Hepatitis A is an acute infection of the liver, which is mostly asymptomatic in children and increases the severity with age. Although in most patients the infection resolves completely, in a few of them it may follow a prolonged or relapsed course or even a fulminant form. The reason for these different outcomes is unknown, but it is generally accepted that host factors such as the immunological status, age and the occurrence of underlaying hepatic diseases are the main determinants of the severity. However, it cannot be ruled out that some virus traits may also contribute to the severe clinical outcomes. In this review, we will analyze which genetic determinants of the virus may determine virulence, in the context of a paradigmatic virus in terms of its genomic, molecular, replicative, and evolutionary features. De Morbis Internis” an illness characterized by episodes of jaundice. Similarly, in ancient China jaundice disorders were also recognized. However, the first accurate reference to epidemic jaundice was documented by Cleghorn [Hippocrates, ≈400 years BC, described in his treaty “Cleghorn  in “EpidAll types of hepatitis firstly occur as acute infections, with or without symptoms. However, while hepatitis B, C and D often develop chronic infections, hepatitis E only does it occasionally, and hepatitis A never becomes chronic.Hepatitis A is an acute infection of the liver, mostly asymptomatic or subclinical among children under 5 years, but usually proceeding with symptoms in older children and in the adulthood . The infA clinical case is defined by elevated serum bilirubin and aminotransferases levels that may be preceded by moderate symptoms including fever, malaise, anorexia, nausea, abdominal discomfort, dark urine and jaundice . The incThe underlying reasons associated to these different outcomes are still not fully understood and are likely related to host factors such as the underlying hepatic diseases and age ,7, and/oHepatovirus A and belongs to the Hepatovirus genus within the Picornaviridae family . This is a recently updated classification responding to the need of include the newly described hepatitis A viruses [The etiological agent of the human hepatitis A is the hepatitis A virus (HAV). Taxonomically, is named  viruses . The HAV viruses . The 5ʹN viruses  of the s viruses . Further viruses . Near th viruses . FinallyHowever, despite this genome organization characteristic of picornaviruses, some traits contribute to the uniqueness of HAV. First, the HAV IRES is distinct among picornaviruses and constitutes the type III model ,18,19, wAnother distinctive point is the occurrence of a single serotype due to severe capsid structural constraints, which prevent the emergence of new serotypes. The effect of codon usage on capsid folding may contribute to these structural constraints and in turn to the antigenic stability ,30.The very special genome composition of HAV is also revealed in its very low GC and GC3 contents, 37% and 26%, respectively, vs the theoretical 50% ,31. AddiHAV exists in a dual phenotype , naked aThe hepatitis A infection is transmitted through the fecal-oral route, and the infection cycle starts with the ingestion of naked particles. These particles are highly stable in the environment, for instance high infectious titers can still be detected after 60 days of desiccation on surfaces at room temperature  or in soHAV infects hepatocytes, but how the virus reaches the liver from the gut is still not understood. Two hypothesis have been proposed to explain how HAV crosses the intestinal barrier into the blood: replication in epithelial intestinal cells or transcytosis through “M” cells ,45,46. RMoreover, naked particles devoid of IgAs, enter the hepatocyte by clathrin- and dynamin-dependent endocytosis in a process facilitated by integrin β1, and traffic to late endosomes where the process of uncoating is initiated . Egress The quasi-enveloped virions are also similarly endocytosed, but instead, they traffic to lysosomes where the envelope is degraded . It has A third cell-to-cell transmission pathway of HAV has been recently described, based on the delivery of capsid-free RNA genomes which are an abundant cargo of the quasi-enveloped virions . The quaFailures at any step of the infectious cycle may lead to an abortive cycle and those happening at the very beginning, such as the entry and uncoating, are particularly deleterious. Accordingly, having diverse entry pathways may be of great advantage to ensure the replication and transmission, particularly for HAV with a very slow translation.Another strategy of HAV to warrant a successful replication is its ability to avoid the antiviral responses. The low CpG content may contribute to avoid the induction of antiviral responses, and it has been described that HAV elicits a very limited type I IFN response . This reHAV is also able to avoid the mechanisms of clearance in the blood. Some glycoproteins on the erythrocyte surface function as decoy receptors, binding pathogens and avoiding them to reach their target tissues . Naked vVirulence may be defined as the damage caused by a pathogen infection, including host morbidity and mortality, i.e. the capacity to cause disease. Virulence is multifactorial and results from the complex interactions between pathogen, host and environment.The “virulence–transmission