Severe acute respiratory syndrome coronavirus (SARS-CoV-2), an emerging Betacoronavirus, is the causative agent of COVID-19. Angiotensin converting enzyme 2 (ACE2), being the main cell receptor of SARS-CoV-2, plays a role in the entry of the virus into the cell. Currently, there are neither specific antiviral drugs for the treatment or preventive drugs such as vaccines.We proposed a bioinformatics analysis to test in silico existing drugs as a fast way to identify an efficient therapy. We performed a differential expression analysis in order to identify differentially expressed genes in COVID-19 patients correlated with ACE-2 and we explored their direct relations with a network approach integrating also drug-gene interactions. The drugs with a central role in the network were also investigated with a molecular docking analysis.We found 825 differentially expressed genes correlated with ACE2. The protein-protein interactions among differentially expressed genes identified a network of 474 genes and 1130 interactions.The integration of drug-gene interactions in the network and molecular docking analysis allows us to obtain several drugs with antiviral activity that, alone or in combination with other treatment options, could be considered as therapeutic approaches against COVID-19. Coronaviruses are a group of single-stranded RNA viruses with a wide range of vertebrate hosts.Coronavirus infection in humans is characterized by a wide spectrum of clinical manifestations ranging from mild influenza symptoms to acute respiratory syndrome with damages in lung function, pneumonia, arrhythmia, or death .Betacoronavirus (β-CoV) is the second of four genera of coronaviruses: alpha, beta, gamma and delta. Over the past two decades, three highly pathogenic human betacoronaviruses have spread from animals to humans: Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) in 2002–2003, MERS-CoV in 2012 and SARS-CoV-2 in 2019. Compared to SARS-CoV or MERS-CoV, SARS-CoV-2 spreads more rapidly making it difficult to control [Recently, a first model of infection has been proposed  in whichSeveral studies reported that the first sites of infection seem to hit pneumocytes and enterocytes of small intestine . The molSARS-CoV-2 is a new virus for our immune system. The IgG antibodies produced by other β-CoV do not recognise this SARS-CoV-2, making this virus currently invincible. A possibility would be the use of immunoglobulin collected from immunized COVID-19 patients to be injected in patients suffering from COVID-19 . In siliFurthermore, immunotherapy approach combined with antiviral drugs detected by in silico analyses may be a stronger treatment until definitive options such as vaccines are available.In this work we propose in silico study to identify genes deregulated in COVID-19 positive patients correlated with ACE2. Then, we performed a network analysis based on protein-protein interactions with the aim of identifying existing drugs targeting SARS-CoV-2 deregulated genes, to be proposed as candidate drugs for COVID-19, alone or in combination with other therapeutic strategies. The protein interactions that emerge from our study could help to decipher their mechanisms of action with respect complex biological processes at the basis of COVID-19 infection and progression. The drugs obtained were also investigated through docking simulations.We collected the gene expression levels of lung tissues infected by SARS-CoV-2 from the Gene Expression Omnibus (GEO) database. Specifically, we selected from GSE147507 2 lung biopsies from postmortem COVID-19 positive male patients and 2 lung biopsies from COVID-19 negative male controls.Furthermore, from the Genotype-Tissue Expression (GTEx) project we collected gene expression levels of lung tissues from healthy subjects .TCGAanalyze_Normalization implementing the EDASeq protocol [We normalized and filtered RNA-seq raw counts using the reference of hg19, following the pipeline of the R/ Bioconductor package TCGAbiolinks . In partprotocol , to perfprotocol .P-values, generated from the differential expression analysis, were corrected using the Benjamini–Hochberg procedure for multiple-testing correction [Differential expression analysis between lung biopsies from postmortem COVID-19 positive male patients and negative male controls was performed on GSE147507 gene expression levels with the R-package TCGABiolinks . Specifirrection . We defiPathway enrichment analysis was performed with the R-package clusterProfiler . In partWe generated a protein-protein interaction network considering the direct connections among differentially expressed genes in COVID-19 using SpidermiR  and we sDrug-gene interactions were derived by the package rDGIdb .p-values of the Pearson’s correlation were considered and only ACE2 and genes significantly correlated  were used for the subsequent analysis.A correlation analysis was performed between ACE2 and the other genes in GTEx data  to obtain the genes co-expressed with ACE2. The corresponding We implemented the fast greedy modularity optimization algorithm for finding community structure of our network . CommuniIndeed, a network can be divided into communities or clusters and each community is characterized by many edges within communities and only a few edges between communities.Docking study explored the binding mode of reported drugs on the 3D model of protease of SARS-CoV-2.Based on the above analysis, the structural coordinates of potential drugs (file SDF) were downloaded from PubChem . DiscoveLigand preparation and molecular modelling are performed using AUTODOCK 1.5.6  followinAfter, the ligand is loaded, AUTODOCK automatically prepares it for molecular docking. The software computes Gasteiger charges and if the charges are all zero, it will add charges , 17.Crystal structures of SARS-CoV-2 main proteases  were obtained from Protein Data Bank. Polar hydrogen atoms were added, and water molecules were removed to the SARS-CoV-2 model.The macromolecules (main protease) and ligands were saved in pdbqt format to be used for docking analysis. The grid parameters in the x, y and z dimensions were set to 90,90,90. Autodock uses the Lamarckian genetic algorithm to search for the ligand exposure with the highest binding affinity. Default settings were used for other parameters following the docking protocol reported in .Binding energies between ligand and main protease (docking scores) are reported in kcal/mol. High negative values suggest a better binding , 17.Protein-Ligand Interaction Profiler was used to visualize the binding interactions of the selected drugs with 3D model of protease of COVID-19 .p-value  were found.We studied the interactions among differentially expressed genes correlated with ACE2. From the analysis of protein-protein interaction network, we obtained a network of 474 genes and 1130 interactions. We queried DGIdb and we obtained 714 drugs that interact with 119 of 474 genes involving 950 interactions.We integrated protein-protein interactions’ information with the drug-gene interactions obtaining a network of 1188 nodes (including 474 genes and 714 drugs) and 2080 interactions. The overview of such network is shown in Fig. The drugs with the highest degree centrality (d.c.) are: CHEMBL1161866 (d.c. = 11), bortezomib (d.c. = 6), puromycin (d.c. = 6), metformin hydrochloride (d.c. = 6), NV-128 (d.c. = 6), ME-344 (d.c. = 6), methylene blue (d.c. = 5), anisomycin (d.c. = 5) and everolimus (d.c. = 5) , APP (d.c. = 143), SUMO2 (d.c. = 106), CUL3 (d.c. = 89), CA2 (d.c. = 72), GSK3B (d.c. = 61), HSPA4 (d.c. = 60), NRAS (d.c. = 53) and MTOR (d.c. = 50) , SUMO2 (d.c. = 53), and APP (d.c. = 41). The drugs with key roles in the network are 7: bortezomib, puromycin, ixazomib citrate, carfilzomib, oprozomib, methylene blue and anisomycin. The drugs with the major number of drug targets in the network are: puromycin, methylene blue and anisomycin. They interact with 5 proteins: RPL11, RPL15, RPL26L1, RPL37 and RPL8 , NRAS (d.c. = 22) and RB1 (d.c. = 10). The drugs involved in this community are 27: metformin, abemaciclib, ribociclib, palbociclib, pictilisib, fluorouracil, trametinib, vorinostat, sirolimus, entinostat, everolimus, cetuximab, crizotinib, sorafenib, afatinib, ethyl nitrosourea, gefitinib, osimertinib, mk-2206, buparlisib, gedatolisib, bgb-283, panitumumab, selumetinib, chembl1229592, vemurafenib and dabrafenib. The drug with the major number of drug targets in the network is palbociclib, and it interacts with 4 proteins: CCND3, ERBB2, NRAS and RB1 , PCNA (d.c. = 14) SMAD4 (d.c. = 14), CDK8 (d.c. = 14) and SMAD2 (d.c. = 14). The drugs involved in this community are 17: veliparib, olaparib, rucaparib, niraparib, talazoparib, talazoparib tosylate, rucaparib camsylate, camptothecin, temozolomide, azacitidine, lomustine, gemcitabine, roniciclib, irinotecan, PHA-793887, AT-7519 and AZD-5438. The drug with the major number of drug targets in the network is camptothecin. It interacts with 4 proteins: MAPK9, HUS1, MGMT and TOP1  against SARS-CoV-2.The list of drugs examined for docking study is described in Table The binding energies of CHEMBL1161866 and ME-344 with the main proteases of SAR-CoV-2 are presented in Supplementary Two drugs (Puromycin and anisomycin) obtained a negative binding energy value as shown in the Table The details of the parameters for the considered conformations are presented in the Supplementary 2.Docking interactions of puromycin with PDB ID 5R82 and anisomycin with PDB ID 5R7Z, which obtained the best binding energies, are presented in Fig. Binding interactions of puromycin with PDB ID 5R82 involve 1 hydrophobic interaction and 4 hydrogen bonds  is higher. This is due to their difficulty in following the recommendations from public health authorities and the difficulty of maintaining isolation during behavioural and psychological symptoms of dementia. In addition, patients with AD often have age and comorbid medical conditions associated with a poor prognosis and mortality from COVID-19 . The devAmong the top differentially expressed mRNAs we also found mRNAs associated with human T-cell leukemia virus 1 infection, Shigellosis, hepatocellular carcinoma, viral carcinogenesis, apoptosis, non-alcoholic fatty liver disease (NAFLD), mTOR signaling, cellular senescence, protein processing in endoplasmic reticulum and cell cycle.Chronic lymphocytic leukemia (CLL) is the most common adult leukemic disorder in the Euro-American population. It is caused by human retrovirus named human T lymphotropic viruses 1 (HTLV-1). HTLV-1 is very similar and relevant to human coronavirus (HCoVs) in animal-to-human transmission event. Although it is not yet fully known how SARS-CoV-2 interacts with host antiviral immunity, similar mechanisms are found from other HCoVs and human pathogenic viruses in other families that are very similar including HTLV-1 and the human immunodeficiency viruses (HIVs) . SeveralShigella spp. The expression of ACE2 is abundant in the epithelia of the lungs as well as in intestinal epithelial cells. In line with this scenario, COVID-19 may be associated with gut microbiota [Virus infection alters the gut microbiome in humans and makes the organism more susceptible to intestinal bacterial infections such as Shigellosis. Shigellosis is an infectious intestinal disease, also called bacillary dysentery, caused by the genus crobiota . The concrobiota , 27.In addition to the lung and intestine, several evidences showed possible implications of hepatic involvement in SARS-Cov-2 infection. Patients with liver diseases, including hepatocellular carcinoma and Non-alcoholic