFMV is transmitted non-persistently by the aphid species M. persicae and Aphis gossypii (cotton aphid) [Convolvulaceae that it infects naturally in this region [Convolvulaceae species Ipomoea spp. , Hewittia sublobata, and Lepistemon owariensis [SPCSV and SPMMV are transmitted semi-persistently by the whiteflies, SPCSV by n aphid) ,99. SPFMn aphid) . The samn aphid) ,187. By s region . Alternaariensis ,188,189.What was responsible for the emergence of SPVD as a major threat to sweet potato production within East Africa? The traditional East African land races that survived since sweet potato first arrived were tolerant of SPVD. What led to the SPVD pandemic in that part of the world was (i) land race displacement by vulnerable high-yielding sweet potato cultivars bred elsewhere and (ii) agricultural intensification aimed at growing more sweet potato to help meet the needs of the burgeoning human population ,187. ThuMusa spp., including dessert banana and plantain) ranks sixth in the world after maize, wheat, rice, potato and cassava, and the fourth most important for developing countries [M. acuminata (genetic constitution AA) and M. balbisiana (BB) [As a world staple food crop, banana  causes a devastating pandemic in banana crops. It constitutes the most damaging virus disease affecting this crop on a global scale and seriously threatens food security in developing countries ,64,192. Pentalonia nigronervosa (banana aphid). This aphid vector spreads BBTV locally within and between banana and plantations. Its natural alternative hosts are limited to three other Musaceae species, Musa paradisiaca, M. textilis and Ensete ventriculosum. These three species are potential sources for BBTV to spread locally to plantations [Pentalonia nigronervosa vector and agricultural intensification to increase production. BBTD is caused by BBTV, which is transmitted persistently by ntations . Phylogentations . What trCitrus species is considered to be in the southeast foothills of the Himalayas from which they spread throughout subtropical and tropical regions of the Indian subcontinent, East Asia, Southeast Asia, Melanesia, and Oceania [Citrus species in different parts of this wider region, especially in Southeast Asia, before being introduced gradually to all continents except Antarctica [Citrus fruits are important for human nutrition. They provide the human body with vitamins, minerals and plant compounds that bestow health benefits, including anti-inflammatory and antioxidant effects. They include orange, mandarin, lemon, grapefruit, lime and pomelo. The center of origin of wild  Oceania . The diftarctica . Citrus tarctica . The exaCitrus tristeza disease (CTD) is the most economically important virus disease of citrus plantations globally. It causes a lethal tree decline syndrome, especially in oranges, mandarins, grapefruits and limes propagated on sour orange rootstocks. At the beginning of the 20th century, death of trees growing on sour orange rootstocks was reported in South Africa, followed by Argentina and Brazil in the 1930s, and shortly afterwards in several other South American countries. Its name ‘tristeza’, which means ‘sadness’ in Spanish and Portuguese, was given it due to the massive destruction it engendered in South American plantations. CTD was first recorded in the 1930s in Argentina and shortly afterwards in Brazil and several other South American countries. Virtually all citrus trees growing on sour orange rootstocks were killed and plantations abandoned in worst-affected regions. By 1959 in Brazil’s Sao Paulo state, 75% of all orange trees  were killed, and similar problems occurred in South Africa, West Africa and California . After 1CTD results from killing of the phloem below the graft union causing foliage symptoms consisting of leaf size reduction and veinal yellowing, leaf drop, twig dieback, wilting and tree death. This tree decline followed by death occurs at different rates. There is no fruit yield with ‘quick decline’, and although some small fruit may form with ‘slow decline’, it still decreases fruit yields drastically. In addition, the related stem pitting decline syndrome (CSPS) severely damages plantations of grapefruit, lime and several orange cultivars, and another related condition, seedling yellows syndrome (CYS), also damages sour orange, lemon and grapefruit plants mostly in nurseries. With CSPS, citrus branches and trunks develop pits and grooves, and gum often appears, and the affected trees are stunted producing fewer, smaller fruit. Thus, tree vigor and fruit yield are both diminished. With CYS, yellowing develops on leaves and branches die back ,102. Closterovirus, family, Closteroviridae). A range of biological CTV strains exist that elicit symptoms which vary in severity from quick tree decline and death to asymptomatic infection, and in type . Symptom severity also differs with citrus cultivar and rootstock type, and is favored by warmer conditions. CTV is transmitted semi-persistently by several aphid species. Its most efficient and globally important vector is Toxoptera citricida (brown citrus aphid). However, although less efficient, A. gossypii is the most important vector in Europe, and two other less efficient vectors are also of importance in other continents, T. aurantii (black citrus aphid) and A. spiraecola (green citrus aphid). Viruliferous aphids spread the virus locally within and between citrus plantations, and, over greater distances when strong winds disperse them. However, more distant CTV spread, including between regions, countries and continents, is almost entirely by distribution of infected planting material  derived from CTV-infected nurseries. Alternative natural CTV hosts are limited to other citrus species and species in related genera, such as Fortunella and Poncirus [The causal agent of CTD, CSPS and CYS in citrus trees is citrus tristeza virus  are also important for human nutrition, not only as a source of vitamins and minerals but also generally as part of healthy eating patterns that reduce the risk developing certain chronic diseases that afflict humanity. They include the Prunus species plums, cherries, peaches, nectarines, apricots, cherries and almonds. Cultivated Prunus species were all domesticated in the northern hemisphere, for example plum and cherry species in Europe, Asia and North America, but peaches and nectarines in China [Like citrus fruits, stone fruits (in China . They suin China . An examPlum pox disease (PPD = Sharka disease) is the most destructive virus disease of stone fruit worldwide. Collectively, its epidemics have caused a devastating pandemic in plum, peach, apricot and nectarine orchards, which, over the last 40 years, has been the subject of numerous reviews ,115,116.M. persicae. These aphid vectors contribute to local PPV spread within and between orchards. More distant spread, including between regions, countries and continents, occurs mostly through the inadvertent distribution of infected planting material  derived from PPV-infected nurseries. Strict quarantine regulations and sole use of healthy planting material are critical measures that prevent its long-distance spread. Apart from occurring in cultivated Prunus stone fruit and ornamental trees, natural infection with PPV also occurs in wild Prunus species [Prunus hosts to plum and cherry within this region. By contrast, as peach and nectarine were domesticated in China, PPV’s spread into them required a new encounter situation when they were planted next to PPV-infected plum or cherry orchards following their introduction to Europe or the Levant. What caused PPV to emerge as the cause of such a destructive global pandemic? The most likely causes are wide-scale inadvertent transportation of PPV-infected Prunus planting material and germplasm to new geographic locations around the world, local spread by aphid vectors and the widespread adoption of vulnerable stone fruit cultivars [The causal agent of PPD is PPV, which is transmitted non-persistently by many aphid species several of which are important vectors, including  species ,115,116. species ,197. Thuultivars ,14. Vicia faba) is described below.Including grain legumes in the human diet is important for balanced nutrition and helping combat food insecurity. In addition, the atmospheric nitrogen they fix is important for soil fertility and achieving sustainable agriculture . HoweverFaba bean necrotic yellows disease (FBNYD) epidemics cause severe yield losses and crop failure in faba bean crops. FBNYD was reported first in Syria in 1986 , but nowNanovirus, family, Nanoviridae), which is transmitted persistently by three aphid species, A. craccivora, A. fabae (black bean aphid) and Acyrthosiphon pisum (pea aphid). Although faba bean is FBNYV’s main host, its alternative hosts include the grain legume crops common bean (Phaseolus vulgaris), cowpea (Vigna unguiculata), chickpea (Cicer arietinum) and lentil (Lens culinaris), several wild and pasture legumes, and several non-legume species belonging to the genus Amaranthus [FBNYD is caused by faba bean necrotic yellows virus  is the most important vegetable crop worldwide [Solanum pimpinellifolium was used as a food. It was then taken north within the Americas reaching Mexico approximately 7000 years ago, where the crop’s domestication continued. In the 16th century domesticated tomato was transferred from Mexico to Europe before being distributed worldwide [Tomato  was first found in 2014 infecting tomato in Jordan so, apparently, it only emerged recently [2Tm-2, so all TMV- and ToMV-resistant tomato cultivars become ToBRFV infected [2Tm-2, a gene that had apparently remained effective against tobamovises during the previous 55 years [Capsicum annuum) plants in which