Liquid-liquid phase separation (LLPS) is a rapidly growing research focus due to numerous demonstrations that many cellular proteins phase-separate to form biomolecular condensates (BMCs) that nucleate membraneless organelles (MLOs). A growing repertoire of mechanisms supporting BMC formation, composition, dynamics, and functions are becoming elucidated. BMCs are now appreciated as required for several steps of gene regulation, while their deregulation promotes pathological aggregates, such as stress granules (SGs) and insoluble irreversible plaques that are hallmarks of neurodegenerative diseases. Treatment of BMC-related diseases will greatly benefit from identification of therapeutics preventing pathological aggregates while sparing BMCs required for cellular functions. Numerous viruses that block SG assembly also utilize or engineer BMCs for their replication. While BMC formation first depends on prion-like disordered protein domains (PrLDs), metal ion-controlled RNA-binding domains (RBDs) also orchestrate their formation. Virus replication and viral genomic RNA (vRNA) packaging dynamics involving nucleocapsid (NC) proteins and their orthologs rely on Zinc (Zn) availability, while virus morphology and infectivity are negatively influenced by excess Copper (Cu). While virus infections modify physiological metal homeostasis towards an increased copper to zinc ratio (Cu/Zn), how and why they do this remains elusive. Following our recent finding that pan-retroviruses employ Zn for NC-mediated LLPS for virus assembly, we present a pan-virus bioinformatics and literature meta-analysis study identifying metal-based mechanisms linking virus-induced BMCs to neurodegenerative disease processes. We discover that conserved degree and placement of PrLDs juxtaposing metal-regulated RBDs are associated with disease-causing prion-like proteins and are common features of viral proteins responsible for virus capsid assembly and structure. Virus infections both modulate gene expression of metalloproteins and interfere with metal homeostasis, representing an additional virus strategy impeding physiological and cellular antiviral responses. Our analyses reveal that metal-coordinated virus NC protein PrLDs initiate LLPS that nucleate pan-virus assembly and contribute to their persistence as cell-free infectious aerosol droplets. Virus aerosol droplets and insoluble neurological disease aggregates should be eliminated by physiological or environmental metals that outcompete PrLD-bound metals. While environmental metals can control virus spreading via aerosol droplets, therapeutic interference with metals or metalloproteins represent additional attractive avenues against pan-virus infection and virus-exacerbated neurological diseases. The recent re-classification of the eukaryotic cellular phenomena of phase separation of protein condensates as the underlying mechanism creating membraneless organelles (MLOs) for cellular compartmentalization is initiated by a liquid demixing program. Liquid-liquid phase separation (LLPS) is an evolved cellular survival strategy mediating stress-triggered environmental sensing and also nucleates the cellular self-assembly processes of biomolecular condensates (BMCs) required for many cellular processes, including signaling, cytoskeletal organization, and transcriptional regulation ,2,3,4,5.Many different viruses employ LLPS to engineer BMCs used for their replication. Various virus family-centric terms and functions are used to describe these ‘viral replication compartments’ , ‘virosomes’, ‘virus factories’ (VFs), ‘viroplasm’, ‘mini-organelles’, ‘inclusion bodies’, and ‘negri bodies’ (NBs) ,28,29,30Gag promotes Gag and Capsid (CA) domain-resistant SGs causing translational arrest [2+-dependent HIV-1 NC domain is responsible for the positioning, trafficking, and packaging of the viral genomic RNA (vRNA) during virus assembly [2+- and ZnF-dependent LLPS condensates in vitro and in living cells [2+-dependent LLPS support a pan-retrovirus-wide model of virus assembly dynamics primed by LLPS [2+-dependent manner [We have recently demonstrated that the Nucleocapsid (NC) domain of HIV-1 pr55l arrest ,51, suppl arrest ,53. Morel arrest . The Zn2ng cells . Measlesng cells . In sili by LLPS . The SARt manner ,59,60,61PrLD-containing proteins are central to dynamic protein interaction network coordinating hubs . Crystal2+ and ZnF motifs within PrLDs [2+-dependent NC condensate model may be extended to represent a fundamental underlying mechanism nucleating virus replication, and an untapped avenue for pan-virus pharmacological targeting. To elucidate a common mechanism controlling and linking LLPS, SGs, neurological aggregates, and virus assembly dynamics, we performed literature surveys demonstrating that these assemblies are promoted and inhibited by cellular, physiological, and environmental exposure to Zn2+ and Cu2+. Our findings are supported by reports that modulation in Cu/Zn ratios and altered expression of proteins maintaining cellular and physiological ion homeostasis are hallmarks of both virus infections and neurological diseases. We and others have contributed to the greater body of literature promoting the theory that towards their own replicative benefit, underlying virus infections induce neurocognitive disorders and may exacerbate neurological diseases [Indeed, a core requirement of replication and budding of bona fide infectious virus particles is the specific binding of structural viral proteins to their cognate vRNA. Since vRNA binding for retrovirus Gag proteins is dependent on Znin PrLDs , we havediseases ,70,71,72http://plaac.wi.mit.edu/), Predictor of Natural Disordered Regions  [http://mobidb.bio.unipd.it/) were used to identify and validate the positions of PrLDs in viral proteins. FASTA input sequences were obtained from the NCBI protein sequence database and analyzed using software default parameters, with VLXT and VSL2 output styles selected for PONDR, and regions having a >0.8 PONDR Score were mapped using Adobe Illustrator software. Mapped ZnF and RRM placements were determined using the NCBI protein sequence database, from supporting references listed in https://www.genome.jp/tools/motif/) and Prosite (https://prosite.expasy.org/) [http://phylot.biobyte.de) [http://itol.embl.de) [Prion-Like Amino Acid Composition  , and thesy.org/) , which wsy.org/) . Phylogrbyte.de)  and Inteembl.de) .This study did not generate new datasets or code. Protein sequences were obtained from the NCBI Reference Sequence Database, with Genbank IDs listed in Disordered metal-binding regions of proteins can be stabilized upon ion-binding for gain of structure and function . We hypoWe were interested to find which other viral proteins with functional equivalence to HIV-1 Gag in vRNA-binding and encapsidation and viral capsid architecture also possessed juxtaposing PrLDs, RRMs, and ZnFs, inducing metal-dependent condensates. In some cases where identification of functional orthologs of Gag was complicated by diverging viruses having vastly different replication intermediate steps, we broadened our inclusion criteria to accept viral proteins that bind viral RNAs, or viral proteins possessing characterized ZnFs.2+-dependence, or from their propensity to multimerize, phase-separate, and bind vRNAs [To map proximities of viral protein PrLDs, RRMs, and ZnFs, NCBI protein database amino acid sequences were analyzed using PONDR and PLAAC algorithms ,78,79,80nd vRNAs ,88,89,90nd vRNAs ,103,104.nd vRNAs ,107,108.2+ binding and pathogenesis. This virus’ ZnFs juxtapose C-terminal PrLDs as we have observed for other viral vRNA-binding proteins, while ZnF mutants have attenuated virus infectivity [The combinatorial features of PrLDs, RRMs, and ZnFs towards a chaperoned RNA-LLPS model nucleating virus assembly that we have recently described for HIV-1 is one that is dependent on the Gag NC domain  and may ectivity .High degrees of disorder thus represent an evolutionary asset providing ancient viruses, encoded by very little genetic material, a replicative advantage and protection against host-factor interference or immune recognition. Indeed, viral nucleocapsid (nucleoproteins) have also frequently been characterized as viral RNA or protein chaperones. Since RBD-containing PrLD proteins are those that nucleate SGs , to repl2+ binds to RRMs of other cellular proteins that phase-separate and assemble into SGs [In our previous work, we observed that although Spumaviruses do not have ZnFs, these, nevertheless, have an abundance of RRMs mapping to the same approximate positions juxtaposing conserved C-terminal PrLDs found for all retroviruses ,124. Theinto SGs . RRM reg2+ binding, mediate conformational changes of NC proteins, permitting binding to coiled nucleic acid grooves.Viral proteins that undergo phase separation bind vRNAs via RRMs or ZnFs within their PrLDs. Many cellular RNA-binding proteins  have arginine-rich, positively charged sequences  that drive LLPS in an ionic strength-dependent manner ,125. Pos2+, Cu2+, and magnesium (Mg2+) are the most common metal ions binding to viral proteins and participate in strand transfer during reverse transcription of the vRNA, nucleic acid annealing and integration, transcription, and vRNA maturation [Among the many roles of metal ions in biological processes, these bridge interactions between distant residues of protein domains mediate protein–ligand interactions and serve as nucleophilic catalysts of enzymatic active sites. Although many biological processes are historically accepted as metal-ion dependent, a growing body of knowledge also demonstrates that metals can be interchangeable with certain cellular mechanisms. Metals are an integral part of viral proteins and play important roles in their survival and pathogenesis. Znturation . The impturation .2+, positively influence phase separation [2+, Ca2+) negatively influence PrLD–RNA interactions and reduce LLPS, as does the metal chelator EDTA [2+ is the presumed metal binding ZnFs, ZnFs also coordinate numerous other metal ions . As thed in [2+ . Copper-d in [2+ . Cu2+ haunctions .2+-binding prevents Aβ protein β sheet conformation and aggregation [2+ binding to Aβ is more kinetically favored, and Cu2+ can bind to previously self-aggregated Aβ [2+-binding, on the other hand, induces spherical Aβ structures different from β sheet structures [2+ concentrations inhibiting Zn2+-induced aggregates [2+ and Zn2+ bind to the same Aβ histidine residues, where Zn2+ precipitates at least two peptides to induce spherical structures, whereas Cu2+ outcompetes Zn2+ to inhibit intra-protein contacts [2+), Zn2+ alone could induce Tau protein condensates, another prime causative agent of neurodegenerative disease deposits [2+ and Cu2+, with both metals inducing structural changes and decreased solubility [2+-binding induces protease resistant variants [Aβ protein has been extensively studied as a prime causative agent of neurodegenerative disease deposits . Importantly, a number of different metal ions associate with monomeric Aβ to induce its aggregation but individually cause measurable variations in the types of Aβ deposits they induce ,133. Indregation ,134 and regation ,136. Mosgated Aβ . Zn2+-biructures . Early egregates . It was contacts . Indeed,deposits . The cellubility  and difflubility ,142, whevariants ,144. Relvariants . Indeed,variants , modulat2+ versus circulating Zn2+ levels in virus-infected patients. Indeed, Zn2+ is critical for innate and adaptive immunity [2+ deficiency correlates with diminished CD4+ T cells, high viral loads, AIDS, and mortality [2+ deficiency, virus replication requires Zn2+ [An interesting conundrum is exposed in the examination of the requirement of viruses for Znimmunity  and its immunity ,149,150,immunity ,152,153.ortality ,158,159,ortality ,163,164.res Zn2+ ,165.2+ is the most common viral protein cofactor assisting numerous replication processes of both RNA and DNA viruses [2+ also confers proper folding and varied functions to HIV-1 Integrase and Tat and Vif proteins [2+ ejectors [Zn viruses . In addi viruses , Zn2+ alproteins ,175,176.ejectors ,193,194.2+ deficiency is also common to infection by numerous other viruses, and Zn2+ supplementation as an antiviral therapy has been clinically tested against many divergent viruses, including herpes simplex virus (HSV), rhinovirus (RV), influenza, human papillomavirus (HPV), HIV-1, and hepatitus C virus (HCV) [2+-deficient