fatty liver disease (NAFLD) (a metabolic disorder due to the accumulation of fat in the liver), can be more susceptible to the serious consequences of the COVID-19. This could be due to reduced innate immunity to the virus, as the liver plays crucial role in innate and adaptive immunity. As previously reported, the expression of ACE2 is abundant also in the epithelia of intestine, and as there is a rich circulation of blood from the intestine to liver, the spread of the virus in the liver is expected. Reduced hepatic innate immune status could contribute to progression of COVID-19 –30.The mammalian target of rapamycin (mTOR) signaling pathway is a conserved serine/threonine kinase and promotes the cellular proliferation, protein synthesis and DNA replication. mTOR has been already associated with the development of influenza by promoting influenza virus replication . The antApoptosis, cellular senescence, protein processing in endoplasmic reticulum and cell cycle are all processes that are altered as consequence of the virus infection. The infected cell promotes an activation of apoptosis and cell senescence, and an inhibition of protein synthesis and cell cycle in order to remove the infecting organism and reduce the spread of the virus.We investigated the role of existing drugs in the protein network finding the drugs with the highest degree centrality in the network: CHEMBL1161866 (d.c. = 11), bortezomib (d.c. = 6), puromycin (d.c. = 6), metformin hydrochloride (d.c. = 6), NV-128 (d.c. = 6), ME-344 (d.c. = 6), methylene blue (d.c. = 5), anisomycin (d.c. = 5) and everolimus (d.c. = 5)  and glutamate dehydrogenase, suggesting a possible beneficial application of this drug on liver infection by SARS-CoV-2. ADH enzymes, highly expressed in liver and colorectal tissues, catalyzed the conversion of alcohol to aldehydes during alcohol metabolism. In particular, ADH could have a role in hepatitis B virus (HBV) and hepatitis C virus infection, being the increased activity of ADH enzymes a clinical marker of liver injury . AlthougBortezomib , a proteasome inhibitor approved against multiple myelomas, showed in several studies its antiviral proprieties. Proteasome inhibitors are encouraging antiviral agents that could inhibit viral entry. The ubiquitin-proteasome system (UPS) is involved in different steps of a broad range of viruses’ replication cycle, including coronavirus. The UPS regulates many different processes, such as the cell cycle progression and apoptosis. In addition, ubiquitination is involved in the host immune response to viral infection regulating antigen presentation –37. The NCT02972723) and Human Immunodeficiency (NCT04500678).Recent studies suggested that a drug already FDA approved for use against diabetes, metformin, may be effective against COVID-19. Metformin inhibits glucose production decreasing intestinal glucose absorption and enhancing glucose uptake by peripheral tissues. People with diabetes mellitus (DM) have poor prognosis and increased fatality in a wide range of viral infections, including SARS and MERS. The link between COVID-19 and DM could be explained by the use of angiotensin-converting enzyme inhibitors (ACEi) and angiotensin-receptor blockers (ARBs) in people with DM. Indeed, ACEi/ARBs act upregulating the receptor for entry of the virus into host pneumocytes. However, Metformin does not interfere with ACE2, and it is not clear how it could act on patients’ outcome , 39. To NCT04376788) involves Methylene blue as potential treatment of patients with COVID-19. Unfortunately, the results of this clinical study are not yet available.Methylene blue is a fluorescent dye used for nucleic acid stain. It has been approved by the Food and Drug Administration (FDA) as treatment of methemoglobinemia. Due to its antimicrobial, anti-inflammatory, and antioxidant effects, Methylene blue has been successfully utilized in vitro. Its antiviral proprieties have been attributed to its ability to intercalate into nucleic acids and inactivate RNA viruses. Virus inactivation with Methylene blue was applied in plasma to mitigate the risk of transmission by transfusion in human immunodeficiency virus (HIV), hepatitis B virus, hepatitis C virus and Zika virus , 41. MetAnisomycin was originally defined by FDA as an antibiotic against protozoa and fungi. The antiviral effect of anisomycin was demonstrated in cell cultures of Dengue and Zika viruses, mainly involving viral macromolecular synthesis . In addiNCT01624948) and hepatitis C virus (NCT01134952).Everolimus is an mTOR inhibitor with well-known antitumor activity in advanced cancer, including kidney and breast cancer. Everolimus showed also in vitro antiviral effects against influenza A virus: in a lethal mouse model of MERS, it delayed death and reduced MERS-CoV infection by ~ 60%. It has also been correlated with a reduced frequency of cytomegalovirus infection in transplant patients –48. FDA Puromycin is an antibiotic able to inhibit protein synthesis. Although protein inhibition could be obtained by puromycin treatment, in cornea C-M virus-infected cells as well as in HeLa cells infected with Newcastle disease virus the viral particle continues to be produced due to viral protein intracellular accumulation. In this way, viral particles could be assembled, also in the absence of continuous protein synthesis . It seemThere is not also a clear connection between NV-128, ME-344 and COVID-19, but we cannot exclude a future possible implication of these drugs in COVID-19 patients.The docking studies estimated that two drugs (puromycin and anisomycin) have potential characteristics of binding to SARS-CoV-2.This study identified, in silico, a group of proteins, drug targets of conventional treatments, within multiple complexes and biological processes that could represent effective mechanism of therapeutic actions also for SARS-CoV-2 infection. The protein interactions, that emerge from our analysis, highlight several drugs, as actionable molecules on proteins of the SARS-CoV-2-specific deregulated gene network; they could be proposed with a rational against the virus, alone or in combination with other therapies.Additional file 1: Supplementary 1. Differentially expressed genes between lung biopsies from postmortem COVID-19 positive patients and lung biopsies from negative controls. Supplementary 2. The details of the parameters for the conformations obtained with molecular docking analysis and the specific binding interactions


Coronavirus 2019 (COVID-19) is currently the center of what has become a public health crisis. While the virus is well-known for its trademark effects on respiratory function, neurological damage has been reported to affect a considerable proportion of severe cases. To characterize the neuro-invasive potential of this disease, a contemporary review of COVID-19 and its neurological sequelae was conducted using the limited, but growing, literature that is available. These neurological squeal are based on the manifestations that the virus has on normal central and peripheral nervous system function. The authors present the virology of the SARS-CoV-2 agent by analyzing its classification as an enveloped, positive-stranded RNA virus. A comprehensive timeline is then presented, indicating the progression of the disease as a public health threat. Furthermore, underlying chronic neurological conditions potentially lead to more adverse cases of COVID-19. SARS-CoV-2 may reach ACE2 receptors on neuronal tissue through mode of the general circulation. The CNS may also be susceptible to an immune response where a “cytokine storm” can manifest into neural injury. Histological evidence is provided, while symptoms such as headache and vertigo are highlighted as CNS manifestations of COVID-19. Treatment of these symptoms is addressed with paracetamol being recommended as a possible, but not conclusive, treatment to some CNS symptoms. The authors then discuss the peripheral nervous system sequelae and COVID's impact on causing chemosensory dysfunction starting with viral attack on olfactory sensory neurons and cells types within the lining of the nose. Histological evidence is also provided while symptoms such as anosmia and ageusia are characterized as PNS manifestations. Possible treatment options for these symptoms are then addressed as a major limitation, as anecdotal, and not conclusive evidence can be made. Finally, preventive measures of the neurological sequelae are addressed using a multidirectional approach. Postmortem examinations of the brains of COVID-19 patients are suggested as being a possible key to formulating new understandings of its neuropathology. Lastly, the authors suggest a more comprehensive neurological follow-up of recovered patients, in order to better characterize the neurological sequelae of this illness. The emergence of the coronavirus and its viral agent, severe respiratory syndrome coronavirus 2 (SARS-CoV-2), has derived into one of the most unprecedented and considerable pandemics of the modern era. The third zoonotic coronavirus this century, its pathogenic nature and large scale adverse medical effects has cemented its status as one of history's greatest public health threats. This virus was first isolated in December 2019 after many patients in Wuhan, China, were diagnosed with pneumonia of unknown etiology . FollowiUntil recently, COVID-19 was investigated as primarily a respiratory illness. However, as the number of cases continue to grow, more reports are starting to show that the virus is capable of not only lung damage, but also neurological damage. In a case series from Wuhan, neurological symptoms were noticed in 36.4% of patients with severe infections  circulate in a diverse array of mammalian and avian reservoirs. CoVs are classified into four genera and are enveloped, positive-stranded, RNA viruses known to have large genomes susceptible to recombination and mutations that lead to the emergence of novel viruses. They can cause respiratory, enteric, hepatic, and neurological diseases  for SARS-CoV-2 ranges from 2.2 to 5.5 . It is widely understood that the mode of transmission of COVID-19 is through direct contact or respiratory droplets from an infected individual. However, indirect contact through fomites can also act as a vehicle of transmission. Fomites are inanimate surfaces that contain the virus (through direct contact), and based on the viability of the virus on to an inanimate surface, it can lead to the rampant spread onto another host. As soon as the virus enters the body, its effects are highly dependent on its viral structure. The viral structure of COVID-19 is contained within the nucleocapsid which is surrounded by a nuclear envelope. The nuclear envelope is derived from the host cell's membrane and embedded within that membrane are glycoprotein spikes, known as S-proteins . It is tThe pathophysiology of neural infections in the CNS from COVID-19 is a phenomenon that is still being characterized. Previous studies have shown that SARS-CoV-2 may reach ACE2 receptors on neuronal tissue through mode of the general circulation. Viremia causes the virus to pass into the cerebral circulation and reach the brain to induce neurotropic effects. The virus may also reach the brain through routes of the nasal cavity, specifically as the virus passes through the cribriform plate. Upon entry into the neural cavity, the virus will encounter ACE2 receptors located on the endothelial lining between the blood capillaries and the brain . The virA recent case of a female patient in Detroit, Michigan showed the possible long-term impact of