its foliage symptoms include leaf vein clearing, mosaic and discoloration, stem browning, and fruit mosaic and distortion [L1, L2 and L3 [Tomato brown rugose fruit virus , petunia (Petunia hybrida) and the weed S. nigrum [ToBRFV has rod-shaped stable virions that reach high concentrations in infected plants and remain infectious for long periods on contaminated surfaces. These properties enable its efficient contact transmission when a healthy tomato plant comes into contact with an infected plant, contaminated soil, contaminated nutrient solutions or a contaminated surface, such as equipment, tools, hands and clothing used during pruning or trellising within protected cropping situations. It is also transmitted by grafting, cuttings ,123,124 . nigrum . Its epiWhat is responsible for ToBRFV’s apparent emergence in 2014 in the Middle East, followed by its rapid geographical dispersal to Europe, Africa, East Asia and the Americas over the last six years? One possibility is that it emerged recently from spread by contact transmission from an unknown infected alternative solanaceous crop or weed host to the introduced crop tomato, and was detected infecting tomato soon afterwards. Tomato seed crops then became ToBRFV-infected and the international seed trade distributed contaminated seed around the world, albeit inadvertently, introducing the virus to other counties and continents. Another possibility is that it was being confused with TMV or ToMV, and had already been present infecting tomatoes in all of these different parts of the world but had gone unnoticed . HoweverPotexvirus, family, Alphaflexiviridae) was isolated first in 1974 from a plant of the indigenous Andean fruit crop pepino (Solanum muricatum) showing yellow leaf mosaic symptoms growing in coastal Peru. When inoculated with PepMV-infective sap, a range of other solanaceous crop species became infected, including tomato [Pepino mosaic virus  and pumpkin (Cucurbita moschata and Cucurbita maxima) were domesticated from their wild ancestors in the Americas in the pre-Columbian era, cucumber (Cucumis sativus) in India, and both watermelon (Citrullus lanatus) and gherkin (Cucumis anguria) in Africa; melons (Cucumis melo) were domesticated twice, once each in Africa and India/Southeast Asia [https://www.quora.com/Which-fruit-is-the-most-popular-and-most-consumed-in-the-world). Cucumber and gherkin are the third most important vegetables, and the tenth most important are pumpkin, squash, zucchini and gourds . Although not ranked among the top ten fruits, melon is also a very important dietary component in many countries. An example of a recent major seed-borne virus disease epidemic of cucurbits is described below.Among the world’s principal cucurbit crops, squash, zucchini  was amongst the earliest plant viruses described. This was in 1935 when it was found to cause a cucumber disease in glasshouse crops in England [Cucumber green mottle mosaic virus , Ecballium elaterium (Cucurbitaceae), Chrozophora tinctoria (Euphorbiaceae), Moluccella laevis (Lamiaceae) and Withania somnifera (Solanacaeae) [CGMMV has stable rod-shaped virions that remain infectious for long periods on contaminated surfaces and reach high concentrations in infected plants. Both of these factors favor its efficient transmission by contact when a healthy cucurbit plant comes into contact with an infected plant surface or with a contaminated soil . Within nacaeae) . Their rnacaeae) . Phylogenetic studies indicate an Asian origin for CGMMV, rather than a European origin, despite its initial detection under protected cropping in England . The onlPotyvirus, family, Potyviridae) is also the causal agent of a global virus disease epidemic that severely damages cucurbit crops in world regions with tropical, subtropical and Mediterranean climates. In cool temperate conditions, e.g., in Northern Europe, it is limited to protected cropping situations. [Another seed-borne cucurbit virus, zucchini yellow mosaic virus , but also at land borders, seaports and airports (border) . SuccessWhen virus pandemics or epidemics are already taking place, integrated virus disease management programs need to be devised to ensure they are controlled efficiently. Devising these requires knowledge of the epidemiology of the causal virus(es) and their disease cycles ,13,49,50To optimize the effectiveness of the control being achieved, whenever possible, new technologies should also be exploited. These include deploying remote sensing in association with precision agriculture to forecast: (i) epidemics on local, regional or continental scales via satellites; (ii) epidemics in individual crops via lightweight unmanned aerial vehicles; and (iii) precisely where to target control measures . The latIn order to manage damaging global virus disease pandemics and major epidemics effectively, collaborative multidisciplinary research networks need to be organized. These require linkages between developed and developing country researchers, including plant virologists, entomologists, agronomists and plant breeders, along with modelers, statisticians, and socioeconomics experts ,13,25. AWhen staple food crops suffer large-scale losses from destructive virus disease pandemics or epidemics, this has major implications for people whose livelihoods depend directly or indirectly upon these crops. It can lead to dire circumstances of hunger and famine, especially in the world’s poorer countries. Similarly, damaging virus disease pandemics or epidemics afflicting non-staple food crops necessary for balanced nutrition have serious implications for human health. This review provides striking examples of the crop spoilage, losses in overall yield and produce quality, and hardship in the human population that arise. The virus disease examples described afflict six of the world’s most important staple food crops , and five non-staple food crops that are critical for balanced human nutrition . Three of the six pandemic examples  are caused by distinct virus complexes and occur in open-field (arable) crops. The other three  are each caused by a single virus and affect plantation or orchard crops. By contrast, none of the seven major epidemic examples involve plantation or orchard crops. Four of these involve open-field crops , and three occur both under protected cropping situations and in the open field . Most of the causal virus(es) of these 13 examples likely coevolved with the principal crop(s) they infect within their respective crop domestication center(s). However, proof of this requires phylogenetic analysis of sufficient virus isolate sequences from relevant crop domestication center(s). This is currently lacking in most instances—the four exceptions being for the causal viruses of PTNRD, RTD, BBTD, PPD. (i)Introduction of vulnerable higher-yielding crop cultivars with MLND, WYDD, RTD, SPVD, BBTD, CTD, PPD and FBNYD;(ii)Agricultural intensification to increase crop yields with MLND, WSMD, RTD, SPVD, BBTD, CTD, FBNYD, PepMD, TBRFD and CGMMD;(iii)Virus recombination events resulting in more virulent virus variants with WYDD, RTD, PTNRD and TBRFD; and(iv)Increased, or more efficient, vector populations with MLND, WSMD, BBTD and CTD.Most viruses responsible for the disease pandemics and epidemics described here became distributed between continents, albeit inadvertently, by long-distance dispersal of virus-contaminated seed or planting material. This occurred via either international trade in plants and plant products or germplasm introductions for plant breeding purposes. Such dispersal mostly involved virus-contaminated propagules of the affected crop rather than propagules of a related crop. In some instances, critically important vectors were distributed along with the virus-contaminated planting material, e.g., with BBTD and CTD. The causal viruses known to have spread by contaminated seed were MCMV, WSMV, PepMV, TBRFV and CGMMV, whereas those known to have spread by contaminated planting material were BBTV, CTV, PPV, PVY-R2, SPCSV and SPFMV. RTBV, RTSV, SCMV and SPMMV also likely spread via infected planting material, but MDMV via contaminated seed. The most important additional factors favoring virus disease pandemic or major epidemic development were: As the world population continues to grow, the need to achieve overall global food security is becoming increasingly urgent. The escalating climate change-induced problems associated with controlling disastrous plant virus diseases threatening food crops constitute a crucially important issue for humankind to resolve. Therefore, achieving effective management of virus disease pandemics and major epidemics that harm not only staple food crops, but also crops essential for balanced human nutrition, is an extremely important goal. Achieving this goal requires international collaboration on a worldwide scale between researchers and other experts in relevant disciplines, extension personnel, plant biosecurity organizations, funding bodies, and policy makers. The objective is to develop a complete understanding of how each pathosystem behaves in different world regions, its disease cycle, its epidemic drivers and its overall epidemiology. This knowledge is essential for the development of biosecurity responses that prevent virus entry to a new region, or, where they fail to do so, eradicate or contain virus disease incursions after their arrival. It is also essential for optimizing the effectiveness of individual control measures. Finally, it is needed to devise integrated virus disease management and extension approaches directly suited to minimizing the deleterious impact of virus disease pandemics and epidemics upon food security and human health in a socially and environmentally responsible way.