plants are also more susceptible to diseases. Zn2+ deficiencies result from infection by turnip yellow and tobacco mosaic viruses, and plants supplemented with Zn2+ have ameliorated responses to pathogenic diseases [2+ was first reported to inhibit RV in 1974 [2+ against coronavirus, encephalomyocarditis virus, foot and mouth disease virus, HCV, HSV, HIV-1, HPV, Rous sarcoma virus (RSV), Semliki Forest virus (SFV), Sindbis virus, vaccinia virus, varicella-zoster virus, HEV, and arteriviruses [2+ concentrations tested exceed safe physiological ranges associated with clinical testing [Znus (HCV) . Zn2+-dediseases ,196,197. in 1974 , and zin in 1974 . Numerouiviruses ,201,202. testing ,204, pro testing ,206.2+ both promotes and inhibits virus replication, while its deficiency is associated with increased viral load, suggesting that there are major gaps in our understanding of how this metal influences viruses. While Zn2+ is required for virus replication, its chelation inhibits viral proteins  and virus replication , and Japanese encephalitis virus (JEV)) [2+ chelators, such as TPEN -1,2-ethanediamine) and 1,6-hexanediol, other drug classes with better safety profiles, including bananins, may be effective against HIV-1 and other viruses, including coronaviruses [2+ ionophores, such as pyrithione and chloroquine derivatives, among others, have also recently been shown to inhibit diverse groups of viruses, such as HSV, DENV, and SARS-CoV-2 [It is puzzling that Zns (JEV)) ,209,210.aviruses ,212. Zn2RS-CoV-2 ,214.2+- and Cu2+-regulating and -regulated cysteine-rich metallothioneins (MTs) [2+ imbalances are also present in human cancers and are also linked to regulation of HPV infection [2+ by the HPV16 E7 protein produces a compact environment leading to its self-assembly into spherical oligomers similar to those found in amyloids, whereas Zn2+ depletion results in loss of its aggregation [2+-dependent soluble agglomerates, whereas the metal chelating agent EDTA stabilizes its monomeric form and destabilizes existing agglomerates [2+ complexes also cause E6 aggregation, inhibiting its function [2+ and be regulated by Zn2+ [Another piece of the puzzle may involve Znns (MTs) . Despitens (MTs) . Metallonfection . As withnfection ,218. Notregation ,220,221.omerates , and whefunction . Recent function , a prote by Zn2+ .2+ and Zn2+-chelation both negatively affect viruses may be explained by differing research aims or experimental models from past reports. If virus biogenesis and assembly is Zn2+-dependent, then pre-formed virus capsid structure may be challenged by higher external concentrations of Zn2+ outcompeting virus-internal Zn2+, just as Zn2+ chelation would. In addition, if metal ions ultimately regulate virus core assembly (and stability) nucleated by metal-induced condensates during late stages of virus replication, then virus cores at early stages of reinfection should also be susceptible to metal ions or their chelation. Indeed, another effective antiviral agent, the zinc-finger-reactive disulfide NSC20625, ejects Zn2+ from JUNV Matrix protein RING finger motif, causing incomplete virion uncoating and release of NC into the cytoplasm [2+ bound to highly conserved ZnFs of influenza virus M1 matrix proteins is described as the determining factor of conformational transition of capsids in acidic environments, leading to their uncoating [2+ cause virus uncoating by destabilizing Zn2+-bound M1 protein [2+, leading to increased virus permeability [Confounding observations that both Znytoplasm . Zn2+ boncoating . Similarncoating . Upon in protein . Poliovieability .2+, ultimately regulating virus core assembly and stability, susceptibility of cell free viruses to environmental metal ions or their chelation should be evaluated. Indeed, virucidal agents ejecting Zn2+ from NC to inactivate HIV-1 have been developed for use as topical microbicides [2+ and Zn2+ also show virucidal activity against HIV-1 and other enveloped viruses [2+ ions and Zn2+ chelators inactivate viruses. Landmark studies relevant to the current COVID-19 pandemic tested a range of surfaces to find that copper alloys and Cu/Zn brass surfaces inactivated replication and propagation abilities of SARS-CoV [2+ is found to synergize with Cu2+ in surfaces and to increase efficacy of brass surfaces with lower percentages of Cu2+, suggesting that these ions destabilize capsids for viral genome destruction [2+ ion that affects shell stability [2+ and is also influenced by Cu2+ [2+ metallopeptidase angiotensin-converting enzyme 2 (ACE2) entry receptor, and high numbers of Zn2+-binding cysteine residues within these regions may also contribute to increasing binding affinity of the S protein to ACE2 [To determine how viruses are affected by exogenous Znobicides . Non-tox viruses . Thus, bSARS-CoV ,230 and SARS-CoV . Norovirtruction ,231. Imptability . Indeed,tability ,233. Imp by Cu2+ . Intrins to ACE2 ,235,236.2+ supports capsid assembly and structure across viruses through its interaction with ZnFs within PrLDs, difficulties in identifying effective therapeutics interfering with ZnFs or PrLDs for the elimination of virus condensates lead us to consider additional ways to control Zn2+-dependent viral condensates by examining upstream physiological or cellular regulating processes. From reports above describing Zn2+ binding site competition by other ions, such as Cu2+, that interfere with the establishment of condensates, below we describe alternative routes to control pathologic forms of LLPS, as supported by ancient evolutionary protein chaperone pathways.Despite the many instances demonstrating that Zn2+) has been used to disinfect fluids, solids, and tissues for centuries. Cu2+ was discovered by the ancient Greeks in the time of Hippocrates (400 BC), who used it to purify water and treat pulmonary disease. For their anti-fouling properties, Cu2+ vats were used to store holy water from the Ganges River, and Cu2+ strips were also used to construct ship hulls by the early Phoenicians. By the 18th century, Cu2+ was widely accepted for clinical treatment of mental disorders and pulmonary diseases. Both early American pioneers and Japanese soldiers of WWII placed Cu2+ coins to sterilize their drinking water, and NASA Apollo flights used Cu2+-based water sterilizing systems. Cu2+ has also historically been used by Africans and Asians to treat skin diseases [2+ is highly abundant and safe, with 1 mg consumed daily and excess copper released by excretion. More modern demonstrations of Cu2+ safety come from widespread and long-term use of Cu2+-based intrauterine devices (IUDs) to which human tissues have low sensitivity [2+-based IUDs correlated with 50% lower incidence of cervical cancers [Copper  [2+ concentrations lower than that required for inactivation by ethanol and where Cu2+ preserved cell viability while completely inhibiting formation of syncytia and virus production [2+ caused its cysteine-dependent oxidation [2+ in a cysteine-dependent manner, which was most notably found to cause its aggregation [Copper is a well-established biocide used against viruses and many other pathogens. In 1964, Cuiophages , followe in 1971 . In 1974nst Cu2+ . In 1992and HSV) ,245. Duroduction . More prxidation . HIV-1 pregation .2+-containing filters also effectively neutralize HIV-1 in medium and breast milk and reduce cell-associated HIV-1 in a dose-dependent manner [2+-containing filters also reduce infectious viral titers of several DNA and RNA viruses, including YFV, influenza A virus, MeV, RSV, adenovirus type 1, and cytomegalovirus [2+ in water pipes synergize with low levels of free chlorine has proven effective in inactivation of poliovirus, bacteriophage MS-2, hepatitis A virus, human rotavirus, and human adenovirus [2+ reduced survival and infectivity of waterborne viruses poliovirus, coxsackie virus types B2 and B4, echovirus 4, and simian rotavirus SA11 by 95% [The use of copper in free flow filters deactivates HIV-1 and West Nile virus, reducing infectious titers of these viruses by 5 to 6 logs . Cu2+-cot manner . Cu2+-coalovirus . Likewisenovirus ,251. The1 by 95% . Finally1 by 95% .2+ alloys can rapidly and effectively kill a wide range of microbial pathogens at a range of temperatures and under various conditions of humidity [2+ alloys is useful in preventing secondary transfer from surfaces in clinical facilities and other closed environments, including long term care facilities, public washrooms, cruise ships, and casinos [2+ in hours, while it can remain infectious on other surfaces for days [2+ surfaces [2+ also possess broad-spectrum anti-microbial and antiviral properties [Non-toxic, hybrid cured coatings containing copper also have virucidal activity against HIV-1 and other enveloped viruses . The rishumidity . Clinicahumidity ,256,257. casinos . As withfor days . This hafor days . Manufacfor days , also insurfaces . Textileoperties .2+ for their functions, while others bind to and control the uptake and delivery of Cu2+ for cellular and physiological processes. Cytosolic Cu-Zn-superoxide dismutase (SOD-1) is a ubiquitous cytosolic homodimeric isoenzyme that scavenges and catalyzes the dismutation of superoxide radicals [2+ and Zn2+ and leading to distorted SOD-1–SOD-1 interactions, leading to formation of insoluble aggregates described above [2+ chaperone [A number proteins are bound by Curadicals . Severaled above —a phenomed above . Intrigued above , entomoped above , and baced above , encode ed above . All chaed above  but may haperone . Modulathaperone ,270. Cu/haperone .2+-binding glycoprotein [2+, accounting for more than half of plasma Cu2+ [2+ from the liver and delivers it directly to cells for the synthesis of 40–70% of Cu2+-binding proteins and enzymes of various organs and tissues [2+ attributed to CP [2+ to the sites of infection to attack invading pathogens with Cu2+ toxicity [2+ homeostasis, may exacerbate the LLPS underpinnings of viral and neurological diseases.Ceruloplasmin (CP) is another Cuoprotein , mostly sma Cu2+ ,274,275. tissues ,277. A ced to CP ,279,280,toxicity . CP exprtoxicity . CP is atoxicity , and remtoxicity . CP is atoxicity ,285. Inctoxicity  and Alzhtoxicity , where itoxicity  and whertoxicity . These a2+ deficiencies associated with several diseases [Imbalances in homeostasis of physiological metals are important disease biomarkers. Plasma Cu/Zn ratios represent a common clinical assessment for Zndiseases . Elevatediseases , contribdiseases . It alsodiseases ,293 but diseases ,297,298.diseases . Signifidiseases ,301 and diseases . Increasdiseases .2+, inducing its beta-sheet conformation and aggregation [2+ and nucleic acids [sc) is induced by HIV-1 infection [2+- or Zn2+ -binding also induces PrP fold variants [2+ and Cu2+ to PrP is accompanied by structural changes and decreased solubility, where Cu2+-binding causes protease resistant PrP conformations [Fatal neurodegenerative prion diseases are caused by proteinaceous infectious particles, or “prions”. Prion disease is caused by the accumulation of abnormally folded isoforms of the cellular prion protein , where the unstructured amyloidogenic region of PrP preferentially binds to Curegation . Recent regation . Antimicic acids ,306. PRNic acids ,310,311,ic acids . PrP is ic acids ,314. Notnfection ,315. As variants ,142, sugrmations ,143,144.2+ and Cu2+ metal ions differentially influence formation of BMCs and protein aggregates of neurological diseases, and then extends to how viral infections induced metal imbalances may exacerbate neurological diseases (We were interested in gaining a better understanding of mechanistic intersections and fundamental features of proteins commonly associated with pan-viral engineered BMCs and neuropathologies. To propose a simplified model illustrating how viral infections may exacerbate neurological diseases, a bioinformatics-coupled meta-analysis system biology framework investigated how cellular, physiological, and environmental metals may govern both essential BMCs and pathological protein aggregates. The model illustrates how physiological Zndiseases A. Such i2+ bioavailability to inactivate viruses, possibly by outcompeting Zn2+-dependent processes during both early and late stages of virus replication. In early stage virus binding, entry, and uncoating, high Cu2+ concentrations may prematurely disrupt pre-formed virus capsid integrity to cause premature vRNA exposure and its subsequent degradation. In