neurological manifestations of COVID-19  while alWhile the more severe symptomatic manifestations of the CNS may include ANE, meningitis, and hemorrhage, the more commonly reported CNS symptoms are vertigo, cephalgia, impaired consciousness, seizures, ataxia, and acute cerebrovascular disease  Table 1Table 1. Treatment of COVID-19 and its CNS manifestations are still ongoing. However, if the coronavirus is breaching the blood-brain barrier, this could complicate the recovery process . The rolCOVID-19, like its pathogenic relatives, could also specifically compromise the activity of the peripheral nervous system. Just like the CNS, PNS manifestations arise following direct contact through respiratory droplets or indirect contact through fomites. However, as an individual starts to touch mucosal membranes, such as their eyes, nose, or mouth, it presents a portal of entry for the virus to disrupt neurological sensations that pertain to these sites. Moreover, just like the CNS manifestations, furin's endoproteolytic activity is maintained in order to activate the virus following its interactions with ACE2 receptors. These consistencies are not just present within the nervous system but along all physiological systems that get affected through COVID-19. However, COVID-19, has a peripheral neural involvement that occurs when ACE2 receptors are interacting with the viral agent. These ACE2 receptors have been found along the tongue, mouth, and nasal passage showing the possibility of the virus targeting multiple neurons and influencing those senses tremendously .It was recently discovered that two cell types in the nose are the most likely initial infection points for viral entry into the PNS. These two cell types are called goblet cells and ciliated cells . Goblet Histological significance pertaining to the PNS involvement was found following analysis of infected olfactory tissue. Professor Carl Philpott, director of medical affairs and research at Fifth Sense, states that while looking at the olfactory tissue microscopically, one will notice the fine hair-like endings of the receptor cells had fallen, thus disabling the cells from receiving olfactory senses from the nose . This isAccording to the American Academy of Otolaryngology—Head and Neck Surgery, otolaryngologists in the U.K have reported that more than two-thirds of confirmed COVID-19 cases in Germany had anosmia (loss of smell) . While iin vivo following CPM administration  of the cranium along with lumbar puncture procedures to collect cerebrospinal fluid would highlight such a neuroinvasive association to the pathogen . For patIn summary, the SARS-CoV-2 pandemic remains one of the greatest threats to public health to date. Although the virus is known to directly invade the lungs, emerging research shows central and peripheral nervous system involvement in the pathology of the disease and clinical worsening in patients. Patients with nervous system involvement as the presenting symptoms in the early stages of infection may be misdiagnosed, and may inadvertently spread the virus. Furthermore, studies suggest that the time it takes for SARS-CoV-2 to advance from initial symptoms, such as coughing, tiredness, and fever, to difficulty breathing, is roughly five days, which is long enough for the virus to enter and damage medullary neurons involved in respiratory control , 55. ThuGoing forward, to elucidate the involvement of the nervous system in the progression of this disease, postmortem examinations of the brain may be a valuable facet in understanding the neurological mechanisms the virus manifests. Currently, few, if any, autopsies are being performed due to the fear of contracting the disease. Furthermore, a more comprehensive neurological follow-up of patients who recovered from the disease may be warranted to screen for long-lasting neurological impacts of the illness and save lives.BF was the primary author contributing to the outline/direction of the paper, literature review, and writing, editing, and overseeing of the paper as a whole including submission process. RJ played a major role in writing and editing the paper. MS, AD, and CC focused their efforts on writing. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Background: Intermittent Theta Burst Stimulation (iTBS) is a design of repetitive Transcranial Magnetic Stimulation (rTMS) and could be a candidate to replace rTMS in the treatment of depression, thanks to its efficacy, shorter duration, and ease of use. The antidepressant mechanism of iTBS, and whether this mechanism is mediated by a modulation of cortical excitability, remains unknown.Methods: Using a randomized double-blind, sham-controlled trial, 30 healthy volunteers received either iTBS or a sham treatment targeting the left DorsoLateral PreFrontal Cortex (L-DLPFC), twice a day over 5 consecutive days. Cortical excitability was measured before and after the 5 days of stimulation.Results: No difference in cortical excitability was observed between active or sham iTBS.Conclusion: Our study does not support any effect on cortical excitability of repetitive iTBS targeting the L-DLPFC. Repeated Transcranial Magnetic Stimulation (rTMS) is a validated non-invasive brain stimulation technique used to treat resistant depression .Intermittent Theta Burst Stimulation (iTBS) is a design of rTMS that uses a very highly modulated frequency to produce a high number of pulses in a shorter time. This increases cortical excitability in a more robust and longer lasting way than rTMS, and in a similar way to High-Frequency (HF) rTMS .Several studies have reported that iTBS is non-inferior to the gold standard 10 Hz rTMS in the treatment of major depression , 4. FurtThe exact neurobiological mechanisms of iTBS/rTMS that lead to an antidepressant effect remain unknown.Some etiologies of depression involve an imbalance of inter-hemispheric neuroexcitability. This imbalance has been identified in the dorsolateral prefrontal cortex (DLPFC) via imaging and EEG studies. This has justified the use of high-frequency (HF) rTMS targeting the left DLPFC to treat major depressive episodes , 7. OtheWe have thus stipulated that the remission of depressive symptoms after an increase in DLPFC excitability via HF rTMS (or iTBS) could also lead to a similar increase in the excitability of the ipsilateral motor cortex. A study by Spampinato et al.  supportsThe aim of this study was to assess the effect of iTBS on cortical excitability in the motor cortex in healthy controls.This study is a randomized double-blind vs. placebo trial designed to assess cortical excitability in the motor cortex before and after ten sessions of iTBS over the L-DLPFC.Thirty volunteers, who had given written consent, were randomly assigned to either an active or a sham iTBS group. Healthy subjects between 18 and 65 years old, with no known history of psychiatric illness, and who had been matched in age, gender and education level, were included. They had to be right-handed, with a BDI (Beck Depression Inventory) score of <8, and an HDRS  score of <8. Healthy female subjects had to confirm that they were not pregnant, and were on oral contraception. The non-inclusion criteria consisted of: being on any psychotropic drug, having any psychiatric disorder on axis I or II of the DSM-IV-TR , suffering from epilepsy, or having any other contraindications for rTMS.All participants gave written consent, and the study was approved by the local ethical committee .Surface electromyograms were taken from abductor pollicis brevis muscles (APB) via electromyographic (EMG) self-adhesive electrodes, with solid gel coated disposable Ag/AgCl electrodes placed on the belly and tendons of the APB muscles.To assess cortical excitability, TMS was performed using a standard 70 mm figure-of-eight coil (MCF-B65 Butterfly Coil) attached to a Medtronic MagOption stimulator. The coil was attached to the head with the handle pointing backwards and laterally, at an angle of 45° to the sagittal plane.EMG activity was amplified 10,000-fold using a Matrix Light amplifier  through filters set at 20 Hz and 2 kHz with a sampling rate of 16 kHz. The activity was then recorded by a computer using SystemPlus EVOLUTION software .The cortical excitability of each subject was assessed at baseline and 48 h after the end of the treatment. The following features of the left and right cortices were assessed: resting motor threshold (rMT), motor evoked potentials (MEPs), and intracortical facilitation (ICF).The rMT was defined as the lowest TMS intensity required to induce an MEP with an amplitude of at least 50 microVolts (μV) in at least five out of ten trials .Motor evoked potentials were defined as the overall reaction of a peripheral muscle, as measured by electromyography (EMG), that were induced via TMS of the contralateral motor cortex . The peaIntracortical facilitation (ICF) was performed using two stimuli with an inter-stimulus interval (ISI) of 15 ms (melliseconds) Intracortical facilitation is believed to be mediated by excitatory inputs from glutamatergic pathways . In our For all these excitability parameters, five trials were recorded with an interval of at least 5 s between each trial. For the rMT and MEPs, results were expressed using the mean and standard deviation of the five trials. For the ICF, results were expressed as the ratio of the average of the five MEPs obtained via the ICF paradigm to the average of the standard MEPs .iTBS was applied using parameters described by Huang et al. , except Using an intensity at 80% of the rMT instead of 80% of the active motor threshold (aMT) allows for higher stimulation power. Two of the most cited studies that have used iTBS in the treatment of resistant depression, namely Blumberger et al.  and ColeThree 50 Hz stimuli were repeated at a frequency of 200 ms for 2 s, which constituted a stimulation train. This train was then repeated every 10 s with a total stimulation duration of 190 s, and a total number of stimuli at 600.A Magstim Super Rapid stimulator  was used with a 70 mm double air film coil. For the control group, a sham coil providing the same acoustic sensation and visual impact as the active coil was used. The sham coil stimulated the skin and subcutaneous tissue of the scalp, giving the subjects the sensation of magnetic stimulation. The coil was positioned over the left DLPFC using Brainsight, an MRI-guided neuronavigation software .