trade-off”’ hypothesis states that virulence is an unavoidable consequence of parasite transmission . Hence, HAV is fecal-orally transmitted and the stability of the virus outside the host is critical for the host-to-host transmission. Coupling of a deviated codon usage with an inefficient IRES results in a very slow and finely tuned translation rate, which in turn determines the protein folding for a highly robust capsid, at the cost of a low production ,27,28. TOverall, it can be postulated that HAV is not particularly virulent because of a strategy to safeguard its host-to-host transmission that is not strictly based on a high replication rate, combined with a very limited induction of cellular antiviral responses. However, changes in this silent dynamics might modify the phenotype of HAV virulence. Mutations resulting in an increase of replication and virus progeny, even at the expense of a less resistant and transmissible capsid, could increase virulence .Figure 3Despite HAV exists as a single serotype, human strains are distributed into three genotypes  and seven subgenotypes  , althougThere are a few studies suggesting the association of some subgenotypes to fulminant hepatitis A cases. The worldwide prevalence of subgenotypes IA, IB and IIIA is of 66%, 14% and 21%, respectively, while the association of these subgenotypes to fulminant cases is of 30%, 30% and 41%, respectively . These dThe Relative Codon Deoptimization Index (RCDI) is a measure of the deviation of the codon usage of a virus with respect its host ,88. The Nevertheless, although these findings deserve attention it should be emphasized that they are very speculative, and conclusions should be read with precaution.In the clinical context, a pure “virulence” factor might be better considered to be one that enhances disease without influencing virus replication. Virus virulence can be measured based on illness, pathological lesions or mortality they induce. Study of the actual virulence and the mechanisms by which infection leads to disease, i.e. pathogenesis, should be studied in animal models. For HAV, these animals include tamarins , chimpanIn HAV, the inefficient IRES and the deviated codon usage separately, and particularly in combination, are responsible for a very slow translation rate. The occurrence of three mutations in the IRES , which induce a change of its secondary structure , signifiThis fast-growing strain of HAV was present at a very low frequency in the mutant swarm of a population long adapted to grow in conditions of moderate transcription shutoff, and was rescued only after a process of competition with a population adapted to grow in conditions of high transcription shutoff . AdditioFew clinical studies have addressed the role of mutations located in the IRES region in the outcome of the hepatitis A infection. A study with twelve patients with severe and benign hepatitis could not identify mutations in the IRES specifically associated with either form of the infection . In contDespite the underlying mechanism would still be unknown, an independent study supported this observation. A deep-sequencing analysis of the 5ʹNCR of viruses isolated from five patients from an outbreak in the men-having-sex-with-men group  revealedIt has been suggested that the VP1-2A and 2C genes, separately and particularly in combination, are virulence factors, as mutations in these genes attenuate the phenotype of a wild-type strain in tamarins . Yet, thAdditionally, when compared to subgenotypes IA, subgenotype IIIA and to a lesser extent subgenotype IB strains show more amino acid replacements in the VP1-2A region, which could be related to their higher virulence. Presently, there are no experimental data in animal models confirming such a possibility, which may be partially explained by the difficulties to grow wild-type viruses in cell cultures, and thus the unavailability of virus inocula of such strains.The actual function of 2C protein, and thus its relationship to virulence, is not well characterized, but sequences of strains from fulminant and severe hepatitis cases show fewer amino acid substitutions than the sequences from acute hepatitis cases . This coVirulence is determined by the invasiveness, tropism, replication, modification of the host defense mechanisms, cell killing and spreading capacities of a pathogen. The host-to-host transmission of HAV is ensured by an outstandingly stable capsid whose solid folding is accomplished by a finely-tuned slow translation controlled by a special codon composition and an inefficient IRES. The consequence is a slow virus replication. Additionally, the HAV infection induces limited antiviral innate cell responses. The combination of a slow replication and a limited antiviral response results in low virulence.However, a small proportion of severe and fulminant cases do occur. The reason of these serious clinical outcomes is probably multifactorial being the result of host and virus factors. The IRES activity may presumably act as a virulence factor, and it is tempting to speculate that the ability to interact with the biogenesis of the pseudo-enveloped particles may also be a determinant of pathogenesis. Infection of patients with impaired innate antiviral responses with strains bearing mutations affecting the mentioned virulence factors may result in severe hepatitis A cases.