late stages of virus replication, high Cu2+ concentrations may interfere with the biogenesis of Zn2+-mediated viral BMCs required for nuclear transcription, export, translation, and trafficking and vRNA packaging by NC proteins. Indeed, many more viral proteins than those analyzed in 2+ [2+ concentrations. High Cu2+ that outcompetes Zn2+ for correct folding, activity, or multimerization of essential cellular proteins as BMCs may rather nucleate their aggregation as insoluble pathological plaques of neurodegenerative diseases (The model first illustrates how virus infections cause increases in both Cu/Zn ratios and expression of ancient antimicrobial prion proteins, such as Aβ protein. For one, this suggests that an evolutionary conserved anti-viral mechanism promotes heightened Cued in 2+ ,175,176.diseases A.2+ supplementation has been successfully used as treatment against many viruses [2+ supplementation is also described as a paradigm-shifting practice for neurodegenerative disease associated with Zn2+ and/or Cu2+ homeostasis abnormalities [2+ for BMC formation regulating many cellular processes, suggests that prolonged altered Cu/Zn ratios may represent an underappreciated and underlying feature of diseases (2+ metalloproteins and transporters are also modified by viral infections [2+ supplementation may assist in resolving its associated morbidities.Indeed, virus infections have been shown to exacerbate neurodegenerative disease phenotypes ,70,71,72 viruses , Zn2+ sumalities . Elevatediseases . In addifections ,326,327.2+. Cu2+ has historically been an easily procured effective antimicrobial and its anti-viral usage is supported by many demonstrations that this metal effectively inactivates all viruses tested. The current COVID-19 pandemic highlights an urgent need for methods reducing virus transmission. Mandatory face masks protect against virus aerosol droplets, but with evidence of SARS-CoV-2’s survival on certain surfaces for hours to days, and with copper surfaces showing the fastest inactivation [2+ coating of reused face masks and shields may be more protective (2+ mediates virus inactivation is by its outcompeting virus-internalized structure-lending Zn2+, leading to premature virus uncoating and degradation of the consequently exposed vRNA (2+ coatings to touch surfaces may reduce transmission of current and future dangerous viruses while targeting drugs or vaccines become approved and available. Considering the global economic arrest caused by COVID-19, simpler strategies such as Zn2+ supplementation or Cu2+ surface coatings may keep future pandemics at bay.The model extends itself to propose countering viral transmission using environmental Cutivation , Cu2+ cootective B. Finallsed vRNA C. Theref


The analysis of the economic burden for childhood poisoning has great value in Morocco where there still exists a paucity of information on the subject. The objective of this study was to explore the economic costs of unintentional and intentional poisoning in children in the region of Rabat-Salé-Kénitra, Morocco.A prospective study of children younger than 15 years with a poisoning diagnosis conducted between March and July 2016 in the Children’s University Hospital of Rabat, Morocco. The source of data for this study was questionnaire that collected information on the costs, the epidemiological and the socio-economic characteristics of childhood poisoning.Eighty-seven patients were interviewed during the study period . The majority of poisoning cases (98.85%) were accidental and 1.15% were intentional. Drugs, snake bites and scorpion stings, carbon monoxide, food, plants, household products, illegal drugs, pesticides, petroleum and industrial chemicals products were caused the poisoning. Of all the hospitalized patients, 77 (88.5%) were admitted to the emergency department and 5 (5.7%) were admitted to the intensive care unit. The average direct medical costs and the average direct non-medical costs of providing poison treatment were USD 127 and USD 30 per child, respectively. Total average direct cost of USD 157 (127 + 30) represented 60% of the national minimum wage per month in Morocco. Total direct medical costs accounted for 80%, as against 20% direct non-medical costs. The mean ± SD length of stay (LOS) for children with poisoning was 2.15 ± 1.87 days with a range variated between 0 day and 10 days.Overall, this study confirms that the costs of childhood poisoning are not negligible costs in Morocco. Therefore, the prevalence and the costs of childhood poisoning can be reduced by monitoring an open communication between parents, the Poison Control Centre of Morocco (MPCC) and physicians in order to increase the vigilance of parents against the risks related to unintentional poisoning that can be prevented with more awareness. Poisoning has an important impact on health and well-being of children. It is associated with high rates of disability, death and significant economic costs , 2. In 2Health is a major element of human capital that impacts the economic outcomes. According to Baker and Stabile (2011), every child has a specific stock of health which decreases by diseases, injuries, mental and physical conditions , while pThe hypothesis assumes that the unintentional and intentional poisonings are related to significant social and economic burdens. Following the Institute for Health Metrics and Evaluation (IHME) contribution to the worldwide burden of diseases, the Global Burden of Disease study (GBD) has shown an overall number of 180,000 deaths in 2017 due to the unintentional poisoning . SmartRiThis study explores the direct costs of poisoning among children from patient perspective in the Rabat-Salé-Kénitra region, Morocco. It attempts to describe the epidemiological characters and the costs of childhood as well as to compare these costs according to its different components and the type of hospital admissions.This is a hospital-based prospective study, the interview took place in the Children’s University Hospital of Rabat, Morocco. In total, one hundred cases with poisoning diagnosis were reached the hospital from March to July 2016 whether hospitalized or not, from which eighty-seven accepted to participate in the study. The face-to-face interview with parents was used to obtain data in order to lead quantitative analysis that describes the epidemiological characters of childhood poisoning as well as direct medical costs  and direct non-medical costs . The Children’s University Hospital of Rabat is the only hospital for children with a regional vocation.During the study period, eighty-seven children younger than 15 years, with poisoning diagnosis attended the hospital were eligible for inclusion in the study. Children were excluded from the study if the parental consent was not obtained. A written consent for participation was individually distributed to parents before the process of data collection in order to ensure confidentiality and anonymity of participants.The ethical standards of the study were approved by the Ethics Committees for Biomedical Research (CERB), Faculty of Medicine and Pharmacy of Rabat; clearance number-IORG0006594. Informed consent was obtained from all participants. The respondents provided signed consent form before their participation to the survey. Each potential respondent was properly informed of the questionnaire objectives, the related aspects to the study and the research procedure. The follow-up survey was carried out during the entire length of hospital stay.The collection of data started from the child’s arrival to their departure from the hospital. In the current study, the children interval of age variate between [0–15[ instead of [0–18[ since during the study period no adolescent with poisoning diagnosis was received to the hospital.The questionnaire aimed to underline the importance of poisoning burden in Morocco. The first section collected the sociodemographic characteristics of patients and their companions . The next section of the questionnaire concerned the clinical data filled out every day during the length of hospital stay. The final section of the questionnaire explored the direct costs incurred during the length of hospital stay. Indirect costs are out of the scope of this study.The average medical cost per poisoning child was calculated based on all the expenditures incurred including . The average non-medical cost per poisoning child was calculated based on all the expenditures incurred including  . MoreoveCosts presented in tables as means with standard deviations (SD). All data were manually recorded before being registered and analysed using the IBM SPSS Statistics version 25.A total of eighty-seven poisoning children attended the hospital during the period of the study . Children less than five years presented 67% of poisoning cases with a sex ratio (M/F) of 1.2. A proportion of 71% of cases occurred in urban areas against 29% in rural areas and they were mainly from the Rabat-Salé-Kénitra region. In this study, patients were mainly of low socio-economic status with a percentage of 64.4% against 32.2% of middle socio-economic status.Table Of all the hospitalised patients, 77 (88.5%) were admitted to the emergency department and 5 (5.7%) were admitted to the ICU. All poisoning cases admitted to the hospital didn’t contact the Poison Control Center of Morocco (MPCC), while 32.2% of parents proceed with self-care of their children, 8% consulted a physician and 1.1% consulted traditional healer.The different laboratory tests administrated according to the patient state were: complete blood count (CBC), biochemical tests, the c-reactive protein (CRP) test, lactate dehydrogenase (LDH), glutamic-oxaloacetic transaminase (GOT), glutamate-pyruvate transaminase (GPT), prothrombin time (PT), bilirubin, reticulocyte test, and triglyceride test. While, the functional and imaging tests included electrocardiography (ECG), brain CT scan, chest radiography and plain film of the abdomen.Table Table Table Of the admitting children, we found that drugs, snakebites and scorpion stings, plants and petroleum products poisonings were associated with the longest LOS which varied between 6 and 10 days of hospital stays.To the best of our knowledge, this is the first study in Morocco that evaluated the direct costs of childhood poisoning by estimating the medical and non-medical costs. In Morocco, all the articles published on poisoning analyse and discuss the morbidity impact of patients and neglect to quantify the economic burden of poisoning.In the results of this study, the average length of stay for children with poisoning diagnoses was 2.15 ± 1.87 days with a range variated between 0 day and 10 days of hospital stay and 24% of all children had a length of stay equal to or longer than 3 days. In an Australian study, Lee and Colleagues reported that the inpatients admitted to a combined general medicine and toxicology services experienced a shorter length of stay than patients not consulted by toxicologists . DiffereThe total burden of poisoning in this research was estimated to USD 13,620. Direct medical costs accounted for 80% against 20% of direct non-medical costs. The average direct medical and non-medical costs of providing poison treatment were respectively USD 127 and USD 30 per child’s . The average direct cost of poisoning USD 157 represented more than half (60%) of the national minimum wage per month in Morocco, and it is almost the same as the cost reported in Nigeria (USD 168) [From this study, 80% of companions were economically inactive . Thus, the average number of work-loss days and the productivity losses associated with the children poison treatment were not estimated. It should be recognized that, in general, the indirect costs of poisoning can merely be equal to the direct costs or more, especially in the case of deaths related to poisoning.In the international literature, different countries mainly in the developed countries have evaluated the cost of poisoning by estimating the direct and indirect related costs. Whereas, our study refers only to the direct costs of poisoning, the importance of the indirect costs has been emphasized internationally and exceed direct costs worldwide. In the USA, medical spending for poison treatment totalled USD 3 billion in 1992 . In CanaThe results obtained in this study reflect only the poison treatment during the length of stay from arrival at the hospital until departure and do not include the indirect costs of parents’ absenteeism from work or the costs occurred before that patients were referred to the hospital and after their discharge; for example, in cases of the psychotherapy sessions that the children needed to get after suicide attempts. Therefore, future study that takes into consideration the indirect costs related to childhood poisoning would be beneficial, as the costs explored in this study might have been underestimated because the indirect costs have not been taken into consideration.Considering that the cost of poisoning has not been estimated before in Morocco, this study showed that the childhood poisoning associated with significant costs. Yet, the prevalence and the costs related to the unintentional childhood poisoning can be prevented by improving the parents’ awareness, parents’ socioeconomic status and parents’ literacy.