*256.MRI scans were performed on a 3 Tesla scanner . A 3D T1-weighted sequence was acquired with the following parameters: 176 contiguous slices, slice thickness = 1.0 mm, repetition time (TR) = 2,300 ms, echo time (TE) = 2.98 ms, field of view = 256http://marsbar.sourceforge.net/)  coordinates: x = −50, y = 30, z = 36) . To do tge.net/) . The ROIt-test.Differences between subjects in active and sham arms for sociodemographic characteristics were compared with a Student's We computed the delta (baseline - after stimulation) for each measurement  and for each group (active or sham).We compared the delta in the active and sham group.The Gaussian Distribution of each variable was evaluated by the Shapiro-Wilk test.Some of the variables did not meet the assumptions for parametric statistics  and is why we chose non-parametric statistical tests (Mann–Whitney U) for some. For the normally distributed variables, we performed t-test instead of ANCOVA because our groups were matched for age and gender, thus there was no confound that we could take into account. All statistical analyses were conducted using Python Statsmodels, an open-source library .There were no significant differences in age or gender ratio between active and placebo groups at baseline .The impact of iTBS and sham stimulation on all endpoint parameters is reported in No difference was found between active and placebo stimulation in the rMT, MEPs and ICF in the right and the left cortex. There was also no trend suggesting a difference between the active and the sham arm.We found no difference in all cortical excitability measurements between active and placebo stimulation in the left or right hemisphere. This negative result was contrary to our expectations.The DLPFC has various anatomical projections on M1. These two regions are believed to work closely together to form fundamental circuitry involved in motor tasks of varying complexity . It is aWe therefore hypothesized that repeated sessions of iTBS over the L-DLPFC would result in increased excitability of the ipsilateral M1.Studies assessing iTBS on cortical excitability are scarce, but can be divided into three types: (i) studies exploring TBS impact on cortical excitability after only one session of stimulation targeting the M1, (ii) studies exploring iTBS impact on cortical excitability after repeated sessions targeting the M1, (iii) studies exploring iTBS impact on cortical excitability targeting the DLPFC (a protocol template for treating depression).Regarding (i), Chung et al.  replicatRegarding (ii), Perellón-Alfonso et al.  reportedTo date (iii), only Cao et al.  has asseTriggs et al.  and SpamBrown et al.  found noOur study is the first to evaluate the effect of iTBS over the L-DLPFC on cumulative cortical excitability in healthy subjects, using a “depression stimulation protocol.” Our results do not support any impact of repeated iTBS sessions applied over the L-DLPFC on the excitability of the M1.The result of our study, especially in view of the scarcity of studies on the subject and their contradictory results, is difficult to explain. Nevertheless, some hypotheses are possible.First, although a DLPFC-M1 connection has been widely reported , 25, 26,Secondly, the stimulated target and coil orientation were probably not optimal to test our hypothesis. Indeed, we targeted an area traditionally used for the treatment of depression, whereas it could have been preferable to vary the target area from patient to patient, to identify a specific area more likely to induce excitability in M1.Thirdly, the hypothesis linking the antidepressant effect of rTMS/iTBS to a change in cortical excitability may not be accurate. This is because the antidepressant action may be mediated by other neurobiological mechanisms that are distinct from a simple modulation of cortical excitability, such as modulation in dopamine and/or glutamate, connectivity between non-motor brain regions, modulation of gene expression, de novo expression of proteins, morphological changes, and/or changes in intrinsic firing patterns of diverse neocortical neurons or intrinsic membrane properties .The conclusion of our study, which does not support any impact of repeated iTBS sessions applied over L-DLPFC on the excitability of the M1 should be taken with caution. Despite its original design, our study suffers from several limitations: first, the relatively small sample size may have resulted in Type 2 errors. Secondly, our methodology for collecting cortical excitability parameters is probably not optimal. In fact, we conducted five trials of each variable, but more trials would be preferable. Using Transcranial Magnetic Stimulation (TMS) to assess cortical excitability  can be considerably affected by interpersonal, intrapersonal and intersessional variability, which reduces the sensitivity and reproducibility of this method. Our results showed a significant interpersonal and intrapersonal variability in cortical excitability between the different sessions that was partly due to the choice of conducting an average of only five trials for each parameter. Biabani et al.  reportedTo the best of our knowledge, the majority of studies using TMS or iTBS in physiology or in therapeutic trials use at least five out of ten positive trials (motor response and/or induction of an EMP with amplitude ≥50 μV) to determine the motor threshold. However, the updated IFCN guidelines Rossini et al.  suggest Studies evaluating the cumulative effects of iTBS (or rTMS) on cortical excitability are rare.Bäumer et al.  reportedAnother limitation is the fact that in our study we did not control the timing of inclusion, stimulation and assessment of excitability in relation to the phase of the menstrual cycle, which could be a limitation of our study. Given the potential influence of female hormones on cortical excitability, one could speculate that rMT and other indices of cortical excitability may vary with different phases of the menstrual cycle. Indeed, and despite heterogeneous studies, female hormones probably impact cortical excitability. Smith et al.  describeDespite these limitations, we believe that our study has strengths, and that its original design would benefit from being replicated and improved.To the best of our knowledge, this randomized double-blind controlled study is the first to evaluate the effect of iTBS over the L-DLPFC on cortical excitability in healthy subjects, using a “depression stimulation” protocol with repetitive sessions. Furthermore, this is the largest trial assessing TBS impact on cortical excitability in healthy volunteers. Our study corroborates the safety and the good tolerability of repeated sessions of iTBS.Even with a higher intensity than that initially used by Huang et al.   for the publication of any potentially identifiable images or data included in this article.NB: wrote the article, contributed to the conception and design of the study, carried the statistical analysis, and contributed to the carrying out of cortical excitability procedures. CL: contributed to the writing of the article and the carrying out of the statistical tests. FT: contributed to the carrying out of cortical excitability procedures. PS-K: contributed to magnetic stimulation, participant monitoring and to the carrying out of cortical excitability procedures. VM: contributed to the design of the study and the methodological follow-up. DJ: contributed to the conception and design of the study, as well as the writing of the article. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Apis and non-Apis bee species and on the discovery of previously unknown bee viruses. The natural transmission of bee viruses will be discussed among different bee species and other insects. Finally, the research potential of in vivo (host organisms) and in vitro (cell lines) serial passages of bee viruses is discussed, from the perspective of the host-virus landscape changes and potential transmission routes for emerging bee virus infections.Numerous studies have recently reported on the discovery of bee viruses in different arthropod species and their possible transmission routes, vastly increasing our understanding of these viruses and their distribution. Here, we review the current literature on the recent advances in understanding the transmission of viruses, both on the presence of bee viruses in  Viruses are omnipresent in practically all life forms, where they pose a potential threat to the health of the organism. This also applies for the viruses found in bees. Most of these viruses were originally discovered in honey bees, either through symptoms or diseases associated with infection. Some honey bee viruses can be propagated in, as well as isolated and purified from honey bee pupae. This enabled the initial characterization of the viruses and the development of diagnostic assays. With these early diagnostic assays, it was shown even then that these “honey bee viruses” could also be detected in other bee species and wasps, although no systematic host-range study has ever been conducted. However, recent studies found that those viruses are much more common and widespread than previously suspected, occurring in numerous other hymenopteran and non-hymenopteran arthropods. Furthermore, there are reports of spillover from and between hymenopteran taxa and non-hymenopteran arthropods. Knowing the different possible virus transmission routes and their potential host-range is key to understanding prevalence, epidemiology and virulence in different bee species. Numerous studies have recently reported the discovery and transmission routes of viruses in different arthropod species, including many bee species. The aim of this review is to provide an overview of the recent advances of the impact and transmission of viruses found in bees. We have divided this review into two main parts. The first part, ‘Natural infections,’ summarizes the current knowledge regarding natural infections and the transmission routes of viruses found in honey bees. The second part, ‘Artificial infections,’ provides an overview of the studies involving controlled experimental virus infections and transmission. The major modes and routes of virus transmission in bees are summarized in Horizontal transmission is the transmission of infectious agents among individuals of the same generation. Horizontal transmission of viruses in honey bees includes transmission to different bee developmental stages via oral and/or body contact. It includes indirect infections through contaminated food (food-borne transmission) and contact with feces; venereal transmission, where virus is transmitted from drones to queens during the nuptial flights or by artificial insemination, and vector-mediated transmission, where transmission is mediated by other organisms (vectors). This vector can either be a mechanical or biological vector. A mechanical vector is defined as an organism that transmits viruses without being infected itself, while in a biological vector the virus replicates in the vector organism before being transmitted.Vertical transmission consists of the transmission of viruses to the next generation, which for honey bees is primarily from queens to their eggs. This transmission can be defined as either transovum or transovarial transmission, depending on whether viruses are transmitted on the egg surface or within the egg, respectively. A second form of vertical transmission is transspermal transmission, if the virus is present inside the sperm, which would be the drone equivalent of transovarial transmission. This has so far not been detected in honey bees.Transmission is defined as the establishment of a new infection in a previously non-infected individual, after acquiring the virus inoculum directly from another infected individual, from a vector or indirectly from the environment. It excludes any passive acquisition or retention of virus inoculum that does not cause infection of the bodily tissues.Apis mellifera. A big part of the recent developments in this field allowed for the expansion in knowledge of virus infections in other bee species. Most of these new developments involve bee species that can either be easily reared or are used for commercial pollination services, such as bumble bees (Bombus spp.), stingless bees , mason bees  and leafcutter bees . This section is therefore divided into five subsections: transmission of viruses in Apis mellifera, distribution of bee viruses in other Apis species, recently discovered bee viruses, transmission of bee viruses in non-Apis pollinators and transmission of viruses in non-bee insects.Most of our understanding about natural bee virus infections came from studies with honey bees, particularly Nosema sp. Bees complete the transmission when attempting to remove these