We call the virus particle causing the COVID-19 disease SARS-CoV-2, a spherical capsid covered with spikes termed peplomers. Since the virus is not motile, it relies on its own random thermal motion, specifically the rotational component of this thermal motion, to align its peplomers with targets. The governing transport property for the virus to attack successfully is thus the rotational diffusivity. Too little rotational diffusivity and too few alignments are produced to properly infect. Too much, and the alignment intervals will be too short to properly infect, and the peplomer is wasted. In this paper, we calculate the rotational diffusivity along with the complex viscosity of four classes of virus particles of ascending geometric complexity: tobacco mosaic, gemini, adeno, and corona. The gemini and adeno viruses share icosahedral bead arrangements, and for the corona virus, we use polyhedral solutions to the Thomson problem to arrange its peplomers. We employ general rigid bead–rod theory to calculate complex viscosities and rotational diffusivities, from first principles, of the virus suspensions. We find that our ab initio calculations agree with the observed complex viscosity of the tobacco mosaic virus suspension. From our analysis of the gemini virus suspension, we learn that the fine detail of the virus structure governs its rotational diffusivity. We find the characteristic time for the adenovirus from general rigid bead–rod theory. Finally, from our analysis of the coronavirus suspension, we learn that its rotational diffusivity descends monotonically with its number of peplomers.Just 11 weeks after the confirmation of first infection, one team had already discovered and published [D. Wrapp  I.SARS-CoV-2, a spherical capsid covered with hollow spikes termed peplomers. Since the virus is not motile, it relies on its own random thermal motion, specifically, the rotational component of this thermal motion, to align its peplomers with targets. From Fig. 1(B) of Ref. 2). Furthermore, this alignment must be long enough for fusion. Once fused, perfusion progresses to infection.Shortly after the confirmation of first infection, one team had already discovered and publishedWhereas much of the prior work on flow and the virus focuses on infection of the organism,The governing transport property for the virus to attack successfully is the rotational diffusivity of the SARS-CoV-2 particle  of Ref. continuity, we mean that the diffusion equation conserves orientation, preserving one and only one orientation per macromolecule. Hassager solves the diffusion equation for the configuration distribution function in small-amplitude oscillatory flows, which, for rigid macromolecules, reduces to the orientation distribution function, ψ.General rigid bead–rod theory proceeds from the continuity equation for the macromolecular configuration, called the θ = π/2) with the long axis of the dimeric receptor along the longitudinal direction. For this special infection opportunity, the probability of finding a peplomer aligned with said receptor is given by [see Eq. (9) of Ref. p.Consider, for instance, a coronavirus particle close enough to fuse with a dimeric receptor.ψ with macromolecular shapes, including those of viruses. In this way, the virus shape confers the transport properties to its suspension, including viscosity, elasticity, and diffusivities, be they rotational or translational. Little is known experimentally about the diffusivity of viruses, especially the rotational diffusivity. For instance, the translational diffusivity of the adenovirus has been measured by photon-correlation spectroscopy.General rigid bead–rod theory connects ab initio calculations from general rigid bead–rod theory on coronaviruses is that the peplomer arrangement is not known. However, we do know that the spikes are charge-rich,Thomson problem, we mean how identically charged particles will organize themselves onto a sphere by minimizing system potential energy. In this work, we are thus using minimum potential energy peplomer arrangements for our coronavirus model particles.One of the challenges of diffusive rotational search of the spike-protein unfolding that accompanies binding.The rotational alignment of the virus particle studied herein is prefusion and not to be confused with the postfusion II.moment of inertia ellipsoid (MIE) whose center is the center of mass and whose principal moments of inertia match those of the virus particle. The MIE thus determines the orientability of the virus particle and thus the virus rotational diffusivity. Our use of moment of inertia ellipsoids is not to be confused with replacing the virus particle with an ellipsoid of revolution, with its own hydrodynamic environment.Using general rigid bead–rod theory, we propose the construction of virus particles from sets of beads whose positions are fixed relative to one another. For example, the SARS-CoV-2 particle geometry is a spherical capsid surrounded by a constellation of protruding peplomers. We understand that the number of peplomers per virus particle differs from particle to particle and seems to decrease with time after inoculation. We suspend our bead–rod models of virus particles into a Newtonian solvent. We begin by neglecting interactions of the solvent velocity fields, be they (i) between nearest beads within the virus particlemi with the position vector of the ith bead ri, where the virus particle center of mass R satisfiesN is the total number of beads and d and mass m, then M…mN, and thus, the center of mass isTo model the virus particle, we locate each bead of mass viral coordinates at the center of mass of the virus, and we orient these Cartesian coordinates such that moment of inertia ellipsoid. For our virus particles, We next install ith bead with respect to the virus center of mass is given byI1, I2, and I3 by [Eqs. (16.7-17) and (16.7-18) of Ref. i is the bead number. We design each virus particle structure by first rigidly connecting nearest bead centers with massless widthless rods. Throughout our work, L is the distance between the nearest bead centers. We then complete the general rigid bead–rod construction by rigidly connecting the remaining bead centers to their nearest neighbors. For the SARS-CoV-2 particle, L is the center to center distance between osculating beads forming the capsid. Although the peplomer is a spike with a bulbous triadic head, in this work, we will model it as a single bead not touching the capsid.The position vector of the axisymmetric, we mean that both the virus particle and its moment of inertia ellipsoid have at least one axis of symmetry.I1 = I2. Our usage of axisymmetric is not to be confused with the common geometric meaning of continuous rotational symmetry about an axis.Since the virus particle structure is axisymmetric, so will be its moment of inertia ellipsoid. By b ≤ 3/5 and 0 ≤ aν ≤ 7/2. The three quantities a, b, and ν thus define completely the differences in linear viscoelastic behaviors arising between different axisymmetric macromolecular structures. Whereas we associate a with the Dirac delta function contribution to the relaxation function, we associate b with the dying exponential.Hassager derives the expression for the dimensionless shear relaxation function for general rigid bead–rod theory,φ is the bead volume fraction, and for osculating beads, where L = d,The relaxation time of the corresponding virus particle suspension can be expressed asDividing Eq. We can then use Eq. dimensionless rotational diffusivityλs. The quantity ν thus defines completely the rotational diffusivity of a virus particle.In this work, we apply these derivations to virus particles, specifically to the calculation of the rotational diffusivity, given by the identity  of Ref. Following EXAMPLE 5.2-6 of Ref. IV.λ0 is given by Eq. d is the diameter of the tobacco mosaic virus , and at T = 298.2 K, Dr = 206 s−1 (or λ0Dr = 1.18 × 10−3). These values compare closely with the value predicted by Eq. N = 12. Furthermore, from the available measurements , the rotational diffusivity range for the tobacco mosaic virus at room temperature (20°C–25°C) isDr = 206 s−1, fitted to complex viscosity measurements . From λ/λ0 and (η0 − ηs)/nkTλ over two osculating beads. From this, we deepen our understanding of the role played by the finer twin truncated icosahedral structure.In this section, we use general rigid bead–rod theory to model the gemini virus as twin truncated icosahedra. To illustrate these twin truncated icosahedra, we construct λ0Dr in b/aν for the gemini virus in η′ − ηs)/(η0 − ηs) descends less sharply than for twin osculating beads. We also learn that for the twin truncated icosahedral structure, η0 − ηs) falls below that of the twin osculating beads. We thus find that the fine structure of the gemini virus matters, raising viscosity and lowering elasticity.By comparing the values of VI.To arrive at the rotational diffusivity of the adenovirus, we follow the method of Sec. An icosahedron has 20 triangular faces, 30 edges, and 12 vertices. Each vertex holds a penton, shared by five edges and five faces. The faces are made of two nested triangular arrangements, the inner triangle being made of six hexons. Each of 120 edge hexons is shared by two faces. Each of the 120 face hexons belongs to a single face, with all six of its neighbors lying in that face.L, with the interparticle distance L equal to the diameter d of the capsomer. The 12 vertices sit on the edges of three rectangles with Cartesian coordinates given by cyclic permutations of  scaled by 5L/2, where Vi of the 12 vertices in Eij = Vj − Vi, where, for example, E0111 = V11–V01.The edges are taken to have length 5ijk arising from a counterclockwise