In context of the End TB goal of zero tuberculosis (TB)-affected households encountering catastrophic costs due to TB by 2020, the estimation of national prevalence of catastrophic costs due to TB is a priority to inform programme design. We explore approaches to estimate the national prevalence of catastrophic costs due to TB from existing datasets as an alternative to nationally representative surveys.We obtained, standardized and merged three patient-level datasets from existing studies on patient-incurred costs due to TB in South Africa. A deterministic cohort model was developed with the aim of estimating the national prevalence of catastrophic costs, using national data on the prevalence of TB and likelihood of loss to follow-up by income quintile and HIV status. Two approaches were tested to parameterize the model with existing cost data. First, a meta-analysis summarized study-level data by HIV status and income quintile. Second, a regression analysis of patient-level data also included employment status, education level and urbanicity. We summarized findings by type of cost and examined uncertainty around resulting estimates.Overall, the median prevalence of catastrophic costs for the meta-analysis and regression approaches were 11% (interquartile range [IQR] 9–13%) and 6% (IQR 5–8%), respectively. Both approaches indicated that the main burden of catastrophic costs falls on the poorest households. An individual-level regression analysis produced lower uncertainty around estimates than a study-level meta-analysis.This paper presents a novel application of existing data to estimate the national prevalence of catastrophic costs due to TB. This type of model could be useful for researchers and policy makers looking to inform certain policy decisions; however, some uncertainties remain due to limitations in data availability. There is an urgent need for standardized reporting of cost data and improved guidance on methods to collect income data to improve these estimates going forward.The online version of this article (10.1007/s40273-020-00898-3) contains supplementary material, which is available to authorized users. Tuberculosis (TB) remains the leading cause of death from a single infectious agent worldwide, with 10 million people falling ill and 1.2 million people dying from TB in 2018 .In recognition of the impact of the costs of illness on households, the World Health Organization (WHO) has highlighted reduction of catastrophic costs due to TB as one of three priority targets for 2020 . Costs dTo help track country progress against this goal and inform programme planning, the Global TB Programme has developed guidelines for the conduct of nationally representative cross-sectional surveys to estimate the prevalence of catastrophic costs . HoweverIn many settings, data on patient costs of TB have been collected as part of trials or other smaller-scale projects; however, recent systematic reviews of patient-incurred costs due to TB observed large heterogeneity in the quality of reporting as well as the methods used to collect cost data and measure income loss , 10, 11.We created an individual-level deterministic cohort model that simulated progression through the TB care cascade in order to estimate the prevalence of catastrophic costs in South Africa Fig. . The modWe collated all research articles reporting any estimates of patient-incurred costs due to TB in South Africa from the Unit Cost Study Repository (UCSR) of the Global Health Cost Consortium . PatientWe reconciled timeframes for cost data by identifying the treatment start date, interview date and recall period for each participant. Direct out-of-pocket costs incurred in each treatment phase (intensive and continuation phase) were categorized as direct medical costs , direct non-medical costs  and food costs . Cost estimates were distinguished by treatment phase (intensive and continuation phase) and by type of healthcare provider, including public healthcare (PHC) facility (study site), another PHC facility (non-study site), private general practitioner, pharmacy, hospital inpatient service, hospital outpatient service, and traditional healer.All data in different studies were collected using adaptations of the Tool to Estimate Patient Costs , and thuAll costs are reported in 2017 US dollars. Data collected before 2017 were inflated using the US consumer price index . Prior tWe tested two approaches to estimate mean and standard error values for direct costs and hours lost due to treatment by household income quintile, HIV status and treatment phase: (1) meta-analysis of summary statistics from the standardized datasets; and (2) regression analysis of the pooled standardized dataset.Our first approach was meta-analysis to calculate pooled estimates of available (study-level) mean values for the above-described cost categories for each treatment phase, by HIV status and household income quintile . Given tOur second approach was to identify a regression model to predict the above-described cost categories for each treatment phase, by HIV status and household income quintile. Firstly, we imputed missing values in the pooled dataset. Where total consultation hours were missing, we used multivariate imputation with chained equations (MICE) to impute these values based on the number of visits by phase and provider type. Total travel hours and total direct non-medical costs were imputed based on number of visits and transport method, as well as demographic variables included in the regression analysis. All imputations used predictive mean matching (PMM), as a non-parametric alternative for imputing skewed data. Imputations generated 20 plausible datasets, which were then used for analysis. The number of missing observations by dataset are listed in ESM Table 3 .Following imputation, we conducted a series of regression analyses to predict the cost of each cost category for each treatment phase. The regression analyses used a generalized linear model (GLM) approach assuming a gamma distribution and a log link to accommodate skewed data . The speFollowing the regression analysis, marginal estimates for each of the above-described cost variables were obtained by HIV status, household income quintile and employment status, with urbanicity and education values held constant at the mean observed among people with TB in the NIDS dataset . To help us interpret the results of the regression analysis using the pooled dataset, we also tested the extent to which cost drivers varied across datasets. To do this, we conducted a regression on the unimputed data for each dataset separately. As there were insufficient observations to do this for the intensive phase, we conducted this test only on observations in the continuation phase.We sampled patient-incurred direct costs and total time per individual in the cohort following a gamma distribution based on the mean and standard error values determined in the analyses described earlier. Individuals that were lost to follow-up before treatment initiation were assumed to encounter only costs of special food and supplements, while those initiated onto treatment were assumed to also encounter direct medical, direct non-medical and indirect costs.Following prediction of total time spent accessing care, indirect costs were estimated using a human capital approach to value the opportunity cost of travel and consultation time whilst seeking care. This was calculated as the total hours lost multiplied by an approximation of individual income per hour, estimated assuming 220 working days per year and an 8-h working day. Indirect costs were assumed to be zero for those who were unemployed; however, we undertook a sensitivity analysis valuing costs for unemployed people using an equivalent individual salary for a similar person. Sensitivity analysis also tested inclusion of all treatment costs for those lost to follow-up. Due to a lack of data, we were not able to estimate indirect costs using an output approach (income loss due to illness), nor were we able to include costs incurred by other members of the household such as guardians or lay caregivers.Catastrophic costs were defined as where the total patient-incurred cost during treatment was greater than 20% of annual household income . For eacThree datasets were obtained and merged –29, resuTables The variation in demographics observed across datasets provided a motivation for pursuing a regression analysis, which allowed inclusion of other explanatory variables in estimation of costs and time associated with accessing care. The results for the regression analysis are listed in Tables Table Including all costs for those lost to follow-up, and valuing time for those unemployed, increased the prevalence of catastrophic costs in quintiles 1 and 2 in both the meta-analysis and regression approaches. Our estimate of the overall prevalence of catastrophic costs using the meta-analysis approach were robust in sensitivity analyses; overall estimates for the regression approach increased slightly in sensitivity analysis.We present estimates of the prevalence of catastrophic costs associated with TB, employing an individual-level cohort model using two approaches to parameterize cost estimates: a meta-analysis approach using study-level statistics and a regression approach using individual-level primary data. Overall, the median prevalence of catastrophic costs was estimated at 11% using a meta-analysis approach and 6% using a regression approach. Both approaches confirmed that catastrophic costs had a higher prevalence among the poorest quintile.Our analysis had several limitations, mostly related to limitations in the available data. Due to limited data availability, the scope of the analysis was restricted to costs incurred by DS-TB patients during treatment. We were also unable to include costs before treatment initiation, costs incurred by other household members or indirect costs associated with lost income due to illness as there were no data on these costs. These restrictions on the analysis are likely to result in an under-estimation of the true prevalence of catastrophic costs. Furthermore, as all studies collected data at the health facility level, we were unable to include any cost estimates for people unable to access care. This may exclude the most vulnerable households impacted by TB, and thus likely results in an underestimate of the economic burden of TB. Finally, we do not include funeral costs for TB-related deaths, as there is limited evidence on these costs.The meta-analysis approach produced higher estimates and wider uncertainty intervals than the regression analysis. This reflects the limitations of parameterization of a deterministic model using available cost and epidemiological data. We designed the model using the best available information on the TB epidemic in South Africa, using HIV status and income quintile to delineate TB prevalence and loss to follow-up rates. There was no available information on TB prevalence or loss to follow-up by urbanicity, unemployment or education level, and thus no way to include these effects in the meta-analysis approach.In contrast, the better-parameterized regression approach allowed us to obtain cost estimates that had been corrected for the additional independent variables of urbanicity, unemployment and education. As these three variables were found to have significant effects on cost, it is therefore not surprising that the estimates obtained through the regression approach resulted in reduced uncertainty. This also