Culicoides sonorensis biting midges are biological vectors of vesicular stomatitis virus (VSV) in the U.S. Yet, little is known regarding the amount of ingested virus required to infect midges, nor how their feeding behavior or age affects viral replication and vector competence. We determined the minimum infectious dose of VSV-New Jersey for C. sonorensis midges and examined the effects of multiple blood-feeding cycles and age at the time of virus acquisition on infection dynamics. A minimum dose of 3.2 logs of virus/mL of blood resulted in midgut infections, and 5.2 logs/mL resulted in a disseminated infection to salivary glands. For blood-feeding behavior studies, ingestion of one or two non-infectious blood meals (BM) after a VSV infectious blood meal (VSV-BM) resulted in higher whole-body virus titers than midges receiving only the single infectious VSV-BM. Interestingly, this infection enhancement was not seen when a non-infectious BM preceded the infectious VSV-BM. Lastly, increased midge age at the time of infection correlated to increased whole-body virus titers. This research highlights the epidemiological implications of infectious doses, vector feeding behaviors, and vector age on VSV infection dynamics to estimate the risk of transmission by Culicoides midges more precisely. Culicoides biting midges (Diptera: Ceratopogonidae) are significant agricultural pests and biological vectors of orbiviruses, orthobunyaviruses, bunyaviruses, and rhabdovi-ruses [Culicoides sonorensis midges play a critical role in the epidemiology of the rhabdovirus, vesicular stomatitis virus (VSV) [vi-ruses ,2. Specius (VSV) ,7,8,9,10us (VSV) . Economius (VSV) , outbreaus (VSV) ,11,13,14Culicoides females rely on the consumption of plant carbohydrates as an energy source, they ingest blood to obtain protein for egg-laying [Culicoides midges use their mouthparts to cut the epidermis and ingest blood and potentially other skin surface contaminates that pool in the wound. This causes significant mechanical damage to the dermis and induces physiological and immunological responses favorable for rapid infection and systemic dissemination of arboviruses delivered during blood-feeding [Vector-borne disease transmission dynamics depend on virus–vector interactions, namely the ability of the vector to replicate and transmit virus, and vector–host interactions, namely the frequency with which the vector encounters a susceptible animal for blood-feeding. Although adult g-laying . Female g-laying . As is c-feeding . Infectious VSV particles have proven difficult to detect in the blood of infected animals with viral RNA readily detected in plasma or serum (RNAemia), possibly due to the action of the vertebrate complement system or other blood factors that interfere with laboratory virus isolation methods . VesiculC. sonorensis midges has been previously investigated in laboratory studies by providing a high titer single infectious BM to newly emerged females [The vector competence of  females ,23. Howe females ,24. Prev females ,25. In m females ,27. Alth females . TherefoCulicoides midges will feed multiple times during their three to six week lifespan sustaining multiple cycles [Culicoides midges share many biological traits with mosquitoes, midges acquiring a VSV infection during their first blood meal may have increased potential for transmission through feeding-related and age-related mechanisms that favor virus amplification [Culicoides-VSV transmission dynamics accurately. The gonotrophic cycle (GC) comprises the time from the ingestion of a BM to the egg-laying event. Under natural conditions, e cycles . The bloe cycles ,31,32 ane cycles ,31,32. Me cycles ,34,35. Ae cycles ,37, the e cycles , and thee cycles , may cone cycles . As Culification . Therefo10 plaque-forming units (PFU) per mL were provided to C. sonorensis females. Immediately after feeding, the ingested virus titer was measured in individual fully engorged midges by plaque assay and cytopathic effect (CPE). All females fed to repletion on each of the six viral dilutions were positive for infectious virus as detected by CPE. However, quantitation of virus titers by plaque assay was only achieved for the higher infectious dose groups . The minimum VSV-BM infectious dose to