feces as part of their cleaning activities.This route is arguably the most common route for bee virus transmission, both within honey bee colonies and between bee species . There aA foodborne transmission pathway involving trophallaxis (mouth-to-mouth sharing of food between colony members) was proposed to exist for Israeli acute paralysis virus IAPV; . LaboratThe hypopharyngeal glands are paired tubular secretory organs in the frontal region of the worker bee head . They seApis mellifera filamentous virus (AmFV) has been detected in honey and pollen  also suggests an oral transmission route. KBV and SBV have been detected in honey, pollen, and royal jelly . Apis med pollen . IAPV wad pollen  while Bld pollen .Nosema apis for infection of adult bees by ingestion  is also considered a form of horizontal transmission, since it still concerns transmission between individuals of the same generation. The close interaction between honey bees with obligate parasites such as endo- and ectoparasitic mites create a scenario where such mites can act as either mechanical vectors  or even biological vectors  for viruses . Picornais woodi  which isis woodi . AlthougVarroa destructor  by feeding on infected pupae and subsequently transmitting the virus to new parasitized pupae . Although replication of intermediate forms for these viruses were detected in varroa mites  profile of these viruses found in mites were different from those found in honey bees. V. destructor-mediated transmission of LSV is also suspected, as the virus is readily detected in the mite, although no causal association has been shown thus far  are also able to parasitise Apis mellifera. Compared with V. destructor, T. mercedesae is much more dependent on the continuous availability of honey bee brood for feeding and reproduction , is a scavenger of honey bee colonies, whose larvae feed on honey, pollen and detritus. It has been shown that SHB can acquire DWV by feeding on DWV-infected brood and bees, by topical contact with DWV-contaminated wax and by exploiting trophallaxis between bees. The occurrence of high DWV titers in SHB suggests that it could be a true host, and possible biological reservoir, for DWV. Since SHB consumes bees, the detection of DWV replication intermediates (negative-strand RNA) is by itself not conclusive evidence for biological-vector status, similar as for varroa, but supports the evidence of quantitative increase in DWV titers  and in all pools of 50 eggs from these queens (N = 86) testing DWV positive. Moreover, For honey bees, vertical transmission involves the transfer of viruses from queens or drones  to their offspring. Several studies reported detection correlated distribution of viruses in queens and their eggs, which implies vertical transmission. For instance, BQCV and DWV were detected in all analyzed queens , BeeMLV, and/or ARV-1/BRV-1.The present knowledge about which bee viruses are transmitted through various transmission routes is summarized in Apis spp.), which share ecological habitats and geographic ranges, particularly in south-east Asia. The most prevalent viruses in Apis species are DWV and BQCV, which for historical reasons were first described in the European honey bee, A. mellifera  (A. florea and A. dorsata) than BQCV  . Neverthhan BQCV . BQCV anA. cerana was first shown in bees from India  technologies  was described and identified as a virus closely related to LSV. ARV-1/BRV-1 was found in Bombus sp.  and Bombini (bumble bees). Both tribes have a social structure similar to honey bees, with a single reproductive queen and non-reproductive worker bees attending the brood and foraging. A social nest structure creates a high contact between nest mates which facilitates virus transmission. Bees of the family Halictidae are widely distributed. This family displays a plasticity of social behaviors ranging from solitary to communal, semi-social and primitively eusocial  parasitize non-Apis bees. However, non-Apis bees are host to a plethora of other parasites, ranging from ecto-parasites to parasitoids, that could potentially vector viruses. DWV has been identified in several species of parasitoid phorid flies, i.e., Megaselia scalaris and Apocephalus borealis, with evidence of replicating virus in the larvae of Megaselia scalaris  may have come with the bee cadaver, rather than from the wasp/ant host tissues. Similarly, replicative strands of the DWV genome were detected in Myrmica rubra  are considered to be Batesian mimics of adult honey bees. They feed on flowers, effectively imitate honey bee behavior  or were acquired passively through feeding on infected bees or contaminated material .This section deals with the deliberate, experimental infection of bees with controlled amounts of virus, using a variety of inoculation techniques, trying to mimic a natural transmission route. This requires a source of relatively pure virus and, ideally, uninfected experimental bees. The success of the infection procedure is tested either through molecular evidence of replication of the inoculated virus, or more commonly a significant quantitative increase in post-inoculation virus titer that can only be attributed to the inoculum. A number of negative control inoculations are therefore also required, to rule out alternative sources of infection or virus titer increase.in vivo (bees) or in vitro (cell cultures) propagation of natural virus isolates , and the virus was restricted to the abdominal organs  established high levels of DWV infection  solution or PBS (7.8 × 105 genome copies/μl). Over 90% larval mortality was achieved by 96 h post infection. When comparing transmission for both DWV and SBV, 10 SBV and 1010 DWV genome equivalents for young larvae , and 106 SBV and 106 DWV genome equivalents for pupae (injection). feeding . Extrapoortality . Electrobee head . Liu et 7 BQCV genome equivalents per larva and 1.4 × 109 genome equivalents per bee) to look for interactions between pathogens (BQCV and N. ceranae) and between the virus and pesticides (9) caused higher mortalities of the larvae but no impact on adult worker survival was observed  or 2 days old workers  was equivalent to about 102 virus particles per bee when preparations were injected into the haemocoel compared to more than 1011 virus particles per bee for feeding and 108 to 109 particles per bee for spraying, illustrating the high virulence of ABPV upon injection.in vitro injection of honey bee pupae with 104 viral genome equivalents of an IAPV-enriched extract resulted in symptomatic infection.9 IAPV genome equivalents in 30 ml sucrose were used to obtain colony infections) .Oral infections with IAPV can be achieved by feeding IAPV-sucrose solution to honey bees in cage experiments and in honey bee colonies . Approxiections) . IAPV-enAn inoculum containing a mixture of viruses, enriched for SBV and IAPV with low levels of BQCV and DWV was able to produce high levels of mortality within 3 days of oral infection in newly emerged honey bees. Further investigation concluded that the bees died from the extremely high titers of IAPV, and not from other viruses .12 viral particles  than larvae exposed to V. destructor  and 99% (LD99) mortality in bees was 362 and 2266 virus particles, respectively. A time course of SBPV replication in pupae showed that the virus was first detected 42 h after injection. No significant differences were found in the overall proportion of pupae that became infected when mites were introduced over a period of 5 or 10 days, irrespective of the season (July or September). The proportion of pupae infected with SBPV declined significantly with each mite transfer over time, with the majority of pupae becoming infected after the first few mite transfers. These experiments suggest that transmission of SBPV does occur during mite feeding. Furthermore, they conclude that because SBPV is highly virulent as it kills the bees before any dramatic increase in virus titer. This results explains why low SBPV titers exist in honey bee colonies even in the presence of V. destructor  originating from nfection . Althougobserved .Bombus and other pollinator species  infections can be performed either by direct injection of the virus in the bee, or through oral administration. In the following sections, we provide a short overview on the application of different infection techniques to look at cross species infection potential.Many studies have shown that viruses first identified in honey bees could also be detected in B. terrestris. Similarly, B. terrestris. The injection of SBPV particles, on the other hand, has a much less lethal effect on B. terrestris.Bombus species, published experimental work is still scarce. B. terrestris result in an infection and lead to 50% mortality 10 days after injection.The micro-injection procedure used in bumble bees is similar to that used in honey bees. Firstly, bees are anaesthetized by putting them in the freezer for 10–20 min, subsequently bees are injected using a micro-capillary in the soft tissue between the first pair of sternites . Niu et Osmia sp. have been used to test cross-species infectivity of DWV through injection. O. cornuta with extracts DWV infected honey bees. The bees were injected into the thorax, and later replicating DWV could be detected in the abdomen of the infected bees but not in their heads.Other than bumble bees, Apis bees. In nature cross-species infection most likely occur due to feeding on flowers contaminated with fecal matter containing virus particles (vide supra for transmission pathways).Feeding virus particles to bee species other than honey bees, more closely resembles the natural transmission process, as vectors injecting virus are not described for non-B. terrestris with KBV and IAPV using 1 × 107 and 0.5 × 107 virus particles, respectively. Similar orders of magnitude were reported for oral infection of B. terrestris with IAPV in other studies , which led to symptoms at colony (fewer emerging workers) and individual level (impaired locomotion and movement speed).Historically, starting with the work of Louis Pasteur on rabies in the 19th century, serial passage of viruses in alternative hosts has been used for attenuating their virulence, in order to develop low-virulence virus strains for use as live vaccines. The principle is that after the serial passage of a virus to another host species, a loss of virulence is experienced when inoculated again into the original host as the virus becomes adapted to the alternative host and less to the original host. However, serial passaging has also been used to select strains with increased virulence . Serial passaging of bee viruses was initially used to identify the infectious agent associated with different disease symptoms in honey bees, most commonly paralysis, and subsequently also to maintain infective virus stocks for experimentation and raising diagnostic antisera. This work started in 1945 with single passages of extracts from paralytic bees  revealed a similar virus dynamic as individual honey bee (Insect-derived cell cultures have advanced our understanding of insect physiology, development biology, pathology, and molecular biology . With thvirology . It willvirology . Recentlvirology . A monollication . The resoney bee . These roney bee .Although much is known about both the natural transmission routes and the artificial propagation of bee and bee-related viruses, much more still needs to be clarified. Most of our knowledge about bee virus transmission and infection has been derived from the most common honey bee viruses, i.e., those that also cause disease. The challenge is to elucidate the infection and transmission strategies of the much larger number of apparently non-pathogenic newly discovered viruses, as well as their biological and ecological importance to their hosts. Many of them will probably also use the most common transmission routes identified for the disease-causing viruses. The developmental stage and tissues targeted by these viruses will be important for both their transmission strategy and their effects on the host. Mechanical and biological virus vectors can breach the anatomical and physiological host barriers to virus transmission, with potentially drastic consequences for host health, virus virulence evolution, and applied vector-virus virulence management . Also imOY, AD, and NC conceptualized the study. OY, NP, AD, JM, DP, and NC investigated the study. OY, NP, AD, and NC contributed to methodology. OY, NP, AD, JM, PC, DP, EA, GS, DS, and NC wrote the original draft. OY, NP, AD, JM, PC, DP, EA, GS, and NC reviewed and edited the manuscript. OY, NP, AD, JM, PC, DP, EA, GS, DS, and NC approved the final version of manuscript to be published.