enumeration of the five neighboring vertices associated with one vertex. We encode this as follows: The notation 01: 02, 11, 06, 05, 09 means faces ijk = 010 211, 011 106, 010 605, 010 509, 010 902. To describe face beads, we need twelve sets of five triplets Vi with i = 1, 2, …, 12 is associated with five triplets ijk and the following 21 points: the vertex itself, Vi, to which we add vectors for the ten edge beads, Ri of the adenovirus capsid beads so that i = 1, 2, …, 252.We associate two edge beads on five edges and two face beads  on five faces with each vertex, so that each of the 12 vertices Oliver, who measured the translational diffusivity of the adenovirus,From general rigid bead–rod theory Eq. , for thes see in  and the rc ≃ 116 nm (between opposing vertices), and the range for the virus radius, made dimensionless with the capsid radius, is given byr/rc = 5/4  ≃ 1. In general rigid bead–rod theory, we approximate the bulbous SARS-CoV-2 triglycoprotein head with a single bead so that 2rb = d. From the available microscopy, we can see that the range for the triglycoprotein head diameter, made dimensionless with the capsid diameter, is given by , we begin by beading a unit sphere for the capsid around which we arrange a constellation of peplomer heads. We get position vectors for these beads by multiplying the 74 point-charge solution to the Thomson problem extracted from Ref. 29 of Ref. In this work, we choose the measured peplomer population Np = 74  over the/(η0 − ηs) curves for the adeno- and coronavirus bead–rod models descend less sharply than those of tobacco mosaic or gemini viruses. From η″/(η0 − ηs), and thus, the lower the dimensionless elasticity. We also learn that adenovirus is spherically symmetrical (I1 = I2 = I3), and thus, it is not associated with η0 − ηs).A.Nc, and fix the peplomer bead number, Np = 74. We model the different capsid beadings, Nc = 16, 32, 64, 128, 256, 510, and thus construct λ/λ0 in Nc) increases the relaxation time. By examining the values of (η0 − ηs)/nkTλ in Nc does not affect the polymer contribution to zero-shear viscosity, since (η0 − ηs)/nkTλ remains 1.5. From λ0Dr, decreases with Nc.In this subsection, we vary the capsid beading of the coronavirus particle, b/aν for all coronavirus capsid beadings in Nc does not change (η′ − ηs)/(η0 − ηs). A coronavirus model thus always gives a nearly constant viscosity .Using the values of 2B.Nc = 256 and vary the peplomer bead population over 10 ≤ Np ≤ 100 to get λ0, are of order 10−4. Specifically, for the measured peplomer population, Np = 74 .In this subsection, we fix the capsid beading of the coronavirus particle at = 74 see , we get enovirus  and fallenovirus  and gemienovirus . The binλc, and the ratio of the first normal stress coefficient to the viscosity at zero shear rate. Specifically, we next explore how λc depends on the peplomer population. Nondimensionalizing λc with the constant λ0, produces the stairstep plot of λ0, over 10 ≤ Np ≤ 100, fall below 10−4. In other words, the elasticity of the coronavirus particles is slight and is not monotonic with Np. Furthermore, at the measured peplomer population of Np = 74  under this projection gives the probability of one spike aligning properly for fusion, Our macromolecular bead–rod model viruses are suspended in a Newtonian solvent. We neglect interactions of the solvent velocity fields, be they between nearest beads  of Ref. r/rc = 5/4 (For this work, for both adenovirus and coronavirus, we chose the ratio Whereas in engineering, the complex viscosity function has a broad diversity of applications including polymer or suspension processing, for virus suspensions, its main use is for determining rotational diffusivity. The uninitiated might expect that our complex viscosity equations for the adenovirus Eqs.  or coronMore broadly, our vision here is that a handbook of general rigid bead–rod virus models be generated eventually from which the transport properties of any included virus might be calculated and then compared. Such a handbook might thus lead us to deepen our understanding of the relation between the rotational diffusivities of virus particles and their intricate shapes.The data that support the findings of this study are available within the article.


The widespread adoption of bead-based multiplexed bioassays requires the ability to easily synthesize encoded microspheres and conjugate analytes of interest to their surface. Here, we present a simple method (MRBLEs 2.0) for the efficient high-throughput generation of microspheres with ratiometric barcode lanthanide encoding (MRBLEs) that bear functional groups for downstream surface bioconjugation. Bead production in MRBLEs 2.0 relies on the manual mixing of lanthanide/polymer mixtures  followed by droplet generation using single-layer, parallel flow-focusing devices and the off-chip batch polymerization of droplets into beads. To streamline downstream analyte coupling, MRBLEs 2.0 crosslinks copolymers bearing functional groups on the bead surface during bead generation. Using the MRBLEs 2.0 pipeline, we generate monodisperse MRBLEs containing 48 distinct well-resolved spectral codes with high throughput . We further demonstrate the efficient conjugation of oligonucleotides and entire proteins to carboxyl MRBLEs and of biotin to amino MRBLEs. Finally, we show that MRBLEs can also be magnetized via the simultaneous incorporation of magnetic nanoparticles with only a minor decrease in the potential code space. With the advantages of dramatically simplified device fabrication, elimination of the need for custom-made equipment, and the ability to produce spectrally and magnetically encoded beads with direct surface functionalization with high throughput, MRBLEs 2.0 can be directly applied by many labs towards a wide variety of downstream assays, from basic biology to diagnostics and other translational research. Spectrally encoded beads provide a convenient platform for multiplexed bioassays, offering fast binding kinetics and many replicates per assay. A recently developed technology, MRBLEs, spectrally encodes hydrogel beads via the ratiometric incorporation of lanthanide nanophosphors. In the present paper, a team from Stanford University led by Polly Fordyce reports a dramatically simplified method for producing MRBLEs beads bearing various functional groups for downstream chemical coupling or on-bead synthesis. Using a ‘jumper cable’ tubing strategy, they route microfluidic channels in 3D without a need for complex fabrication techniques to create multi-nozzle droplet generators. Using these simple single-layer microfluidic devices, they create beads with 48 unique spectral codes bearing carboxyl and amine groups for downstream coupling with over 1000-fold increase in throughput. Finally, they demonstrate that MRBLEs can be simultaneously spectrally and magnetically encoded. However, spatial arrays suffer from relatively slow kinetics (as bait molecules are immobilized on a planar surface) and typically require relatively large amounts of sample1.The recently developed ability to identify molecules across a cell’s genome, transcriptome, and proteome has dramatically increased the need for technologies capable of detecting interactions between biological macromolecules at scale to understand interactome networks. Multiplexed bioassays, in which binding is assessed between a single ‘bait’ molecule and many possible “prey” interactors, can reduce the number of experiments required to explore a potential interactome space and thus speed the pace of discovery. Pioneering examples of these assays used spatial arrays to test thousands to millions of potential interactions in a single experiment5. Spectrally encoded beads, which are embedded with ratiometric combinations of fluorescent 12) or luminescent materials , provide a particularly convenient format for multiplexed assays and have already been used for a wide variety of applications25. To date, Luminex multianalyte profiling (xMAP) technology represents the most widely used spectrally encoded bead-based technology. Luminex xMAP beads are commercially available, are compatible with flow cytometry26 and are widely used for a variety of bioassays27. These beads consist of magnetic polystyrene microspheres that encapsulate distinct proportions of red and infrared fluorophores, each of which comprises a unique spectral code. However, these beads also suffer from a variety of limitations. First, the use of fluorescent dyes for encoding limits the possible coding space to 500, and code sets typically contain <100 codes. Second, hydrophobic polystyrene beads cannot detect low-affinity interactions due to widespread nonspecific binding mediated by hydrophobic interactions28.Multiplexed bead-based assays provide an appealing alternative, with near fluid-phase interfacial kinetics, many replicates per experiment, opportunities for quality control, and the ability to flexibly couple different probes and targets across experiments30. Hydrogel beads are comprised of a cross-linked, hydrated polymeric network made up of one or more hydrophilic monomers, providing near fluid‐phase kinetics at functionalized surfaces as well as high-efficiency molecular loading32, and microfluidic droplet generators have been used to produce and polymerize hydrogel beads with high throughput33. However, the use of fluorescent organic dyes to create spectral codes in most cases has limited the number of unique spectral codes to <1008. Moreover, such encoded microspheres are often incompatible with harsh organic solvents, precluding their use in a variety of solid-phase synthesis applications. While QD-based spectrally encoded hydrogel beads have been well established12, QD-based encoding