infect midge midguts (30.8%) was 3.2 log0 PFU/mL a. The mi0 PFU/mL b.10 PFU/mL. Additionally, whole-body Ct values were analyzed to determine their best fit to the exponential curve of the bodies to estimate infection or of the heads to estimate dissemination. Regression analysis indicated that the best-fit model for whole-body Ct followed the logistic curve of heads with a steeper slope from growth to plateau phase after ingesting BM titers above 7.2 log10 PFU/mL  values, was used to estimate VSV dose-response for midgut infection (bodies) and dissemination  10 days after ingesting a VSV-BM a. Virus 0 PFU/mL a. The stL of VSV a. A non-0 PFU/mL b.p = 0.028 and 0.002, respectively) (p = 0.007). Similarly, the VSV-BM + 1BM and VSV-BM + 2BM groups had a higher percentage of positive midges as detected by CPE in comparison to age-matched midges receiving only the initial infectious VSV-BM (p > 0.05) due to high variability between individuals. Mean titers detected by plaque assay of original homogenates were 3.7 and 3.5 log10 PFU/mL for 8 dpi VSV-BM and VSV-BM + 1BM, respectively, and 4.6 and 4.2 log10 PFU/mL for 12 dpi VSV-BM and VSV-BM + 2BM, respectively.Virus titers and infection rates of midges that fed on a single infectious blood meal (VSV-BM) were compared with age-matched midges that received one (VSV-BM + 1BM) or two (VSV-BM + 2BM) subsequent non-infectious blood meals a. Virus ctively) a. Higherctively) b  (p > 0.05)  were compared with age-matched midges that received the infectious VSV-BM as their first meal b. At 8 d > 0.05) . Likewis > 0.05) b. Mean tp < 0.0001) (p > 0.05) (Virus titers and infection rates of midges infected shortly after emergence (younger) and midges infected 5 to 8 days after emergence (older) were compared at 8 dpi c. Virus  0.0001) a. Howeve > 0.05) b. C. sonorensis is limited to studies on vector competence [C. sonorensis midge infection and dissemination, examined the effects of prior and subsequent blood-feeding and the effects of age on VSV infection and dissemination. These results were compared to the traditional laboratory infection methods where newly emerged C. sonorensis females are fed with a single, high titer infectious blood-meal (VSV-BM). Current understanding of the VSV infection dynamics within mpetence ,23, bitempetence , and nonmpetence . The phy10 PFU/mL, and disseminated infections were found in midges fed on blood meals ranging from 5.2 to 8.2 log10 PFU/mL. Additionally, logistic modeling estimated that the probability of 50% of midges becoming infected (OID50) and becoming infectious (ODD50) by day 10 required infectious doses of 5.8 log10 PFU/mL and 6.3 log10 PFU/mL, respectively, suggesting a dose-dependent infection as observed in previous studies [The first determinant of vector competence evaluated in this study was the capacity of a newly emerged midge to become infected (VSV+ bodies) and have a disseminated infection (VSV+ heads) 10 days after feeding on 10-fold serially diluted infectious blood meals. Previous studies suggested that disseminated VSV infections correlate with salivary gland infection and transmission potential [ studies , and wit10 PFU/mL are encountered in vesicular fluids, at the margins of damaged tissues, and in the copious amounts of saliva shed by symptomatic animals [Culicoides midges likely ingest VSV from intact skin surfaces contaminated with saliva and vesicular sera, and from the vesicular lesions themselves. Based on our minimum infectious dose study, these reported viral levels would be adequate to initiate midgut and disseminated infections in more than 50% of midges that feed to repletion. Although high levels of viral RNA can be detected by RT-qPCR in the blood of pigs experimentally infected with VSV-NJ [In VSV-infected livestock, 6 to 9 log animals ,41. By ih VSV-NJ , viremiah VSV-NJ ,43,44. Hh VSV-NJ . It is uh VSV-NJ , would bC. sonorensis midges ingest a volume of 100–150 nL [10 PFU/mL may result in fewer than one virion being ingested, lowering the chance that midges will become infected. A previous study with intrathoracic injections of C. sonorensis midges with 200 nL volumes of bluetongue virus at 3 log10 PFU/mL resulted in only 35% of midges becoming infected [Culicoides midges feed opportunistically in swarms, with