explains the reduced estimated overall prevalence using the regression approach. Rural setting and higher education level both had a negative effect on direct costs, neither of which were accounted for in the meta-analysis.Although the regression approach was better parameterized, our analysis indicates that better information is still needed on the household-incurred costs due to TB. Our findings on significant determinants of patient-incurred costs identified in the regression approach are largely supported by the existing literature , 36, 37;Given these uncertainties, our analysis indicates that access to patient-level data is vital for researchers looking to extrapolate existing cost estimates to national settings, at least until the cost function can be better defined. The importance of improving data sharing is being increasingly recognized by publishers and funders , 44, notOf course, primary data collection also comes with some degree of uncertainty. Although primary cost data are increasingly being made available, for example through mechanisms such as the Global Health Cost Consortium’s UCSR , the widDespite the above-discussed uncertainties, this type of model could be useful for researchers and policy makers. A cohort model such as the one presented in this paper can estimate the national prevalence of catastrophic costs due to TB and the uncertainty around these estimates, and can identify the comparative impact of TB-related costs on different sections of the population. It also has potential to inform certain policy decisions; for example, Verguet et al.  use a siThe usefulness of the type of analysis presented in this paper depends on the objectives of the analysis. This analysis may be sensitive enough to capture major movement towards the End TB goal of zero catastrophic costs due to TB; however, it is likely not sensitive enough to capture small changes from year to year—especially in settings where the cost function is still unknown or differs substantially in different settings. Ongoing primary data collection through national surveys is likely still necessary to facilitate annual reporting and programme management until the availability of cost and epidemiological data improves, and the cost function is better identified. However, while probably not providing quite as robust an estimate of catastrophic costs as a national survey, this type of analysis can complement, enrich and add depth to findings from the national surveys, especially for certain groups.This paper presents a novel use of existing data to estimate the prevalence of catastrophic costs due to TB . We findSupplementary file1 (DOCX 520 kb)Below is the link to the electronic supplementary material.


The coronavirus disease 2019 (COVID‐19) caused by the severe acute respiratory syndrome coronavirus 2 is now being a disaster, as of November 3, 2020, the World Health Organization have reported 46 million cases and 1.2 million deaths globally.We have collected the outbreak data from the field epidemiological investigation, the basic information and clinical characteristics were collected using EXCEL, and all throat swabs were collected during the investigation. All data generated or analyzed during the study were included in this manuscript.Mycoplasma pneumoniae, and Chlamydia pneumoniae, using the NxTAG Respiratory Pathogen Panel  according to the manufacturer's protocol.The throat swabs were tested for influenza A virus, influenza A (H1) virus, influenza A (H3) virus, influenza A (H1N1) pdm2009 virus, influenza B virus, respiratory syncytial virus, parainfluenza virus , bocavirus, human metapneumovirus, HRV, adenovirus, human coronavirus , Legionella, χ2 test and found that the rate of patients with runny nose was statistically higher in the HRV positive patients than that of in the HRV‐negative patients  of which were positive for rhinovirus (Table 7.87% of Most “common colds” were caused by HRV, which was reported to be the most common pathogen of upper respiratory tract infection, and also a common copathogen in patients with respiratory tract infection.The authors declare that there are no conflict of interests.Concept and design: Di Wu, Jianyun Lu, and Zhangyu Sun. Acquisition, analysis, or interpretation of data: Qun Liu and Tiantian Wu. Drafting of the manuscript: Di Wu and Qun Liu. Experiment: Lan Cao and Qing Zeng. Statistical analysis: Di Wu. Supervision: Zhicong Yang.


The glioblastoma multiforme (GBM) is one of the deadliest tumors. It has been speculated that virus plays a role in GBM but the evidences are controversy. Published researches are mainly limited to studies on the presence of human cytomegalovirus (HCMV) in GBM. No comprehensive assessment of the brain virome, the collection of viral material in the brain, based on recently sequenced data has been performed. Here, we characterized the virome from 111 GBM samples and 57 normal brain samples from eight projects in the SRA database by a tested and comprehensive assembly approach. The annotation of the assembled contigs showed that most viral sequences in the brain belong to the viral family Retroviridae. In some GBM samples, we also detected full genome sequence of a novel picornavirus recently discovered in invertebrates. Unlike previous reports, our study did not detect herpes virus such as HCMV in GBM from the data we used. However, some contigs that cannot be annotated with any known genes exhibited antibody epitopes in their sequences. These findings provide several avenues for potential cancer therapy: the newly discovered picornavirus could be a starting point to engineer novel oncolytic virus; and the exhibited antibody epitopes could be a source to explore potential drug targets for immune cancer therapy. By characterizing the virosphere in GBM and normal brain at a global level, the results from this study strengthen the link between GBM and viral infection which warrants the further investigation. We employed a data driven approach to identify virus species from the GBM NGS sequencing data. These virus could be potential novel therapeutic targets for cancer therapy. However, assembly of the viral genome has always been challenging due to the fast evolving and fragmented nature of the viral genome.In this study, we applied metagenomics approach to characterize the virosphere of GBM at a global scale and observed some novel viruses previously isolated only from nonhuman organisms. We also observed that the contigs matching the genome sequences of the herpes virus only make up a small portion of the whole viral genome. In addition, we identified some novel sequences with no known annotations. Further analysis showed that these sequences have the signature for antibody epitopes. These findings will provide novel avenues toward future GBM research and therapies.22.1https://www.ncbi.nlm.nih.gov/sra), Gene Expression Omnibus (GEO: https://www.ncbi.nlm.nih.gov/geo/), and PubMed literature to collect NGS studies relating to GBM and normal brain tissues. We also identified a set of samples infected with known viruses as our “positive controls” to test if our assembly approaches can detect these viruses from the sequencing data. We limited our study to the data generated from Illumina sequencing platform and the RNA‐seq data were downloaded from SRA database. The list of accessions for the source data are shown in Supplemental File 1.We searched the NCBI Sequence Read Archive (SRA: 2.2The raw reads in each study were first trimmed and checked using Trimmomatic (version 0.36)For the samples with known virus infections, the MEGAHIT was used for contig assembly, and the resulting contigs were compared with the reference viral genomes Figure . For bra2.3The contigs with length over 500 bp were annotated to known viruses references in both protein and nucleotide databases at NCBI via BLASTThe number of reads contributed to the assembly of each “viral” contig from each sample was calculated to ensure the assembly quality Figure  by mappi2.4https://www.ncbi.nlm.nih.gov/orffinder/). The minimal ORF length was set as 75, with any sense codon and standard genetic code applied. For each of the putative protein‐coding contigs, we applied TMHMM Server v. 2.0 to predict transmembrane domains.https://www.iedb.org/home_v3.php).The shared contigs that have no annotation from the above analysis are view in Venn diagram Figure .76 The33.1To validate our approach, we tested six samples with known viral infections as positive controls to evaluate our methods for viral sequence assembly. These six samples include Human herpesvirus 5 (double stranded DNA virus), Enterobacteria phage phiX174 (single stranded DNA virus), HBV (double stranded DNA virus with reverse transcription), Zika virus , Tick‐borne Encephalitis virus , and Influenza A virus H1N1 . These viruses cover major categories of different types of viruses to ensure the validity of our approach.After trimming and mapping the reads to human genome, the unmapped high‐quality reads from the positive controls were assembled via MEGAHIT. The assembly results from each virus infected samples were compared to its corresponding reference sequences. We observed that for each positive control, the assembled contigs can cover over 90% of the reference genome of the corresponding virus Figure . Five as3.2We collected 111 GBM and 57 healthy brain data sets from eight different projects. This large number of datasets ensures the quality of contig assembly . In total, there are 6609 M (Million) raw sequencing reads for GBM and 2681 M for healthy brain. The low‐quality reads and reads that map to human genome were then removed to yield 210.0 M high quality reads for GBM and 115.4 M for health brain. For each group, reads were pooled together and assembled with MEGAHIT and Trinity, respectively. Using N50, N90 and the number of contigs as a criteria, MEGAHIT performed better than Trinity in assembling the GBM RNA‐Seq reads  at NCBI as well as Swiss‐Prot. Among the 95 642 contigs assembled from GBM samples, 93 228 can be annotated by nt database, 55 200 are annotated by nr database, and 47 070 contigs are annotated by Swiss‐Prot database. Only 959 contigs cannot be annotated by neither of the three databases ; k141_34855 (length = 739); k141_14851 (length = 753); k141_47897 (length = 501)) that were annotated as EBV, the only herpes virus to be found with moderate length of contigs. However, the synteny analysis showed that they are mapped to the same small region of the EBV reference genome Figure . In cont3.4http://www.cbs.dtu.dk/services/TMHMM/) analysis to predict the transmembrane domains. Significant transmembrane domains were found in 31 unknown contigs from GBM and three unknown contigs from health brain. Among these transmembrane contigs, we found that the linear B‐cell epitopes were enriched and analyzed. Some of the contigs, such as k141_31618 assembled from 22 out of 110 GBM samples and k141_77976 from 33 of GBM samples, contains putative antigen epitopes , as this virus was first reported in invertebrate.The presence of EBV in gliomas has always been controversy.A number of contigs cannot be annotated by any databases we used. It is possible that those contigs are artificial or formed from artificial sequences such as vectors or contaminations. However, we observed that various samples from different projects have reads that can cover more than 60% of the contig. For example, over 60% of the length of contig k141‐31618 can be covered by the reads originated from 22 studies from six out of seven projects in the GBM group, making it evident that contigs like this are not contaminations but rather originated from a valid source. Transmembrane analysis and antibody epitope prediction show that significant amount of those contig sequences has antibody epitope sequence signature, suggesting a potential to be used as drug targets for cancer immune therapy.The authors declare that they have no competing interests.ZY, WJZ, and ZA conceived and designed the study, ZY and LZ performed the analysis, XY, LZ, NZ, ZA and WJZ made revisions, ZA and WJZ supervised the project. All authors support the publication of the manuscript.Supplementary MaterialClick here for additional data file.Supplementary MaterialClick here for additional data file.Supplementary MaterialClick here for additional data file.Supplementary MaterialClick here for additional data file.Supplementary MaterialClick here for additional data file.