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Coronaviruses (CoVs) assemble by budding into the lumen of the intermediate compartment (IC) at the endoplasmic reticulum (ER)-Golgi interface. However, why CoVs have chosen the IC as their intracellular site of assembly and how progeny viruses are delivered from this compartment to the extracellular space has remained unclear. Here we address these enigmatic late events of the CoV life cycle in light of recently described properties of the IC. Of particular interest are the emerging spatial and functional connections between IC elements and recycling endosomes (REs), defined by the GTPases Rab1 and Rab11, respectively. The establishment of IC-RE links at the cell periphery, around the centrosome and evidently also at the noncompact zones of the Golgi ribbon indicates that—besides traditional ER-Golgi communication—the IC also promotes a secretory process that bypasses the Golgi stacks, but involves its direct connection with the endocytic recycling system. The initial confinement of CoVs to the lumen of IC-derived large transport carriers and their preferential absence from Golgi stacks is consistent with the idea that they exit cells following such an unconventional route. In fact, CoVs may share this pathway with other intracellularly budding viruses, lipoproteins, procollagen, and/or protein aggregates experimentally introduced into the IC lumen. Enveloped animal viruses have been frequently used as models to study the secretory process, since the viral membrane proteins hijacking the host cell’s transport machineries follow the same intracellular itineraries as their endogenous cellular counterparts. Accordingly, after their synthesis in the endoplasmic reticulum ER, see , membrancisternae, although frequently found in their vicinity [cisternae, typically occurring at their dilated rims [Assembly by budding into the IC lumen is a general hallmark of CoVs. Originally, electron microscopic (EM) studies of cells infected with mouse hepatitis virus (MHV) demonstrated the intracellular assembly mode of these viruses. It was initially concluded that CoV budding takes place in association with the major organelles of the secretory pathway, the ER and/or the Golgi apparatus, although virus particles were also seen in smooth-walled vacuoles of unknown character. Notably, no budding was observed at the PM . To detevicinity . Moreovevicinity . During vicinity . Interested rims ,10. cis-Golgi [cis-Golgi resident and a master regulator of ER-Golgi communication and Golgi organization [Subsequent studies showed that the pre-Golgi compartment where CoV budding takes place corresponds to the IC, which had been introduced as a novel sorting station in bidirectional ER-Golgi trafficking ,12,13. Ais-Golgi . Notablyis-Golgi . Besidesis-Golgi ,19. Of tnization ,21,22,23nization . The twonization . cisternae, virus budding also occurs at p58/ERGIC-53-containing peripheral and central IC elements [Although budding at the IC is frequently brought up as one of the distinctive features of CoVs, it seems likely that their assembly or release strategies are shared by a number of viruses that bud into the lumen of endomembrane compartments. The IC has been implicated in the complex envelopment of large DNA viruses, such as herpes and vaccinia virus ,26,27 anelements . Moreoveelements , resulteelements . Similarelements . Moreoveelements ,36. cis-Golgi membranes [Flaviviruses, such as hepatitis C virus (HCV) and Zika virus, bud into the lumen of a modified ER-derived compartment—or an “ER-Golgi hybrid compartment” —and are embranes ,13, is rembranes . Finallyembranes ,41. In summary, the above studies clearly indicate that the assembly and/or exit strategies of CoVs are shared by other intracellularly budding viruses. In particular, the late steps in the replication of bunyaviruses and CoVs may in fact be more similar than has previously been considered, as also indicated by the suggested incorporation of p58/ERGIC-53 into both types of viral particles . HoweverThe pleomorphic CoV virions display considerable size heterogeneity, ranging from 100 to 160 nm in diameter A. They cIn analogy to viruses budding at different PM domains of epithelial cells , the intcis-Golgi region [trans-Golgi/TGN [cis-Golgi localized COPI  revealed motifs in its first transmembrane segment that dictate its retention to the IC/i region ,57. Howeolgi/TGN ,58,59. Oolgi/TGN ,60. The COPI See -coats coThe observed differential localization of the M protein illustrates that the retention of CoV envelope proteins does not suffice to determine the site of budding, although it is most likely a major requirement ; therefoInterestingly, studies carried out with IBV have provided evidence for the existence of two functional pools of the E protein in CoV-infected cells. An oligomeric pool participates in virus assembly, while a monomeric pool—evidently by interacting with a host component(s)—neutralizes the luminal acidic pH of secretory compartments. Paradoxically, the activity of this monomeric pool not only supports virus release but also induces Golgi disassembly ,73,74 , where they are thought to be sorted into specialized post-Golgi carriers for subsequent delivery to the cell surface [A common denominator of these two transport mechanisms for CoVs is that they both require the structural integrity or functionality of the Golgi stacks. For example, Golgi perturbation by monensin inhibits the release of the CoV transmissible gastroenteritis virus (TGEV) . Thus, a surface   implicated here in CoV egress see  are knowHow about other viruses budding intracellularly? Although there are conflicting reports on the effect of BFA on flavivirus release, some studies suggest that it inhibits productive infection only when added early after the start of infection, but loses its effect if added later, during maximal virus assembly and release ,42,106. The endosomal recycling apparatus—consisting of both peripheral REs and the pericentrosomal endocytic recycling compartment (ERC)—has been shown to support the assembly and release of many viruses ,108. IndIn conclusion, the observations regarding the effect of BFA on virus egress and the role of the endosomal recycling system and Rab11 in this process collectively support our model on a Golgi bypass route for virus release  that—bescisternae. As shown by studies of IBV, this morphological change is most likely the outcome of one of the activities of the E protein, which by interacting with host protein(s) neutralizes Golgi pH [cisternae could also be caused by the luminal accumulation of large-sized virus particles [cisternae, the E protein activity could bring about neutralization of the acidic IC elements and REs [Obviously, the above presented models on virus release illustrate the early stages of CoV infection, preceding virus-induced Golgi fragmentation and disassembly of the cisternal stacks  9,72,80,115. TheGolgi pH , and thearticles . The proarticles   ,127,128.isternae ,132,133.cis-Golgi localized transport machinery, including Rab1 and its partners GM130 and GRASP65 [Interestingly, however, studies of flaviviruses have yielded results supporting the general idea that intracellularly budding viruses hijack pre-existing pathways and transport mechanisms that are normally employed by physiological large cargo . For exa GRASP65 . Initial GRASP65 ,139; howcisternae, possibly employing the same transport machinery—such as COPI, Golgi tethers and SNAREs—that mediates intra-Golgi trafficking by traditional small transport vesicles [The actual mechanisms for Golgi passage of large cargo remain poorly understood. As discussed above, the rim progression hypothesis postulates that the “megavesicles” containing artificial protein aggregates bud from and fuse with the ends of Golgi vesicles . The provesicles . Alternavesicles . cis-Golgi localized luminal aggregates could reach the trans-Golgi by being successively incorporated into a series of large transport carriers. The model in S. cerevisiae based on cisternal maturation [trans-Golgi and endosomal characteristics [Surprisingly, the megavesicles carrying protein aggregates were shown to establish a “fast track” across the Golgi stacks . Indeed,turation ,144,145.turation  traditioeristics . Previously, we have suggested that the noncompact zones alternating with the cisternal stacks in the Golgi ribbon are occupied by pleomorphic IC elements and REs, which by operating in the biogenesis of the Golgi stacks dynamically join them into a continuous structure. We further argued that these “linker compartments” also communicate with each other, establishing a passageway for bidirectional trafficking across the Golgi ribbon that bypasses the cisternal stacks . Here, wThe study of Ghosh and coworkers further showed that, instead of following the Golgi-dependent constitutive secretory route to the extracellular space, β-CoVs employ the endo-lysosomal system for their cellular egress . HoweverFinally, a more general note concerning the use of viruses as models in cell biology. Since the mid-1970s, infection of cultured cells with various enveloped viruses was increasingly employed to study both the endocytic mechanisms operating during virus entry and the secretory apparatus that viruses exploit to gain exit from their host cells ,148,149.The ongoing COVID-19 pandemic, triggering an intensive search for pharmacological treatments to combat the disease—particularly, through repurposing of existing drugs—has been a clear reminder of the great importance of detailed knowledge on the different phases of the virus life cycle. To find out how a particular intracellularly budding virus harnesses the secretory mechanisms of its host cell for its own purposes one should focus on the early stages of infection—using as intact virus as possible—thus allowing the viral structural proteins to undergo unperturbed assembly in the host cell environment and acquire the post-translational modifications that they would naturally receive. However, to overcome the general effects of virus infection on the host cell—affecting over time the operation of membrane organelles and transport pathways—it can also be of interest to follow the assembly and release of VLPs or fluorescent virus particles by employing live cell imaging . The res