reported feeding rates of 110 bites per minute and with the collections of 281 fed females from a single animal after only a 10-min interval [10 PFU/mL VSV from an infected animal, a significant number of midges in a feeding swarm will become infected [Estimating that 0–150 nL ,21, feedinfected . Thus, einfected . Howeverinterval ,47,48,49Leishmania-infected sandflies [Aedes aegypti mosquitoes. Conversely, in Anopheles gambiae mosquitoes, a subsequent blood meal either did not affect the number of Plasmodium oocysts in terms of parasite survival and growth or negatively impacted oocyst development [C. sonorensis midges, suggesting that successive non-infectious blood-feeding may enhance the vector’s likelihood of transmission either as a result of higher titers or potentially shorter EIPs as seen in mosquitoes, and confirming the importance of feeding behavior on vector-virus infection dynamics. Subsequent blood meals may induce extra midgut expansion and increase the number of micro-perforations in the basal lamina over time, enhancing the likelihood of virus escape [Culicoides midges may ingest several blood meals to maximize the number of egg-laying cycles throughout their lifespan [An additional determinant on vector competence measured in this study was feeding on non-infectious blood meals after ingestion of an infectious blood meal. Subsequent blood-feeding events have been shown to decrease the extrinsic incubation period (EIP) and increase the proportion of infectious vectors, as seen in andflies  and in Zandflies , dengue-andflies , and chiandflies ,32 Aedeselopment . Our ress escape ,32. Addis escape ,51,52. Ilifespan ,16. Thuslifespan .Escherichia coli [C. sonorensis midges fed on a previous non-infected blood meal may have elevated resistance to VSV infection via an increase in the number of circulating hemocytes which are then stimulated by the second (infectious) blood meal. Thus, although bloodmeal-induced micro-perforations in midgut epithelium can facilitate the escape of viral progeny and enhance disseminated infections [Given the potential for enhanced distention and porosity of the midgut epithelium as a consequence of blood-feeding ,32, incrhia coli . Thus, wfections ,32, thesfections ,56,57. Culicoides midges. Based on investigations of other Dipteran vectors, we hypothesize that immune-related response activation in some of the target tissues  decreases with aging, allowing increased VSV replication rates. However, the effects of immunosenescence on the competence of Dipteran vectors vary between vector species and pathogen pairing. Older Ae. aegypti had significantly higher dengue-2 virus infection rates at early time points [Culex mosquitoes and Tsetse flies are also less capable of becoming infected with parasites they vector [Ae. trivittatus and Ae. aegypti are less capable of becoming infected with filarial nematodes [Culex tritaeniorhynchus mosquitoes exhibited less susceptibility to oral infection with West Nile Virus [Lastly, we showed that increasing midge age results in significantly higher VSV titers without altering the infection rates. Studies in mosquitoes have shown that the number of hemocytes and strength of the immune response progressively declines with age ,54. Thise points , but thie points . Black fe points , and oldy vector ,58. Moreematodes ,60, and le Virus . This agle Virus .2. Vero MARU cells  grown in 199E media containing 2% FBS, 100 ug/mL strep, 100 units/ml Pen, and 0.25 ug/mL amphotericin B at 37 °C with 5% CO2 were used for detecting and titering infectious virus from midge samples as described below. Stock virus  was grown in porcine epithelial cells  with Eagles MEM containing Earle’s salts  containing 2% FBS and 100 U penicillin/streptomycin sulfate at 37 °C with 5% COCulicoides sonorensis midges (AK colony) maintained by USDA, Arthropod-Borne Animal Diseases Research Unit at the Center for Grain and Animal Health Research in Manhattan, KS, USA. Adult midges were maintained at 25 ± 1 °C and 75 ± 5% RH in environmental chambers with a 13:11 light: dark cycle and offered 10% sucrose solution ad libitum.All experiments were performed using colonized 2, and fully engorged blood-fed females were sorted from unfed and partially fed and maintained in cardboard cages with egg