suffers from potential chemical instability, short luminescence lifetimes, and photoblinking due to their complicated photophysics35. In addition, tightly packed QDs inside the bead matrix can undergo energy transfer and reabsorption36. As a result, the demonstrated code spaces for QD-based approaches have been limited to ≤10, far short of the number in theoretical simulations. Furthermore, concerns about potential biotoxicity can hinder their broad application.Recently developed spectrally encoded hydrogel beads provide an appealing alternative that has been used for a variety of biomedical and sensing applicationsmicrospheres with ratiometric barcode lanthanide encoding) that spectrally encodes beads via the ratiometric incorporation of lanthanide nanophosphors (Lns)14. Lns have narrow and well-separated emission spectra, making it theoretically possible to generate code sets with 105-106 unique members13, and we previously demonstrated the ability to produce and discriminate >1100 codes14. In addition, MRBLEs can be subsequently functionalized for on-bead solid-phase peptide synthesis38. However, prior MRBLE production pipelines were complex, requiring two-layer microfluidic devices with integrated valves and extensive custom pneumatics control hardware14, limiting their widespread adoption. In addition, the production throughput was slow , making bead generation for assay development inefficient and labor intensive.Our group recently developed a novel technology  for the efficient high-throughput generation of spectrally encoded and magnetic beads bearing various functional groups capable of downstream chemical coupling or on-bead synthesis. MRBLEs 2.0 relies on the manual mixing of Ln/polymer mixtures followed by droplet generation using single-layer, parallel flow-focusing (FF) devices and off-chip batch polymerization of droplets into beads, thereby simplifying and enhancing the throughput of bead production. To facilitate downstream analyte coupling, MRBLEs 2.0 localizes copolymers bearing functional groups typically used for bioconjugation to the surface of the hydrogel matrix during droplet generation and covalently crosslinks these polymers in place during bead polymerization. Using the MRBLEs 2.0 pipeline, we demonstrate the ability to generate monodisperse MRBLEs (CV < 7%) containing 48 distinct well-resolved spectral codes (<0.01% probability of code misassignment) with high throughput . To highlight the utility of these MRBLEs in downstream assays, we conjugate amine-functionalized oligonucleotides and entire proteins to MRBLEs bearing carboxyl groups with estimated bead loading densities of 1039.MRBLEs 2.0 requires only 2 syringe pumps, a low-cost microscope, a flood UV illumination source, and an easily fabricated single-layer microfluidic device. To allow parallel microfluidic droplet generation, the device uses PEEK tubing as ‘jumper cables’ to provide three-dimensional routing of flow channels . Within the device, these aqueous polymer and fluorinated oil streams meet at four parallel droplet generation nozzles, where changes in interfacial tension cause the aqueous polymer to break off and form monodisperse droplets within the oil stream , these Ln/polymer mixtures and a fluorinated oil solution (HFE7500 + 2% w/w Ionic Krytox surfactant) are simultaneously introduced into a microfluidic droplet generator using syringe pumps at specific volumetric flow rates Fig. . Within eam Fig. . “Jumpeream Fig.  and subseam Fig. . Between14. Polymerized MRBLEs remain highly monodisperse, with a measured median diameter of 52 µm and an overall CV of 7% , all droplets from a given code are transferred to a single well of a multiwell plate and the entire plate is exposed to flood UV illumination, allowing the simultaneous polymerization of all droplets into solid beads. This pipeline typically produces 3,000,000 MRBLEs/20 min, enhancing throughput by >1000-fold compared to prior production methodsUsing MRBLEs in downstream multiplexed assays requires the ability to distinguish embedded codes from one another with high confidence. To test the robustness of spectral encoding and determine the maximum likely coding capacity achievable with high-throughput production, we designed a target matrix of 48 distinct codes comprised of ratios of dysprosium (Dy), samarium (Sm), thulium (Tm), and europium (Eu) Lns . We then mixed these ratios off-chip via simple manual pipetting, generated droplets from these mixtures, polymerized the droplets, washed extensively with solvents , and imaged the resultant MRBLEs to quantify observed Ln intensity ratios and compare these measured ratios to the desired target ratios.). Ln intensities can be extracted after linear unmixing using the normalized spectral reference of each Ln across all Ln emission channels and can be assigned to specific codes  with emissions collected across 9 Ln emission channels  established that beads composed of a 21.4% v/v poly(ethylene glycol) diacrylate (PEG-DA) matrix were homogeneously polymerized after UV flood exposure without detectable Ln aggregation   to the MRBLE surface at high density and without crosstalk or analyte dissociation over long storage timescales. Covalent coupling is particularly robust and typically relies on two main functional groups: -COOH (carboxyl groups) and -NHps) Fig. . Carboxy2 moieties were covalently coupled to the PEG-DA polymer chains comprising the MRBLE hydrogel matrix via Michael addition after bead synthesis38. However, this method was relatively slow (requiring ~24–48 h) and susceptible to the inhomogeneous distribution of free acrylate groups on the bead surface, leading to heterogeneity of coverage during coupling. To address these problems, we developed a novel functionalization approach that leverages solubility differences between –COOH– or –NH2-terminated comonomers and the photoinitiator (LAP) to drive the polymerization of comonomers at the surface of the hydrogel matrix only  are soluble in HFE 7500 oil, while LAP is only soluble in water. The exposure of LAP-containing aqueous droplets to UV drives a polymerization chain reaction that begins in the aqueous PEG-DA microsphere core and ultimately reaches the oil/water interface to drive the cross-linking of oil-soluble interfacial comonomers to the hydrogel bead matrix  10-UA condition suggested that ~107 to 108 oligos bind to the surface of each bead, consistent with prior estimates of MRBLE peptide loading capacities38.To demonstrate this technique and quantify coupling efficiency and variability for DNA conjugation, we synthesized MRBLEs bearing -COOH functional groups at the surfaces and directly coupled fluorescently labeled Alexa 647), NHtry Fig. . Images try Fig. . Bright-try Fig. . The com, NHtry FTo demonstrate that carboxylated MRBLEs can also be used for the covalent coupling of entire proteins under aqueous conditions, we tested the ability to attach streptavidin molecules to MRBLE surfaces via their primary amines. To visualize the attached streptavidin, we then incubated beads with biotin-FITC conjugates, washed them three times and imaged them Fig. . As with2-functionalized MRBLEs for solid-phase peptide synthesis by (1) polymerizing MRBLEs with P-4-E  added to the oil phase, (2) incubating these functionalized MRBLEs with biotin  in either the presence or absence of required coupling reagents, (3) incubating with DyLight 650-labeled streptavidin, (4) washing, and then (5) imaging to quantify the amount of bead-bound fluorescence . In addition, magnetic beads have particular advantages for bead-based separation, improving bead retention during rinsing and the removal of excess supernatant via the application of a magnetic field and facilitating more stringent washing. Finally, magnetic beads can also aid in loading beads into microwells for high-throughput experiments .3O4 nanoparticles by modifying a previously published coprecipitation method41 to include extra PAA in the precursor solution, thereby enhancing the control of nucleation and nanoparticle wrapping as well as preventing nanoparticle aggregation. PAA wrapping also prevents potential reductions in luminescence from Lns caused by direct contact with magnetic nanoparticles42, as the thickness43 and electrostatic repulsion of PAA increase interparticle distances beyond those that lead to quenching42. The resultant Fe3O4 nanoparticles were ~46 nm in diameter and appeared optically transparent  Fesee Fig. . All thrsee Fig. , suggestN, where N represents the number of FFs). To address this, droplet generation devices containing multiple Y-shaped splitters have been widely used to increase throughput47. Herein, we designed and fabricated an additional device that combined exponential droplet splitting with “jumper cables” to boost droplet production rates without increasing device area. In this device, a large droplet  is initially split to form two smaller droplets upon encountering two bifurcated channels  , the measured droplet production rates averaged 106/min at aqueous and oil flow rates of 1500 µL/h and 5400 µL/h, respectively, representing an ~3-fold increase over a linear production device with four FFs .Leveraging MRBLEs for broad use requires the ability to rapidly and economically produce large numbers of beads. While increasing fluid flow rates can boost droplet production within a narrow range, large increases in flow rates drive a transition in droplet formation from dripping to turbulent jetting regimes, yielding very polydisperse beads. As an alternate approach, throughput can be increased by simply adding additional parallel flow focusers (FFs) to the ‘jumper cable’ synthesis device described above Fig. ; howeverels Fig. . These ses) Fig. . As withFFs Fig. . The