Curruca communis) and Culex mosquitoes in the Netherlands, 2020Detection of West Nile virus in a common whitethroat (" by Sikkema et al, published on 8 October 2020, corrections were made on request of the authors: The sentence “Mosquitoes can acquire the virus by feeding on a viraemic bird and can transmit it to other birds, animals or humans following an intrinsic incubation period” was changed to “Mosquitoes can acquire the virus by feeding on a viraemic bird and can transmit it to other birds, animals or humans following an extrinsic incubation period”. The affiliations for Tjomme van Mastrigt were corrected from 4,8,9 to 3,8,9. These changes were published on 27 October 2020.In the article “


The choice of a group decision-making rule is one of the most important political issues. Buchanan and Tullock have provided a framework for analyzing the optimal k-majority rule from the perspective of “methodological individualism.” They proposed the concept of “external costs” and “decision costs” and argued that the optimal k-majority rule takes place where the sum of these two costs–“total costs”–is minimized. Despite the fact that the approach is widely accepted as a tool for dealing with public decision-making rules, the study of formalizing these two costs in a quantitative manner has been relatively rare. We propose a systematic way of modeling these costs considering the assumptions mentioned by Buchanan and Tullock. We find that the resulting shape of the graphs is generally similar to that of the Buchanan-Tullock model, except for some minor details. Then, using this analytical model, we investigate several factors that could affect Buchanan-Tullock’s two costs and the optimal k-majority rule. We show that “clustering of disadvantages”  and “loss aversion”  could increase external costs in Buchanan-Tullock’s model. These factors can result in a separation between the theoretical and actual optimal k-majority rules. Meanwhile, some recent developments in information and communication technologies can not only decrease decision costs, but also increase the same costs simultaneously through amplified “group polarization” . If the effect of the former is not the same as that of the latter, this leads to a difference in optimal k-majority rules as well. These discrepancies bring us to the dilemma of “public choice before public choice.” Since the dawn of history, human beings have formed communities to prevent harm and achieve cooperation. While the decision maker and the affected are the same in individual decision-making, this is not true in group decision-making. Mankind has continued searching for the ideal group decision-making rule. Many communities have chosen “simple majority rule” as the best decision-making method. However, a simple majority rule conflicts with the modern notion of the free and equal human beings, given that all opponents are affected by a bill even when there is just one more proponent . WickselThis approach is theoretically desirable, but has two weaknesses in reality. One is its high cost and the other is the room for disagreement by strategic actions. Accordingly, it is necessary to find a different approach. Some thinkers have pursued a way of defining a social welfare function that would be considered ideal from a moral standpoint. Rawls is a prime example. Rawls assumed that when a rational person is in the situation of “veil of ignorance,” he/she must choose “maximin principle” as his/her code of conduct . This prThe Calculus of Consent [k value. Like any other cost-based approach in economics, Buchanan and Tullock insisted that the optimal k-majority rule should be at the point where the value of “ interdependent costs” or “total costs” for short, which is the sum of external and decision costs, is minimized. In other words, this optimal point is where the absolute value of the marginal external costs (MEC) equals that of the marginal decision costs (MDC). Otherwise, when we change the k-value, a decrease in one cost is different from the increase in the other, which means it is not at the optimal point.Buchanan and Tullock addressed these problems by introducing the concepts of “external costs” and “decision costs” in  Consent . They deex ante, the contradiction between simple majority and unanimity rules can be resolved. This is because enacting legislation does not necessarily have to follow a unanimous rule while achieving a unanimous agreement under a constitution. In addition, unlike Rawls, they thought that the members of a community do not necessarily adopt a single social welfare function. Departing from the moral and metaphysical assumptions, Buchanan and Tullock respected the diverse preferences of each individual. Their idea that we should decide public choices not through a holistic view but by an aggregation of individual preferences is called “methodological individualism.” They stressed that if liberal and rational individuals exchange their preferences in each legislation by “log-rolling,” which means the trading of votes, the final state may be Pareto optimal and there is no discrepancy between economic and political rationalities. The study of non-market decision-making, including political one, using economic methodologies is called “public choice theory” [The Calculus of Consent since they wrote the book. In particular, James M. Buchanan won the Nobel Prize in economics in 1986 for his contribution to public choice theory [Buchanan and Tullock thought that if all community members agree with the best k-majority rule  theory” . The twoe theory –10.Since the pioneering work of Buchanan and Tullock, researchers have conducted a number of studies on the theory of public decision-making rules. Kafoglis and Cebula extended the Buchanan-Tullock model to consider group size and preference heterogeneity . BoudreaIn this paper, we propose a systematic formulation of external and decision costs in a more unified manner. To this end, we note that decision costs are proportional to the probability of not passing a proposal. Decision and external costs are symmetric because the latter are proportional to the probability of passing a proposal. We add one more component to this here. Some factors affecting these costs that Buchanan and Tullock did not reflect in their model will be considered in our model in the following discussion.Buchanan and Tullock also recognized the existence of these factors, but probably thought that their effects would cancel each other out. This idea is also a general concept of neoclassical economics. However, if each of them is not a simple error (noise) but a kind of systemic human behavior (bias), we should consider it in modeling public decision-making rules. Adding these factors to Buchanan-Tullock’s model provides a more realistic optimal k-majority rule, which is different from the one in their original model. In this regard, we discuss some examples in terms of the two costs. First, we will examine the “clustering of disadvantages”  and “loss aversion” , regarding external costs. Second, we will examine some recent rapid developments in information and communication technologies (ICTs) and following amplified “group polarization,” in terms of decision costs . We point out that the separation of the optimal points brings us to the dilemma of “public choice before public choice.”N individuals trying to design a public decision-making rule. The rule determined at the constitutional stage affects all of the votes during the parliamentary stage. It is assumed that all individuals are so rational that they agree on the optimal k-majority rule, where total costs are minimized. This presupposition is one of the core assumptions of Buchanan and Tullock’s theory. For convenience of discussion, the optimal point is assumed to be single.Let us consider a group of p or refuse to accept it in favor of the status quo with the probability of 1–p. We suppose a situation in which voters do not know specifically whether they would approve or reject each bill at the constitutional stage, and the only thing they know is its probability distribution. This is named as “veil of uncertainty” by Buchanan and Tullock. Strictly speaking, they did not mention exactly what the meaning of “uncertainty” was in The Calculus of Consent. In economics, uncertainty refers to the state in which an actor is fully ignorant or roughly knows the probability distributions [p is a constant at 1/2 in our current model. This means no ex ante information, which is “the principle of equal a priori probability.” This supposition will be further refined and generalized below.In our model, all people offered a specific bill have two alternatives. It is assumed that they favor the proposal with the probability of ibutions . We chooibutions . Applyins is a random variable representing the number of “yea” votes in N Bernoulli trials. Then, the probability that the proposal will pass under a given k-majority rule can be written asFor this social system, the number of “yea” votes follows the binomial distribution given byThe probability of not passing the proposal can be expressed in the same way as in These two probabilities are shown in To be more exact, “external costs” and “decision costs” are expected values because they occur after the votes, not at the rule-making stage. In this regard, Dougherty and Edward  defined Here, we assume that the relative weight of the expected external and decision costs is the same. Under some circumstances, an individual might place more weight on one cost than on the other. However, such a change does not affect the overall logic structures, except for a few details.k > 50, so that it is no longer a logit function. As a result, the value of the expected total costs, the sum of expected external and decision costs, is minimized at k = 58.The two costs are shown in e1, to show this effect. The expected external costs with the additional parameter e1 can be expressed asThese formulas are not perfect because everyone’s costs are considered to have an equal unit value. Under this condition, the assumption of diverse preferences introduced by Buchanan and Tullock is nullified. In the real vote, there may be a difference in the intensity of preferences for each proposal. Therefore, we now examine several subtleties mentioned by Buchanan and Tullock that the model from the binomial distribution did not reflect. Suppose a specific proposal with the k-majority rule has been passed. In e1 has a constant value greater than or equal to 1, and if k = N, the entire term containing e1 equals 1 (perfect collective intelligence). As we saw earlier, there is no expected external cost in this case.The parameter d1 to reflect it. The expected decision costs with parameter d1 can be represented asNow, consider the opposite situation of persuading those who disagree to pass the same bill under the given k-majority rule. The smaller the number of remaining opponents, the more difficult it will be to persuade each of them. This is because, like game theory, we think and act strategically. However, if the k-value is low, there are many candidates to persuade, so their power or the possibility of strategic behavior is not that high. Therefore, we introduce an additional parameter d1 also has a constant value greater than or equal to 1, and if k = 0, the entire term containing d1 equals 1 (non-strategic situation). Similar to the previous analysis, there is no expected decision cost in this case.The parameter k < 40 in k increases, as shown in k = 58, which is the same as the one in e1 and d1 were set to 1.1.With the refining parameters included, the two costs from Eqs  and 7)  7) are se2 and d2 are used to represent these effects on the external and decision costs, respectively, asA similar