cups.Blood meals (BM) consisted of a 1:1 mixture of defibrinated sheep blood  and VSV stock virus  or sheep blood alone . Midges were allowed to feed on an artificial membrane feeding system  for 60 m10 PFU/mL. VSV titer ingested by individual females was quantified immediately after feeding (time zero). Eight fully engorged midges from each virus dilution were collected individually in 500 µL of antibiotic medium  and stored at −80 °C until further processing. The remaining fully engorged females were sorted into cardboard cages, maintained for 10 days, collected individually with heads separated from bodies  in 300 µL of TRIzol  for reverse transcriptase quantitative PCR (RT-qPCR), and stored at −80 °C until further processing.To test the effects of infectious dose on VSV infection, 1-to-3-day-old females (3 replicates) were allowed to feed on six serially-diluted infectious VSV-BMs containing titers ranging from 3.2 to 8.2 log10 PFU/mL). Fully engorged females were sorted into two cardboard maintenance cages with egg cups. At 4 and 8 days, after GC-1 and GC-2, one cage was provided with a BM. As above, fully engorged females were selected and placed in new cages after each feeding. From each cage, on days 8 and 12, midges (n = 44 per group) were collected individually in TRIzol or antibiotic medium and stored until processing, as above.To test the effects of subsequent BMs on VSV infection a, 1-to-310 PFU/mL). Fully engorged females were sorted, maintained for eight days on 10% sucrose, sampled (n = 32 per group) as above, and stored at −80 °C until further processing.To test the effects of a prior BM on VSV infection b, 1-to-310 PFU/mL) and held for eight days. Simultaneously, a second group of 5-to-8-day-old females , that had been maintained on 10% sucrose solution, were provided a VSV-BM. Fully engorged females from both groups were selected, sorted, maintained for an additional eight days, then sampled (n = 32 per group) and stored until processing, as above.To test the effects of age on VSV infection c, 1-to-310 of ssRNA VSV ng and the linear regression (y= −3.30578x+11.02683) allowed determination of viral genomic equivalents per midge [Frozen TRIzol midge samples were thawed on ice, two 2.4 mm stainless steel beads  were added, and tubes were homogenized by shaking at 3.1m/s with a Bead Mill Homogenizer (Omni Inc.). Samples were centrifuged at 12,000× g for 6 min to pellet debris. Total RNA was extracted using Trizol-BCP , and RNA extracts were analyzed using TaqMan Fast Virus 1-Step MasterMix  in an RT-qPCR assay detecting the L segment as previously described [g for 6 min to pellet debris. Observation of cytopathic effects (CPE) after two passages was used to indicate infectious virus within that sample [To isolate infectious virus, frozen midges stored in 500 μL antibiotic media were thawed on ice and individually homogenized as above. Samples were centrifuged at 12,000× Data were pooled from the independent replicates of each experiment. Infection rates were calculated by dividing the number of positive midges by the total number of midges tested by RT-qPCR. Dissemination rates were calculated as the proportion of viral-RNA positive heads out of the total number of positive bodies assayed. Non-parametric tests  were used to compare Ct values and the proportion of infected midges. Non-linear regression analyses were used to evaluate the results of the oral dose experiment. GraphPad Prism version 9  was used for statistical analysis and the creation of graphs.Culicoides midges more accurately.This work emphasizes the importance of incorporating vector biology in understanding virus–vector interactions for VSV transmission dynamics and predictive modeling. Our results showed relatively high oral infection rates even with low blood meal titers, and enhanced virus titers in both older midges, and in midges fed on subsequent blood meals after VSV infection. Understanding these variables reinforce the ideal management practice of preventing/controlling midges before they have an opportunity to feed on a host. This research highlights the importance of considering the epidemiological implications of vector biology and feeding behavior to estimate the risk of VSV and likelihood of transmission by