resFFs Fig. .Fig. 5Hi48, T-junctions50, flow focusers56, and discontinuous step generators50). However, the need to route fluidic channels containing dispersed and continuous phases to and from many different droplet generating nozzles without channels intersecting one another presents fabrication challenges. In the past, this has been addressed by fabricating complex 3D wafer designs (for discontinuous step generators) or assembling devices from multiple PDMS layers with laser-cut or punched “vias” connecting the channels between layers (for T-junctions and flow focusers). Here, we adapt prior work that used tubing to connect different PDMS functional “modules”58 to create “jumper cables” that connect different channels within a single-layer device architecture. This strategy significantly reduces the fabrication complexity required to flexibly route channels in three dimensions while still minimizing fluidic dead volumes. Using this strategy, we demonstrated the ability to produce 150,000 MRBLEs/min (linear amplification) or 450,000 MRBLEs/min , representing a pellet volume of ~1 mL/h or 3 mL/h and a 1000- to 3000-fold increase in throughput from prior work14.Developing multiplexed bead-based assays with the potential for broad translation and adoption requires the ability to produce large batches of spectrally encoded beads at low cost. Large bead batches make it possible to distribute beads widely for use in other labs during initial assay development and optimization and further minimize experimental noise resulting from batch-to-batch variation. Increasing the number of droplet generators within a device provides an obvious way to boost production rates, and this approach has been used successfully for a variety of different glass or PDMS droplet generation devices  by adding multiple comonomers to the oil phase during droplet generation, facilitating the production of beads for single-cell profiling methods such as CITE-seq66 or ECCITE-seq67.The utility of spectrally encoded beads in downstream assays also depends critically on the ease with which analytes can be coupled to beads. Streptavidin- or antibody-conjugated hydrogels are often used to recruit and display biotinylated or epitope-tagged proteins, but these conjugations often rely on nonspecific absorption of biomolecules on the bead surfaceThe simultaneous magnetic and spectral encoding strategy demonstrated here enhances the ability to remove a portion of molecules after coupling or synthesis to quantitatively assess bead loading or synthesis quality. As an example, biomolecules could be coupled or synthesized directly on a mixture of spectrally encoded beads and unencoded magnetic beads, making it possible to remove magnetic beads after reactions for elution and downstream characterization.70, in-air jetting droplet generators71, or glass-silicon chips recently shown to produce droplets at rates exceeding 5.5 billion droplets/min51. In this way, the production of Ln-encoded MRBLEs at the industrial scale could become feasible, opening up a wide range of new bead-based multiplexed assays.In future work, the simple encoded bead production and functionalization pipeline presented here could be integrated with various liquid handling robots and high-throughput droplet generation technologies for a variety of downstream commercial and translational applications. Lns, PEG-DA polymer, functionalized comonomers, and photoinitiators are all commercially available and relatively inexpensive, facilitating the development of low-cost assays. In addition, the ability to mix Lns and polymer before introducing solutions into devices for droplet generation makes it possible to automate this process for the production of large code sets using commercial liquid handling robots capable of mixing viscous solutions . These mixed solutions could then be introduced into devices optimized for ultra-high-throughput droplet production via centrifugal methods72. Briefly, microfluidic molding masters were created by (1) coating 4” test-grade silicon wafers  with a single layer of SU-8 2050 negative photoresist, (2) soft baking, (3) exposing this SU-8 layer to UV light passing through a printed transparency mask (designed in AutoCAD (Autodesk); printed at 50,000 dpi by Fineline Imaging), (4) postexposure baking, and (5) developing away uncured photoresist using SU-8 developer  according to standard manufacturer instructions. These molding masters were then used to cast single-layer droplet generators composed of a 1:5 ratio of poly(dimethylsiloxane) crosslinker:base , and these resultant devices were assembled as described in the https://osf.io/jvnpc/).All devices used in this paper were designed and fabricated via standard soft lithography protocols14. In general, premixed formulas  were generated by varying the ratios of three monomer master mixtures, each containing different Lns. All aqueous master mixtures contained purified water with 21.4% v/v PEG-DA  and 5% v/v YVO4:Eu (50 mg/mL). The “Dy”, “Sm” and “Tm” master mixtures also contained 16.3% v/v YVO4:Dy (50 mg/mL), 16.3% v/v YVO4:Sm (50 mg/mL) and 16.3% v/v YVO4:Tm (50 mg/mL), respectively. Each formula was prepared based on the reference ratio listed in Table 2). We recommend direct UV polymerization after the production of every two codes to reduce any potential droplet breakage. After polymerization, we washed the beads with 2 mL of dimethylformamide  for 20 s, with 2 mL of dichloromethane  for 10 s and with 2 mL of methanol (Thermo Fisher Scientific) for 20 s and then resuspended them in either 1 mL of 1X phosphate-buffered saline (PBS) (Thermo Fisher Scientific) with 0.01% (v/v) Tween-20 (Sigma-Aldrich) (PBST) for the aqueous EDC chemistry and biotinylation of NH2-MRBLEs or in dimethyl sulfoxide  for oligonucleotide conjugation. To make magnetic MRBLEs, we added 10% v/v of the Fe3O4 nanoparticle solution (150 mg/mL) to each recipe. Detailed protocols for the synthesis of Lns and magnetic nanoparticles are provided in the Mixtures of PEG-DA polymer, Lns, and LAP photoinitiator were prepared largely as described previously73. We then incubated the reaction at room temperature for 16 h on a shaker. After this incubation, we neutralized the conjugation reaction by adding 50 µL of 500 mM ethanolamine solution (prepared by adding 30.2 µL ethanolamine (Sigma-Aldrich) to 968 µL DMSO) to each reaction and incubating for 1 h. Following conjugation, we washed beads three times with 200 µL of PBS containing 0.1% (v/v) Tween 20. We then combined all beads into a single Eppendorf tube, resuspended them at 100 beads/µL in PBS with 0.1% (v/v) Tween 20, and stored them at 4 °C.To prepare for oligonucleotide conjugation, beads were rinsed three times with 200 µL of DMSO by spinning down the beads into a pellet and removing the supernatant. After the final spin, we resuspended the beads in DMSO at a concentration of 10,000 beads per 170 µL of reaction volume. We assembled the “conjugation reaction” by combining 20 µL of EDC (Sigma-Aldrich) from a 300 mM stock solution prepared by dissolving 58 mg EDC in 1 mL of DMSO; 20 µL of 1-hydroxy-7-azabenzotriazole  from a 60 mM solution prepared by dissolving 9.2 mg of HOAT in 1 mL of DMSO; and 20 µL of diisopropylethylamine  from a 300 mM solution prepared by adding 32 µL of DIPEA to 968 µL of DMSO. We then incubated beads in this EDC, HOAT, and DIPEA solution for 15 min on a shaker (or rotator at 15 rpm) to ensure adequate mixing. After 15 min, we added 10 µL of a 100 µM stock solution of oligonucleotides modified at the 5’ end with amines and a carbon spacer (the “5AmMC12” modification from Integrated DNA Technologies) and modified at the 3’ end with Alexa 647 dye and an additional 20 µL each of EDC, HOAT, and DIPEAFor streptavidin-coated MRBLEs, we washed 150 μL of carboxy MRBLEs and resuspended them in 200 μL of MES buffer (pH = 4.5) supplemented with 0.01% (v/v) Tween 20. Next, we added 200 μL of freshly made EDC solution  into the bead solution and incubated the entire reaction for 3.5 h at room temperature on a rotator. The O-acylisourea intermediate on the beads is unstable in aqueous solutions; thus, the bead mixture was subsequently immediately washed with 1 mL of borate buffer (pH = 8.5) supplemented with 0.01% (v/v) Tween 20 (coupling buffer) and resuspended in 400 μL of coupling buffer. To conjugate streptavidin, we added 16 μL of 1 mg/mL streptavidin  in borate buffer to the mixture and rotated the whole slurry overnight at 4 °C. After incubation, we quenched the reaction by adding 10 μL of 0.25 M ethanolamine in borate buffer and incubating the solution on a rotator for 30 min at 4 °C. The final product was washed three times and resuspended in 200 μL of PBST buffer for further use. To test the conjugation efficiency, we incubated 20 μL of streptavidin MRBLEs with 0.5 μL of FITC-biotin  for 1 h on an end over end rotator at 30 rpm, washed with 1 mL of PBST three times, and then imaged. In our experience, beads can be stored at 4 °C for ~6 months without a loss of streptavidin binding efficiency.MRBLEs coated with amine groups using 0.09% (v/v) pent-4-enylamine were stored in PBST and extensively washed with DCM, methanol, and DMF prior to biotin conjugation. For each conjugation reaction, we combined ~10,000 beads with 39 mg of biotin, 24 µL of N,N’-diisopropylcarbodiimide  and 56 µL of DIPEA in 400 μL of DMF twice overnight, rotating at room temperature. After conjugation, we washed beads serially with 1 mL each of DMF, methanol, DCM, DMF, water and PBST. After washing, we passivated ~2000 beads with 5% BSA PBST for 1 h at room temperature on a rotator at 30 rpm; the other ~8000 beads were stored at 4 °C. We then exchanged the 5% BSA PBST solution to 2% BSA in PBST by washing