The novel coronavirus (SARS-CoV-2) currently spreads worldwide, causing the disease COVID-19. The number of infections increases daily, without any approved antiviral therapy. The recently released viral nucleotide sequence enables the identification of therapeutic targets, e.g. by analyzing integrated human-virus metabolic models. Investigations of changed metabolic processes after virus infections and the effect of knock-outs on the host and the virus can reveal new potential targets.in-vitro experiments.We generated an integrated host–virus genome-scale metabolic model of human alveolar macrophages and SARS-CoV-2. Analyses of stoichiometric and metabolic changes between uninfected and infected host cells using flux balance analysis (FBA) highlighted the different requirements of host and virus. Consequently, alterations in the metabolism can have different effects on host and virus, leading to potential antiviral targets. One of these potential targets is guanylate kinase (GK1). In FBA analyses, the knock-out of the GK1 decreased the growth of the virus to zero, while not affecting the host. As GK1 inhibitors are described in the literature, its potential therapeutic effect for SARS-CoV-2 infections needs to be verified in https://identifiers.org/biomodels.db/MODEL2003020001.The computational model is accessible at  As it is shown that The methods used in this article are based on the article by 2.1Analogous to the biomass production or maintenance function in prokaryotic or eukaryotic metabolic models, the VBOF is a pseudo-reaction simulating the production of virus particles. It comprises nucleotides, amino acids and associated energy metabolites required for the production of the virus particles. Due to a lack of knowledge, the stoichiometric information of the virus envelope, and the dynamic information of lipids are not included in the VBOF. Hence, virus entry or exit cannot be modeled. The generation of the VBOF was performed following the seven steps described by Step 1: Virus genome and protein information. The recently published virus genome and protein sequence were obtained from the NCBI database  genomes via a complementary negative ssRNA. The nucleotide counts of the positive strand can be taken from the genome sequence. The nucleotides of the negative strand can be calculated by counting the complementary nucleotides of the positive strand. Both nucleotides are summed up to receive the total nucleotide count.SARS-CoV-2 has a total of 12 proteins, four structural proteins, and 8 NSPs. Structural and NSPs are not expressed equally, and this ratio needs to be considered during the calculation of the VBOF. To this date, no information about the copy number of structural proteins in Coronaviruses is reported.n System  that chaStep 2: Nucleotide investment. The nucleotide count of the virus genome and its replication intermediate are summed per nucleotide. The genome copy number (Cg) was assumed to be one. According to Aller et al., the moles of nucleotides were converted into grams of nucleotides per mole of the virus. After similar calculations of the amino acids and the calculation of the total molar weight of the virus based on nucleotide and amino acid content, the stoichiometric coefficients of each nucleotide in the VBOF were calculated.Step 3: Amino acid investment. Analogous to the nucleotide investment, the stoichiometric coefficient of each amino acid was calculated. Instead of the genome copy number, a copy number for structural and NSPs is required. As already mentioned, the copy number of structural proteins is not reported to date. Therefore, we repeated all analyses described in this article for copy numbers of structural proteins Csp=200, 500, 800 and 1200. With the information of the total count of each amino acid, weighted by the copy number of structural proteins, and the inclusion of the respective molar mass, the total viral molar mass was calculated  to eventually calculate the stoichiometric coefficient for every amino acid.Steps 4 and 5: Adenosine triphosphate (ATP) requirements, and pyrophosphate liberation. The calculations of the ATP requirements for the polymerization of amino acids and the calculation of pyrophosphate (PPi) liberation from the nucleotide polymerization were performed as described by Aller et al. based on the results of the previous steps. The constants et al. Steps 6 and 7: Total viral molar mass and final construction of the VBOF. The total molar mass of SARS-CoV-2 and the final VBOF were calculated and constructed in accordance with Aller et al.iAB-AMØ-1410 was utilized. This GEM was published in 2010 by Mycobacterium tuberculosis. The cell-specific GEM was constructed based on the human metabolic reconstruction Recon 1 , except for the metabolites for energy requirements. Then, the fold-change (FC) of the normalized amino acid or nucleotide was calculated:i over nucleotides or amino acids and k over all biomass precursors (except for energy requirements). Subscripts H and V represent the use of either the host or virus biomass function. Positive values imply higher usage of nucleotides or amino acids in the virus compared to the host, while negative values imply a lower usage.The human alveolar macrophage model iAB-AMØ-1410 model paved the way for the analysis of metabolic changes between host and virus-optimized states. To identify an optimal state, an objective function that is optimized needs to be defined. In general, every reaction in the GEM can serve as objective function. However, biologically meaningful objective functions depend on the research question. For analyses of the growth or survival of the studied organism or cell, biomass production or maintenance reactions can be introduced into the model as pseudo-reactions. In industrial settings, the production of a specific metabolite might be of interest, and hence its production reaction can be set as the objective function. The defined objective function is then optimized with flux balance analysis (FBA). FBA is a mathematical approach using linear optimization to analyze the flow of metabolites through a metabolic network while optimizing for an objective function  is a method to identify alternate optimal solutions. In FVA, the maximum and minimum possible flux for each reaction in the network is evaluated while constraining the objective value equal or close to the optimal flux  has an objective coefficient of 1. However, one can also set the objective coefficient of several reactions to 1 to optimize for both reactions. Hence, the objective coefficient of both biomass functions from host and virus was set to 1 to optimize for both functions. Last, we used COBRApy’s  and when the growth rate of the virus was below the growth rate of the host-optimized state.The first approach is a reaction knock-out that is already implemented in COBRApy. et al. The corresponding minimum (F–) and maximum (F+) flux values from the FVA of the host (H) and virus (V) optimization and their relation define the minimum  and RNA nucleotides, components for the energy requirements and others, such as fatty acids or phospholipids. In contrast, the VBOF only consists of amino acids, RNA nucleotides and the components for the energy requirements. The stoichiometric coefficients of amino acids and nucleotides are compared by calculating the FC. The result is visualized in Csp). The copy number of structural proteins for some viruses, such as Alpha- and Flaviviruses, is known and ranges from 180 for Flaviviruses  protein, membrane (M) protein, nucleocapsid (N) protein and the spike (S) protein  the host cell is not infected and the metabolic system is optimized for the already included maintenance biomass reaction (host optimization). This scenario reflects the normal physiological state of human alveolar macrophages; (ii) the host cell is infected by the virus and is optimized solely for the production of the virus particles (virus optimization). The host optimization results in a biomass maintenance flux of 0.0267 mmol/(gDW⋅h), while the virus optimization returns a flux of 0.0147 mmol/(gDW⋅h). When defining both the host maintenance and the virus biomass function as objective functions by assigning both an objective coefficient of one simultaneously, the model only optimizes for the host maintenance reaction while the flux through the VBOF is zero. Last, the optimization result of host and virus metabolism was compared using a new constraint. With varying percentages of the host maintenance reaction and the VBOF on the objective expression, the effect on the respective biomass maintenance or growth function was investigated. The model does not predict an equilibrium state, where both the host maintenance reaction and the VBOF are active. As displayed in The flux distribution for the host- and the virus-optimized states were compared using FBA and FC calculations. For 256 reactions , a FC was calculated. As expected from the stoichiometric analysis, reactions related to amino acid and nucleotide metabolism were altered. The main portion of changed reactions consists of transport reactions. However, also several reactions from other subsystems, such as the steroid, fatty acid synthesis and central metabolism, are altered. An overview of altered reactions concerning their FC is given in FBA and FVA can be used to identify metabolic targets for antiviral strategies by comparing the host- and virus-optimized states after alterations in metabolic pathways.The results from the stoichiometric differences and metabolic changes in alveolar macrophages after virus infections with coronavirus SARS-CoV-2 indicate alternative host- and viral-optimal states. The diverging flux distribution in the two states provides an opportunity for the identification of potential antiviral targets. To identify potential antiviral targets that limit virus production, we implemented two different analysis methods: (i) reaction knock-outs and (ii) host-derived enforcement. The reaction knock-out revealed exactly one reaction , whose knock-out reduces the flux of the virus biomass to zero, while maintaining the host biomass maintenance at 100%: The guanylate kinase (GK1) reaction that converts ATP and guanosine monophosphate (GMP) to adenosine diphosphate and guanosine diphosphate (GDP):This reaction is also observed as a potential target in the second analysis method, the host-derived enforcement. In this approach, the reaction fluxes are constrained to ranges derived from FVA. With flux ranges outside of the optimal state of the virus, the virus production is perturbed while the host maintenance is not affected. For structural protein copy numbers between 500 and 1200, we identified four possible targets, including the GK1 that reduced the virus growth flux to below a threshold of 80% of its initial value. Further potential targets concern the availability of the amino acids L-isoleucine (I), and L-lysine (K), either via the alteration of exchange reactions, or, in case of L-isoleucine, also via the L-isoleucine transporter. In contrast to GK1, where the flux through the reaction is down-regulated (or in case of the reaction knock-outs completely knocked out), the alterations of the mentioned exchange, and transport reactions go into the other direction: the uptake of the amino acids is enabled or even enforced, leading to the host’s maintenance while decreasing the growth of the virus to 50% of its initial growth. The enforcement of exchange reactions can be reached by a sufficient supply of the respective amino acid.If the host has enough L-lysine in its environment, the maintenance of the host cell is ensured, while the growth of the virus is diminished. We repeated the analysis that evaluates the effect on the growth rates based on the percental