but separate logic, as we introduced in Eqs  and 7),,7), can e2 = 1.1, and d2 = 1.1. The resultant curves in Now, we will generalize our model assuming that voters can have different probability distributions depending on the quantity and quality of information that they have at the constitutional stage. Each community may have different levels of information, but all members in the same community share similar contents. We also assume that the utility is a random variable and has a different value for each extraction. In this way, individual expected utility can be modeled with a certain distribution, and each utility is extracted from the given distribution function. No matter what type of probability distribution you choose, it is in line with our discussion. If each extraction is independent, individual preferences at the parliamentary stage are not always the same. This assumption of “independent and identically distributed” virtually describes the internal and external independence of preferences in neoclassical economics. In addition, this attempt does not fundamentally violate the assumption of “veil of uncertainty.” In principle, simulation methods should be used for this approach. However, to keep it simple, we use mean values from distributions to reflect these effects in analytical expressions.i of society has his/her utility μi ranging from 0 to 1. The utility is randomly drawn from a predetermined distribution. The larger the utility is, the greater the tendency to agree with the proposal is. If an ith individual’s utility is greater than 0.5, he/she will be in favor of the proposal. Otherwise, he/she will choose to maintain the status quo. We thus obtain the probability of the ith individual’s vote for yea asLet us consider a case in which each member The number of individuals voting for yea is then given byi, whose utility is μi. He/she would perceive his/her losses caused by passing a proposal as 1 − μi. Even those who approve the bill could expect this type of loss according to their own utilities. From this point of view, we propose redefining expected external costs as expected losses per voter. Instead of using each realized utility from repetitive simulations, we approximate utilities with the mean value of some types of distribution functions. To distinguish the utilities of proponents from those of opponents about an issue, we calculate the mean values of their utilities separately. Although this approximation seems to be crude, we note that the errors would be negligible as the number of individuals increases.We now redefine the expected external costs using an individual’s utility. Let us consider individual μi equals 1. Except for it, even those who approve a bill need to be more convinced to be perfect proponents. Thus, we can redefine expected decision costs as the average losses  per voter. It is 1 − μi once again. The expected external and decision costs can be expressed asLikewise, we can redefine the expected decision costs. The ideal state of proponents is achieved when f(μ) is an arbitrary distribution function for utility.Here, μ ≤ 1. The mean losses μ to 0 ≤ μ ≤ 1. Mean losses for opponents k value is 58, which is the same as that in Figs We perform some calculations on two types of utility distributions: uniform and normal distributions. First, consider a uniform distribution that is defined for 0 ≤ from Eqs  and 13)μ ≤ 1. ThWe can see that the results of the present model are generally in good agreement with those of Buchanan and Tullock’s theory. Specifically, in our model, as the k-value increases, the external cost function decreases, the decision cost function increases, and the shape of the total cost function is convex. These are the essential characteristics of Buchanan and Tullock’s model. In addition, as we mentioned, our model is derived not from simulations but from analytic results. Although our model is approximated in some cases and slightly complicated by introducing some parameters, the core of Buchanan and Tullock’s model remains and it does not depend on a specific single parameter.Several factors affecting the two costs of group decision-making can be examined using the new analytical model introduced in the previous discussion. In the following section, we examine two factors regarding external costs and one factor regarding decision costs. The former concerns the clustering of disadvantages  and loss aversion  that can affect external costs, which can lead to the difference between theoretical and actual optimal k-majority rules. The latter concerns the conflicting impact of ICT  that can affect decision costs, which can result in the same kind of difference between theory and reality.All individuals belong to groups with different influences on a variety of social problems. Buchanan and Tullock supposed that each voter is frequently involved in distinct interest groups for each issue, so that one voter may have little power in one proposal, but he/she can have greater influence in another. They thought these effects would cancel out each other by mutual political agreements known as “log-rolling.” Is that true in reality? Rather, once the “veil” is removed, groups that exert a strong influence on one agenda are more likely to exert significant influence on other issues. Therefore, an individual who once belonged to a low-power group continues to predict that his/her opinion will be opposed to that of society. Wolff and De-Shalit expressed this as the concept of “clustering of disadvantages” . If thisec to represent the extent of the clustering of disadvantages effect. The parameter greater than 1 gives an additional weight to external costs. In Eq  is basedWTP) in decision-making is always the same as willingness to accept (WTA) of the choice. There is one major problem with this: Homo economicus always has all the information regarding his/her choices. However, this contradicts Buchanan and Tullock’s aforementioned assumption of “veil of uncertainty.” In addition, if we assume that he/she has perfect information, the concept of “rational ignorance,” which is widely used in the field of public choice, becomes useless. To avoid this problem, we use the concept of rationality as “bounded rationality” in the field of behavioral economics. Behavioral economists have found that humans do not usually have perfect information, and often depend on heuristics (system 1), which could lead us to cognitive bias [We have treated optimal public decision-making rules under the assumption that all voters are “rational.” Buchanan and Tullock did not specify exactly what rational decision-making meant, and the conception of “rationality” can be considered in several ways. First, we may take the view of “homo economicus” which is commonly used in the public choice theory. It is also a typical decision-maker in neoclassical economics. Homo economicus has “perfect” rationality, willpower, and self-interest so that he/she does not regret his/her own decision . This meive bias . Bias isive bias . In theiive bias . Note thλ and α are parameters controlling the loss aversion effect. The values of the parameters used are α = 0.88 and λ = 2.55, estimated by Tversky and Kahneman [Therefore, the irrational aspect of human behaviors that we have not considered in our model could make a difference. Let us consider an example of land expropriation. When a government decides to expropriate private land, the owner of the land can demand just compensation which is stipulated in related laws. However, in this situation, what is “just compensation”? If we accept the existence of loss aversion, the land owner may think that just compensation means WTA. However, the others who vote for that the land should be expropriated maybe regard just compensation as WTP. The optimal k-majority rule Buchanan and Tullock derived is divided into the “subjective” optimal rule that the owner of land supports and the “objective” one that the others support. In our model, the first idea leads to the effect of raising external costs overall when the government passes a specific bill. It can be expressed asKahneman .A graphical representation of this effect is illustrated in Vragov and Kumar investigated the effect of information and communication technologies (ICTs) on the costs of group decision-making . These aHowever, much uncertainty still exists about the relationship between technologies and the effects of communication among citizens. Recent advances in ICTs have not only facilitated better negotiations but also brought about more polarization of group opinions than ever. This effect is well-known as the “echo chamber” or “filter bubble” –30. The di as the “dialogue effect,” which symbolizes the effect that much communication makes us more cooperative, and dp as “polarization effect,” which symbolizes the effect that excessive discussion confined to only the same-minded groups makes us more prejudiced. If the former effect is greater than the latter, the decision cost function shifts to the right, and the optimal k-value increases as depicted in We define We have introduced a new theoretical framework for calculating the expected external and decision costs associated with public decision-making. Our analytical model is based on a binomial formula and supposes that the two costs are proportional to the number of members associated with a specific proposal. We have confirmed that our theoretical model yields results that are similar to those of the Buchanan-Tullock model in a number of situations. The main advantage of our new method is that it does not depend on a specific parameter like previous studies did. Our model is also advantageous for introducing multiple variables that represent assumptions not covered in previous works. At the same time, our method is analytic, so it does not require any simulation methods. Although we approximated by using mean values of continuous utility distributions to incorporate continuous distribution into analytical models, the benefits of sophisticated quantitative group decision-making models can outweigh such a small shortcoming. Further research could be conducted to test and improve our findings in the real world.In addition, we applied our framework to a couple of examples of reality. Buchanan and Tullock’s theory did not clearly mention the meaning of “rationality,” so we can consider various conceptions of it. Then, there can be separations between theoretical and actual public choices. In terms of external costs, we found that the existence of clustering of disadvantages  and loss aversion  makes external and total costs and the optimal k-value higher than Buchanan and Tullock thought. Meanwhile, in terms of decision costs, some rapid developments of ICTs can increase or decrease decision and total costs and the optimal k-value compared to those of Buchanan and Tullock’s original model. We handled a case in which technologies make communication smooth or group thinking more extreme. In these situations, we should make a choice of the socially desirable k-majority rule between the two optimal points. This brings us to the dilemma of “public choice before public choice,” because such a (meta) public choice should be decided before a public decision-making rule is decided, which is when the voters are in the veil of uncertainty. As we previously mentioned, public choice theorists, including Buchanan and Tullock, generally believe political choice can be explained entirely by economic analysis. However, if the dilemma exists in reality, they cannot be completely independent of political decision-making.