and resuspension, added 1 µL of 1 mg/mL DyLight 650-tagged streptavidin (Abcam), and incubated for 30 min at 4 °C on a rotator at 30 rpm. Finally, we washed all beads three times with PBST and imaged them as described below.13. Briefly, beads were excited using a Xenon arc lamp  with an automated filter wheel  containing filters designed to pass 292 nm light  paired with UG11 absorptive glass  and were imaged for lanthanide emission at 9 distinct wavelengths using nine emission filters  and captured images using an sCMOS camera (Andor) . We then extracted the most probable Ln ratios associated with each bead via linear unmixing relative to a series of Ln reference spectra44. The binding of Alexa 647-labeled DNA oligonucleotides, FITC-biotin, and DyLight 650-labeled streptavidin was detected via excitation using a SOLA light engine  and visualized using Cy5, eGFP, and Cy5 filter cube sets, respectively. For imaging beads with more than 2 codes, we recommend dyes excited at higher wavelengths , as fluorescent dyes excited at shorter wavelengths  may be excited by deep-UV light and emit light in the Ln channels, complicating decoding. However, emission from the Ln-encoded beads under SOLA light engine excitation was not observed in the fluorescent dye channels used here, as shown in Figure SBead imaging was performed on a Nikon Ti microscope with a custom UV transilluminator largely as described previouslyEditorial SummarySI figures and SI methods_clean versionMovie S1Movie S2Movie S3


Before the emergence of SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2), the causative agent of the current COVID-19 (coronavirus disease 2019) pandemic . The sciCells, Zika virus and host interactions, thirteen articles that address diverse aspects of ZIKV–host interactions from basic science to clinical research are presented  in E protein that is associated with ZIKV infectivity. To illustrate the importance of these E protein residues in ZIKV infection, the authors swapped them between the Brazilian epidemic strain BR15 and placed them with the corresponding amino acids from the pre-epidemic African strain MR766. The BR15 virus containing the E-152/156/158 residues from MR766 was less infectious in vitro than the parental virus. In contrast, the MR766 virus containing amino acid residues 152/156/158 from the Brazilian strain was more infectious. As changes to this region of E protein did not affect virus binding to host cells, virus-induced cell death, or the interferon response, the authors speculated that they may be important for infectivity by promoting viral membrane fusion inside infected cells. These findings may contribute to the design of innovative strategies to control ZIKV infection at early stages.Bos et al.  showed tFinally, regarding T-cell immunity to ZIKV, the pre-existing immunity to the related flavivirus, dengue virus (DENV), has been discussed as a risk factor for immunopathological responses . Pardy aWe hope that this Special Issue will be of wide interest, particularly to those researchers focused on different aspects of ZIKV–host interactions. It is expected that findings from these studies will contribute to a better understanding of the vector and human host cells interacting with ZIKV, and will serve as the basis for novel diagnostics, antiviral therapeutics, and vaccines.


Viruses represent important test cases for data federation due to their genome size and the rapid increase in sequence data in publicly available databases. However, some consequences of previously decentralized (unfederated) data are lack of consensus or comparisons between feature annotations. Unifying or displaying alternative annotations should be a priority both for communities with robust entry representation and for nascent communities with burgeoning data sources. To this end, during this three-day continuation of the Virus Hunting Toolkit codeathon series (VHT-2), a new integrated and federated viral index was elaborated. This Federated Index of Viral Experiments (FIVE) integrates pre-existing and novel functional and taxonomy annotations and virus–host pairings. Variability in the context of viral genomic diversity is often overlooked in virus databases. As a proof-of-concept, FIVE was the first attempt to include viral genome variation for HIV, the most well-studied human pathogen, through viral genome diversity graphs. As per the publication of this manuscript, FIVE is the first implementation of a virus-specific federated index of such scope. FIVE is coded in BigQuery for optimal access of large quantities of data and is publicly accessible. Many projects of database or index federation fail to provide easier alternatives to access or query information. To this end, a Python API query system was developed to enhance the accessibility of FIVE. While the sharp reduction in the cost of sequencing over the past 15 years   continues to promote such efforts through the Science and Technology Research Infrastructure for Discovery, Experimentation, and Sustainability (STRIDES) Initiative, which “provides cost-effective access to industry-leading partners to help advance biomedical research” . These p7 reassembled contigs, from 2953 SRA entries, were produced  data from diverse biological sources. SRA serves as an umbrella for a variety of sequencing experiments  from different platforms  and applications. The first collaborative attempt to annotate and index SRA datasets in bulk was conducted as part of the inaugural of this series of events, the Virus Hunting Toolkit (VHT) . This fiIn contrast to traditional sequence data databases, federated indices do not store raw sequence data. Instead, federated indices store the results from different sequence analyses. For example, to identify putative novel viruses in SRA contigs, a federated index would store and link results from several analyses on these contigs, allowing researchers to quickly identify SRA contigs of interest without the need to perform the same extensive underlying analyses. Therefore, the output of FIVE is a collation of analysis results from different methods indicating if and where a sequence contains viral or virus-like signals, and not a single similarity score to a known virus sequence or structure. This novel resource indexes sequences, metadata, and hyperdata from the VHT-1 hackathon, sparse databases, and online resources. More than 2953 SRA entries assembled during VHT-1  were reak-mer distances. These methods were added to the extensive information in our previously published SRA annotations ), and otations . Known v as ViPR  or NCBI k-mer size = 6. A subset of 728 datasets was used to compare RPS-tBLASTN and Mash [hclust (stats package v3.5.3) [ade4 R package (v1.7.-15) [RF.dist function in the Phangorn R package (v2.5.5) [dendextend package (v1.13.4) in R (v3.6.2) [To improve the contig annotation index in FIVE, we evaluated 2953 datasets from VHT-1 ,13 againand Mash  to asses v3.5.3) . Correla1.7.-15) . Normali(v2.5.5) , and ent(v3.6.2) . Additio(v3.6.2)  was explA schematic of both pipelines can be seen in −10 and coverage threshold length ≥ 50 nucleotides.We matched CDD entries against the dataset using RPS-BLAST, with 6-frame translation (RPS-tBLASTn) . We filthmmscan) for searching for domains in short reads.In addition to RPS-BLAST and Mash, we tested the feasibility of domain detection using HMMER  directlyFIVE included an index for virus–host pairing (which viruses can infect which organisms). Initially, we federated a reference dataset of experimentally confirmed viral–host pairings, generating 22,896 known virus–host pairings from the NCBI Virus Variation Resource database  and suppThe federated index was expanded upon using CRISPR spacer connections. A large-scale CRISPR spacer database was generated from the initial federated index and four datasets generated using distinct sources: The first dataset of CRISPR spacers was CRISPRCasdb  as a test case for the integration of viral genome diversity into FIVE  (commit 48c4661), a nascent genome graph builder, as a backbone for FIVE’s own implementation . In shorlable at ). HIV-1 lable at , with roNetworkX (v2.4) [To balance sequence diversity with representative HIV-1 sequences, we used 170 HIV-1 reference genome sequences from the Los Alamos National Laboratory’s HIV Sequence Database  . We indexed data generated by working groups during the second codeathon (“Virus Hunting in the Cloud 2.0”) in a relational database format on Google Cloud’s BigQuery, called Federated Index of Viral Experiments (FIVE). This index can be visualized as distinct silos of the data generated by each of the analysis pipelines presented in this manuscript. Data was parsed , loaded One of the most important aspects of an index is accessibility. BigQuery is a relatively new framework and hence it was decided to generate a series of queryable actions, answering the most frequent research questions. To facilitate this research, a Python-based API was developed. This API, called viral-index (v0.0.3) , is freeA broad and detailed explanation of each method section can be found in the corresponding GitHub projects . Each prWe ran the RPS-BLAST pipeline on contigs derived from 2953 public sequencing datasets assembled in VHT-1  . Hit distribution varied enormously; some contigs had multiple CDD hits, peaking at 22,560 hits , but the majority of contigs (77.3%) had one unique CDD hit. The most common CDD was CDD:222853 .Among the 55,503,968 contigs that we searched against the viral specific CDDs , 10% of k-mer length of k = 21 (for both of its input sketches) retrieved almost no hits, so a length of k = 6 was chosen arbitrarily. We could not assess other k values (which may have yielded better estimations) due to