involvement of the two biomass functions within the setting of L-lysine excess. ILEtec). Enforcing a minimal import of L-isoleucine via this transport has the same effect as the other three targets and results in a diminution of 50% of the virus growth. All targets obtained from the host-derived enforcement for the structural protein copy numbers Csp=500, 800 and 1200 are summarized in Analogous to L-lysine, the availability of L-isoleucine and L-tyrosine has a positive effect on the growth of the host while decreasing the growth rate of the virus by 50%. The last potential metabolic target for antiviral strategies using the host-derived enforcement approach concerns one transport reaction of L-isoleucine  P5-(5’-guanosyl) pentaphosphate (Ap5G) to be a potent, bi-substrate inhibitor of GK1  and retroviruses (HIV-1)  signaling process. Dynamic analysis and numerical simulations led to the suggestion that a balance between viral replication and IFN-induced regulation is responsible for the dynamic behavior of virus-triggered signaling and also for antiviral responses  and lack a biomass maintenance function. Following the protocol for generati9.5 ng high-quality genome-scale metabolic reconstructions by Thiele and Palsson, meaningful models of human bronchial and AEC could be generated . These models can then be used to verify the potential antiviral targets that were found in alveolar macrophages.As enerated . HoweverThe integrated host–virus model suggested the supplementation of L-isoleucine and L-lysine as a potential target for antiviral therapies, as well as the inhibition of the GK1. Since compounds that directly inhibit GK1 are already known, their evaluation and verification in cell culture experiments are required for fast responses to the current spread of SARS-CoV-2 worldwide.This work was supported by the Deutsche Forschungsgemeinschaft (German Research Foundation) under Germany’s Excellence Strategy—EXC 2124—390838134 and was supported by the German Center for Infection Research.Conflict of Interest: none declared.https://identifiers.org/biomodels.db/MODEL2003020001.The computational model is accessible at


Phaeocystis globosa) can be discriminated from bacteriophages using flow cytometry, facilitating its use as a representative model system. Of all the non-host organisms tested, anemones, polychaete larvae, sea squirts, crabs, cockles, oysters and sponges significantly reduced viral abundance. The latter four species reduced viral abundance the most, by 90, 43, 12 and 98% over 24 h, respectively. Breadcrumb sponges instantly removed viruses at high rates (176 mL h−1 g tissue dry wt−1) which continued over an extended period of time. The variety of non-host organisms capable of reducing viral abundance highlights that viral loss by ambient organisms is an overlooked avenue of viral ecology. Moreover, our finding that temperate sponges have the huge potential for constant and effective removal of viruses from the water column demonstrates that natural viral loss has, thus far, been underestimated.Viruses are the most abundant biological entities in marine environments, however, despite its potential ecological implications, little is known about virus removal by ambient non-host organisms. Here, we examined the effects of a variety of non-host organisms on the removal of viruses. The marine algal virus PgV-07T (infective to  Via infection and mortality of their microorganism hosts, viruses have the ability to regulate not only host population dynamics but also to drive biogeochemical cycling and carbon sequestration within marine systems6. As lytic virus infections of microbial hosts inevitably result in the death of the host cell, any decay of infectious virus particles is likely to have important repercussions for hosts as it results in lower encounter rates and thus reduced infection levels. Despite marine viruses influencing fundamental biological processes3, research investigating virus decay (e.g. loss of infectivity) and particle loss has primarily focused on abiotic factors such as UV radiation and temperature (loss of infectivity) and adhesion to clay particles and aggregates 11. However, biological factors resulting in removal of virus particles from the water column is still understudied  was chosen as a model system because it can be clearly discriminated (and enumerated) from co-occurring bacteriophages using flow cytometry, based on fluorescence after staining with the nucleic acid-specific dye SYBR Green I26. Phaeocystis globosa strain G (A)  is the host of the double stranded DNA virus PgV-07T  and is an ecologically important primary producer with a wide distribution in temperate  seas during spring and summer31. Algal cultures were grown in Mix-TX medium32 at 15 °C and a light:dark cycle of 16:8 h. Cultures were transferred weekly to keep the cells growing exponentially. The virus PgV was produced by infecting exponentially growing hosts and allowing complete lysis to occur during the following week. Prior to experimental use, the lysate was cleared from most of the lysed P. globosa cell debris by centrifugation  at 2450 × g for 30 min at 15 °C. The supernatant containing the PgV was carefully pipetted off and stored at 15 °C until experimental use (max. 2 days). Filtration of the supernatant resulted in reduced PgV abundances in the filtrate and hence we chose to use unfiltered supernatant for our experiments. The infectivity of the PgV batches used for the experiments was determined by most probable number (MPN) endpoint dilution33. Percentage infectious viruses  was near 100%, ensuring that potential removal of PgV by non-host organisms was not a selective process for either infectious or non-infectious virus particles.The marine phytoplankton virus PgV-07T 26 with modification as described by Mojica et al. 201434). In short, stored samples collected during the experiment were thawed and diluted in sterile 0.2 µm filtered TE buffer , stained with the nucleic acid-specific green fluorescent dye SYBR Green I (Invitrogen-Molecular Probes) for 10 min in the dark at 80 °C, after which PgV was enumerated using a BD FACSCanto™ flow cytometer . The trigger was set on the green fluorescence for the detection of stained PgVs. Data were processed using FCS Express 4 software (De Novo Software).PgV was enumerated according to the protocol by Brussaard 35. Organisms such sponges, anemones and sea squirts are found on hard surfaces such as harbor walls and are typically subtidal but can also be found in some intertidal areas. Both sponges and sea squirts are capable of filtering large volumes of water (300 L h−1 and 200 mL min−1 respectively37) and retaining nano-sized particles40. Polychaete larvae and copepods are pelagic, feeding on prey <53 µm in size41. Copepods live in coastal and upwelling regions and switch prey between algae and ciliates under 60 µm in size with clearance rates of <86 m1 d−l)43. The species used, i.e. anemones (Actinia equina), barnacles , cockles (Cerastoderma edule), crabs (Carcinus maenas), mussels (Mytilus edulis), oysters , sea squirts (Styela clava) and adult copepods  and polychaete larvae  were all collected between spring and summer from the coastal area along the island of Texel (the Netherlands). Breadcrumb sponges  for the first experiment (Exp. 1) were collected along the same coast, but to prevent impacting the local sponge community too much the sponges for subsequent tests were collected from the Oosterschelde (southern Netherlands) and transported to the NIOZ in cool boxes. After collection, all organisms were gently cleaned to remove any visible epibionts. Before being transferred to sterile 100 mL polystyrene pots for the experiments, the cleaned organisms were starved for 24 h in flow-through aquaria (80 × 40 × 40 cm) at 15 °C with a light:dark cycle of 8:16 h.Non-host organisms tested for their ability to reduce viral abundance were chosen based on their geographic distribution coinciding with that of the algal host-virus model system and included organisms of varying feeding mechanisms  as well as range in size . Bivalves such as oysters, mussels and cockles are found in intertidal-subtidal areas and are considered bioengineers, altering substrate type, filtering vast amounts of water and removing particles <250 µm in size6 mL−1), and the second treatment with only the lysate to assess for adherence to the pots. In addition, an extra control with only the test organism in culture media was used to assess for the introduction of viruses by the test organism. As these controls indicated that no viral particles in the same size range as PgV were introduced, they were left out of further analysis. Each of the two regular treatments was replicated six times. All trials took place in a single climate room kept at 15 °C. During the experiment, samples (1 mL) were taken using sterile pipettes (one for each replicate) and placed into 1.5 mL Eppendorf tubes containing 20 µL 25% glutaraldehyde  for 30 min at 4 °C, flash-frozen in liquid nitrogen and stored at −80 °C until further analysis . Samples were taken before the test organisms were added (pre-test or PT), 15 min after the animal were adapted to the pot (T0), after a period of 3 h (T3) and then after 24 h (T24), with the exception of crab, cockle, barnacle and mussel treatments where samples were taken before the test organisms were added (pre-test or PT) and after a period of 3 h (T3).Experiment 1 (Exp. 1) assessed a variety of organisms for their ability to interfere with a marine virus-host transmission pathway by removing infectious virus particles from the water. Sterilized 100 mL polystyrene pots with screw cap  were used during the experiment as aquaria. Prior to the experiment the pots were sterilized using 6 M HCl for 1 h, followed by rinsing in deionized water and then finally washed in 90 °C deionized water to remove any traces of HCl. The experiment consisted of two types of treatment: the first with the non-host organism  in 80 mL of the PgV lysate (around 1 × 106 mL−1) was added (‘spiked’) at 20 min intervals for 6 h to avoid depletion of viruses by the sponges. Samples (1 mL) were taken using individual sterile pipettes prior to and immediately after adding viruses to the system to allow for the calculation of clearance rates. Treatments consisted of test organism spiked with PgV, and two controls . All treatments were replicated four times but due to the mortality of one sponge during the experiment, only three replicates were used for statistical analysis. Samples were processed and stored as in Exp. 1.Breadcrumb sponges were shown to significantly decrease PgV abundance (Exp. 1) and so Experiment 2 (Exp. 2) was designed to assess the ability of the sponges to continuously remove PgV over a longer time period. The experimental set-up was similar to Exp. 1, but PgV . For Exp. 1, the effect of the presence of non-host organisms on changes in PgV abundance over time, was statistically tested by comparing changes in PgV abundance from one sampling time to the next, between the treatment where a non-host organism was present and the control treatment. A univariate analysis of variance (ANOVA) was used when samples were only taken at two time points , whereas a multivariate analysis of variance (MANOVA) was used when samples were taken at more than two occasions . The univariate test statistic is the F-value, the multivariate test statistic is Pillai’s trace. Significances indicate that viral abundances over time differed among treatments. As there are only two treatments (with and without non-host organisms), the tests are in fact equivalent to the t-test and to Hotelling’s TCl is volume of water cleared of suspended particles per unit of time; V is the volume, K is the number of individual