time restrictions.In parallel, we tested the (meta)genomic distance estimation tool Mash  ,49 on prA representative subset of 728 datasets was used to compare the Mash and the RPS-BLAST pipelines. Out of 728 datasets, the Mash pipeline had an order of magnitude fewer hits  than with RPS-tBLASTn . The Mash pipeline was found to be substantially less sensitive than the RPS-tBLASTn analysis, with an average recall of 15.3% and a precision of 37.0%. Despite the low recall value, Canberra distance matrices retrieved from the Mash datasets and RPS-tBLASTn datasets were strongly correlated . Additionally, hierarchical clustering implied that, despite the loss of global structure in the dataset (as shown by a Robinson–Foulds distance of 0.91 and an entanglement of 0.2), the Mash pipeline can be used as a fast tool to quickly identify datasets containing roughly similar viral domains see . While t−3. On a node with 64 2.30 GHz cores, a total of 136 peptide sequences per minute were scanned for domain detection. In order to improve comparability, the same procedure was applied to search domains in a real, non-simulated sequence dataset . A total of 1000 randomly selected reads were extracted, translated into the six possible frames, and scanned for domains using the HMMER pipeline. For this dataset, 125 peptides were searched per minute, detecting 109 domains. Despite its ability to detect viral domains in short sequences, the HMMER pipeline performance did not scale well enough for datasets containing millions of reads over the course of three codeathon days. In order to deploy the HMMER pipeline for such large datasets, we propose the following: (i) collapsing identical or near-identical sequences to reduce redundancy; (ii) splitting translated frames yielding truncated peptides into distinct peptides, using stop codons as peptide boundaries; and, (iii) filtering amino acid sequences by length (≥50 amino acids) to decide whether to accept them as queries for domain detection. Applying these premises might still not be enough to yield reliable resources over the course of an event like a codeathon but might be sufficient to yield results under reasonable research time .The HMMER pipeline (hmmscan against Pfam-A v33.0) was applied to a simulated viral dataset of short reads. A total of 75 herpesvirus-specific domains were detected, with e-values ≤ 1 × 10To establish a baseline for known virus–host pairings, we federated several resources to act as references for experimentally confirmed and inferred interactions for the FIVE. In addition, we included queryable datasets designed to detect putative viral elements and linkable to a putative host. These putative pairings included phages and prophages mainly, which may help to understand prophage variability in well-categorized host systems. We federated existing databases providing information on hosts  and the identity of confirmed viral pairings from PhagesDB and the NCBI Virus Variation Resource database. The aforementioned resources were combined, expanded, and standardized to produce a comprehensive virus–host pairing index, containing 44,975 virus–host pairs  that can be queried from FIVE.Identified CRISPR spacers from four datasets  were curated and compiled into a comprehensive CRISPR spacer database, with 1 million unique spacer sequences linked to a formalized host taxonomy. CRISPR spacers were compared against the 29,847 unique viruses with known hosts identified in the virus–host pairing index. In addition, the CRISPR spacers were compared against 2953 raw datasets selected from NCBI’s SRA used in VHT-1 [k-mers from multiple sources like full viral genomes or virus segments. Examples of our HIV-1 genome graphs are shown in k-mer graph nodes. This metadata was later included in the individual graph nodes (not shown). The sequences were assembled into a graph using the Python3 package NetworkX and stored as a GraphML file that can be visualized and further analyzed using free open-source software such as Gephi [To demonstrate the ability of FIVE to index a wide variety of data types, we made genome graphs with HIV-1 reference genomes. Genome graphs facilitate the analysis of the diversity of a set of closely related sequences by counting and connecting as Gephi  or Cytosas Gephi ,53.Contig annotations and graph data derived from protein domain recognition, viral-specific HMM, virus–host pairing, and HIV-1 genome variability were produced in tabular format (as detailed at the end of each previous sections). Each table was loaded into the FIVE BigQuery Google Cloud index  to be quA range of different functions are implemented in the viral-index API module that enable easy access to FIVE. The viral-index can search the federated indexes by SRA run ID, virus and host taxonomy ID, and CRISPR spacer sequences  get_SRAs_where_CDD_is_found and (ii) get_domains, whereby users can retrieve (i) specific SRA studies where a virus-specific domain is present and (ii) the viruses that may contain a domain, respectively. The former function can be used to get a snapshot of virus domains in several SRA studies analyzed in VHT-1 [get_spacer_seqs function enables the users to fetch all spacer sequences present in the spacer datasets for a given taxonomy ID. The get_metadata_from_spacer_seq function retrieves spacer ID, spacer sequence, GenBank accession, and taxonomy identification of organisms where the given spacer sequence is present. An example, showing how to use the viral-index to retrieve all viruses that infect pigs, is provided at [Virus–host pairs can be searched using the in VHT-1 . Two addvided at .viral-index API enables researchers to interrogate increasingly sophisticated biological questions from FIVE through the multi-layer information available in this federated database indexes.It is important to note that data integration is becoming the norm; powerful analyses can be performed when it is possible to interlink data generated and enriched with multiple layers of known and novel information. The During this three-day continuation of the VHT codeathon series (VHT-2), a new integrated and federated viral index was elaborated. This Federated Index of Viral Experiments—FIVE—integrated new functional and taxonomy annotations, novel virus–host pairings, and for the first time, introduced virus genome diversity as genome graphs. Additionally, FIVE contains a federation of annotations and pairings from pre-existing sources. As per the publication of this manuscript, FIVE is the first implementation of a virus-specific federated index of such scope.Several metagenomic annotation pipelines were developed and tested, building on top of the foundations laid out in previous editions. Three pipelines for annotation of viral contigs through protein domains were proposed: (i) RPS-tBLASTn, (ii) Mash, and (iii) HMMER. Results showed differences in recall, accuracy, and speed between RPS-tBLASTn and Mash. RPS-tBLASTn may have been more computationally expensive than Mash, but it had better recall and overall accuracy. Additionally, as evidenced from VHT-1 , HMMER sWe made an additional effort to expand and federate not only the annotation tools for viral datasets, but also its taxonomical pairing with a given host. This original work expanded the number of known viral–host taxonomical pairings by 129% over VirHostNet 2.0 (release 1/2019) , by intek-mer settings and (ii) adding multiple metadata values to virus genome diversity graphs. Proper attachment of metadata is crucial for indexing datasets and ensuring that data is findable, accessible, interoperable, and reusable (FAIR) [k-mer nodes to component sequence annotations will further enhance the possibility to mine the structural information represented in graphs and to connect it to biological function. In the current state, creating genome diversity graphs is a convoluted process involving iterative testing of multiple parameters and visual inspection of their resulting graphs. Visualizing multiple metadata layers simultaneously becomes challenging and, ultimately, the manual analysis of multiple and complex graphs becomes an unfeasible task. It will be important to develop automated assessments of virus diversity graphs to adjust construction parameters, develop visualization methods for multiple metadata values, and create methods to automate graph analysis. Input from viral genomics is needed in order to standardize a genome diversity graph format.One of the last challenges during the codeathon was to start developing a pipeline and indexing strategy for virus genome diversity. It is known that genome graphs can be used to efficiently summarize known virus genome diversity; thus, as a proof-of-concept, we decided to build an HIV-1 genome diversity graph to index the variability into a federated index, such as FIVE. The two main challenges were (i) finding appropriate e (FAIR) . Metadatde facto used by a larger part of the research community (possessing basic scripting abilities). Accessibility and ease of implementation are often the limiting factors for the broad use of public resources. A graphical user interface (GUI) is under discussion to further broaden accessibility. The final aim is to link FIVE to other widely used viral and host resources, such as those supported NCBI, centralizing the resource and improving its connectivity to other services. Efforts are underway to maintain FIVE through annual updates and continuous federation.Annotations and metadata from the different projects were integrated into the FIVE BigQuery index and later made queryable, making it the first implementation of a virus-specific federated index that is easily accessible and queryable. As per the development of a Python-based API, the FIVE can be