sponges and N0 and Nt are the virus cell abundance at time 0 and time t, respectively44. In this study one piece of sponge  was used per treatment. Clearance rates were calculated for the period after clearance had stabilized, i.e. from 1–5.5 h into the experiment, unless otherwise stated. Linear regressions were subsequently used to test for density dependency by comparing the viral abundances directly before spiking against clearance rates for that specific 20 min period.For Exp. 2, sponge clearance rates were calculated for 20 min intervals using the viral abundances directly before spiking and the viral abundance immediately after the aquaria were spiked with viruses. Sponge clearance rates were determined using the following formula:5Emiliania huxleyi viruses mL−1 , with a clearance rate of 50 mL ind−1 day−1 15 suggesting that removal of viruses by tunicates may be species-specific. The presence of oysters and crabs resulted in significant reduction of PgV abundances over the 24 h experimental period, i.e. <12% and 90% respectively within 3 h. In other studies, after exposure to water containing human enteric viruses, bivalves and crabs showed internal accumulation of the viruses, with recovery of viruses from tissues such as the digestive tract49. This indicates that both decapods and bivalves have the potential to take up viruses from their surrounding environment and, collectively, significantly contribute to the reduction in viral abundances. Moreover, studies have shown that after uptake by bivalve species viral particles released via fecal matter are inactive and thus suggesting that the digestion process renders the particles non-infective51. In other marine macroparasite-host systems crabs and oysters have been shown to effectively reduce the number of free-living trematode parasites18. While the infective cercarial stages of trematode parasites are considerably larger  than virus particles, our study demonstrates the extent of their interactions and their potential to alter a wide range of infectious diseases within a system. Besides the existing studies focusing on the concentration of viruses and their impact on the human food chain we are, to our knowledge, the first to report the removal of non-human viruses by marine shellfish.Ten marine non-host species were tested to assess their ability to reduce abundances of the model virus PgV. As expected, the control treatments containing only PgV showed no significant decline in PgVs over time and controls containing only the non-host organisms did not display virus enrichment. All non-host organisms, except barnacles, copepods and mussels, significantly affected PgV abundances Table , Fig. 2,56, as well as dissolved organic matter (DOM)57. According to the standard practical classification and operational definition, particles <0.2 µm are included in the DOM pool, i.e. the DOM particle size range also includes the nanometer range of virus particles. In this study we used only algal virus PgV-07T but, given the size range of prey sponges can take up, we would expect comparable removal rates of other viruses . Natural seawater viruses, dominated by typically smaller-sized bacteriophages (20–60 nm range)58, have been reported to be removed by a tropical sponge14. Furthermore, even the very small influenza viruses can be effectively removed by bivalves51. While not the focus of this study, the overall removal of virus particles is likely to have selected consequences for nutrient cycling via the direct loss of viruses  and the indirect alterations in biogeochemical cycling resulting from host cell lysis3. The effective removal of ecologically relevant virus concentrations by the temperate breadcrumb sponge highlights that the filtration activity and the removal of viruses from the surrounding water by non-host organisms is compelling. The virus removal rates shown here are much higher than previously reported for other sponge species. For example the tropical sponge Negombata magnifica reduced viruses with an efficiency of 23 ± 3%14. Discrepancies in sponge efficiency between their and the present study may be a result of anatomical differences between the sponges used; here we used a temperate sponge from the order Halichondrida, whereas Hadas and coworkers (2006) used a tropical sponge from the order ‘Poecilosclerida’. Furthermore, the sponge species used in our study has fluctuating removal efficiencies depending on the season. This study was conducted during the period when the sponges exhibit their highest energy demand (April and August60). Importantly, the differences between the studies illustrate that the contribution of sponges in controlling viral abundance in marine systems is thus far substantially underestimated. Such high reductions in viral abundance are likely to have ecological consequences with knock-on effects for the algal host and virus-host contact rates.The strongest removal of PgV was caused by the sponges, not only by removing the most viruses but also because removal of viruses from the system began instantly Fig. . The Bre6 PgV mL−1 to avoid complete depletion in the surrounding water . Given that virus clearance rates reported thus far  are lower than in our study, the ecological importance of virus loss by sponge activity is most likely extremely underestimated. Previous studies on viral and DOM loss by sponges have primarily used tropical sponge species63. To our knowledge, we are the first to show that a temperate sponge species is efficient at removing viruses and thus it is likely that other temperate sponge species are also able to reduce viral abundance with equal or better efficiency.To test the sponges’ ability to consistently remove viruses over time, we spiked replicates with PgV every 20 min for up to 6 h with approx. 2.5 × 10ter Fig. . Removal −1 Fig. . During gVs Fig. . Initial−1 g sponge dry weight and the average sponge dry weight per replicate was 1.76 ± 0.99 g, the average clearance rate was 77 mL (range between 34 and 121 mL) within the 15 min sampling period. Although a higher time resolution is recommended it was logistically constrained by the sample processing time required immediately after taking the samples. By utilizing a controlled experimental design and setup such as the one used in this study we are able to show that the loss of viruses was a direct effect of the sponge and not an artefact due to interactions caused by the presence of other organisms within the system. Furthermore, unlike Hadas et al. 200614, by using a controlled experiment and filtered water we can also conclude that there was no other potential ‘food’ in the water, potentially supporting previous suggestions that viruses act as a nutrient source and a part of the sponge pump64.Theoretically there might have been the possibility that sponges filtered the suspension volume (80 mL) more than once, however given that the mean clearance rate was 176 mL hP. globosa have been shown to contribute considerably to bloom demise and, depending on the environmental conditions, viral activity may even prevent bloom formation66. Thus any reduction in contact rate between PgV and its P. globosa host results in reduced virally induced mortality of the algal host, subsequently promoting a longer blooming period or shifting the share of loss factors from viral lysis to grazing68. Sponges have a crude ability to remove particles based on size73 and it is well documented that bacteria and phytoplankton are a major part of sponge prey75. This could influence host population dynamics directly  and indirectly . This complex form of transmission interference may thus reduce contact rates between viruses and their hosts even further . Therefore, the presence of a non-host organism may be a ‘double edged sword’ affecting the abundance of both viruses and hosts. We conducted a pilot experiment to compare simultaneous removal of PgV, P. globosa and bacteria, and, indeed, all three types of particles were efficiently cleared (within 24 h) by the breadcrumb sponge. The bacterial clearance rate of 535 mL h−1 g tissue dry wt−1 falls within the rates published for other sponges (10–5000 mL h−1 g tissue dry wt−1)76. The P. globosa clearance rate (68.2 mL h−1 g tissue dry wt−1) was in the middle range of published rates for phytoplankton76. Substantial variation in clearance rates for both phytoplankton and bacteria do occur which may be caused by factors such as initial particle concentration, time of year, temperature, species and individual sponge behavior77. Regardless of these differences, our study demonstrates that the virus clearance rates are within the range of the acknowledged natural food particles, bacterial and algal cells.Reductions in natural virus abundance by such high degree as shown in our study, have the potential to impact local microbial host population dynamics as the virus-host contact rate drops due to the decline in virus abundance. For PgV specifically, viruses infecting 78, as well as in tropical79 and deep sea reefs81, collectively they are highly likely to continuously interact with the viruses in the water column creating strong localized differences in viral abundance. For example, the overall effects of sponges on viruses here in the Netherlands is likely to have less of an impact than in other regions such as coral reefs or harbors where the majority of surfaces comprise of hard substrate. In such locations sponge coverage can be high, e.g. ranging from 45–70 m−2 (Indonesia82) to>1400 individuals m−2 (Ireland83), therefore, in locations such as relatively enclosed bays, the effect of sponges on virus removal is probably vastly underestimated. For example, in a hypothetical hard-substrate bay measuring 10 km × 10 km × 0.01 km, an average sponge density of 1,400 sponges m−2, and a sponge clearance rate of viruses of 5 mL h−1 (Exp 2 this study), an estimated 7 × 109 L h−1 is cleared of viruses by the sponges. That equates to 1.7% of the bays water volume every hour. Given stable conditions (e.g. stagnant water), all the viruses in the bay could, hypothetically, be cleared within 3 days. Although such estimates should be approached with caution, the above estimation underlines the enormous potential of sponges as virus loss factor whereby the ultimate ecological impact depends on the local conditions such as tide phase, current strength, local sponge cover, other non-host organisms potentially removing viruses from the water column , as well as host presence and abundance.Given that sponges can be found in high densities in coastal regions, such as harbors86 all of which should be tested for their effects on transmission interference. An interesting follow up experiment would be to expand on the experiments investigating the effect of sponges on the contact rates of host and virus when both are present. Considering that even temperate sponges effectively remove viruses from the water column (this study), the global ecological consequences are expected to be considerable and hence, there is a need for further investigations to determine how, when and to what extent non-host organisms affect virus-host dynamics.In conclusion, our results stress the notion that a wide range of non-host organisms and in particular sponges, have the potential to reduce nano-sized pathogen abundance via transmission interference. It is very likely that there are many more species capable of removing viruses from the water column. It is also likely that there are a variety of factors which facilitate or impede the removal of viruses, such as stratification or mixing of the water column altering contact rates, ambient temperatures affecting non-host organism feeding rates, free-floating clays and aggregates to which viruses may adhereSupplementary material.