This enhances reactivity to drug-associated cues and is required for the expression of incubation. Additionally, incubation of cocaine craving is associated with loss of the synaptic depression normally triggered by stimulation of metabotropic glutamate receptor 5 (mGlu5), leading to endocannabinoid production, and expressed presynaptically via cannabinoid receptor 1 activation. Previous studies have found alterations in mGlu5 and Homer proteins associated with the loss of this synaptic depression. Here we conducted coimmunoprecipitation studies to investigate associations of diacylglycerol lipase-α (DGL), which catalyzes formation of the endocannabinoid 2-arachidonylglycerol (2-AG), with mGlu5 and Homer proteins. Although these interactions were unchanged in the NAc core at incubation-relevant withdrawal times, the association of DGL with total and phosphorylated Ca2+/calmodulin-dependent protein kinase IIα (CaMKIIα) and CaMKIIβ was increased. This would be predicted, based on other studies, to inhibit DGL activity and therefore 2-AG production. This was confirmed by measuring DGL enzymatic activity. However, the magnitude of DGL inhibition did not correlate with the magnitude of incubation of craving for individual rats. These results suggest that CaMKII contributes to the loss of mGlu5-dependent synaptic depression after incubation, but the functional significance of this loss remains unclear.Relapse is a major challenge to the treatment of substance use disorders. A progressive increase in cue-induced drug craving, termed incubation of craving, is observed after withdrawal from multiple drugs of abuse in humans and rodents. Incubation of cocaine craving involves the strengthening of excitatory synapses onto nucleus accumbens (NAc) medium spiny neurons via postsynaptic accumulation of high-conductance Ca Cue-induced drug craving progressively intensifies or incubates after withdrawal from extended-access drug self-administration, augmenting relapse vulnerability. Incubation of cocaine craving in rats is accompanied by robust plasticity in the NAc, including strengthening of excitatory synapses via postsynaptic AMPAR plasticity and the loss of endocannabinoid-dependent synaptic depression. Our results identify a novel mechanism that may account for this loss of synaptic depression, namely reduced activity of DGL, the enzyme that produces the endocannabinoid 2-AG, along with increased physical association of this enzyme with CaMKII, an association predicted to reduce the enzymatic activity of DGL. These findings expand our understanding of mechanisms underlying cocaine-induced alterations in endocannabinoid-dependent synaptic depression.Relapse is often triggered by exposure to drug-associated cues even after prolonged abstinence . During Incubation involves adaptations in regions related to motivation and reward including the nucleus accumbens NAc; . The NAcA well characterized form of LTD in the NAc is triggered by metabotropic glutamate receptor 5 (mGlu5), leading to the generation of endocannabinoids (eCBs) that act as retrograde messengers to stimulate presynaptic cannabinoid receptor 1 (CB1R), resulting in the reduction of glutamate release from excitatory afferents in the NAc . The besNAc synaptic depression, evoked electrically or via pharmacological activation of mGlu5, is abolished 1 d after acute noncontingent cocaine , 14 d af2+/calmodulin-dependent protein kinase II (CaMKII) and DGL were increased at incubation-relevant withdrawal times. This was accompanied by decreased DGL activity, as predicted by studies demonstrating that CaMKII phosphorylation reduces DGL activity  to assess associations between DGL and other components of the signalosome and also assayed enzymatic activity of DGL in the NAc core. Compared with saline controls, associations between total and phosphorylated Caactivity . Howeveractivity .Guide for the Care and Use of Laboratory Animals. Male Sprague Dawley rats (Envigo) weighing 275–300 g were housed three to a cage under a reverse 12 h light/dark cycle with food and water available ad libitum. One week after arrival, rats were implanted with a jugular catheter under ketamine/xylazine anesthesia . Before surgery, rats received an injection of the analgesic Banamine  to minimize postoperative discomfort. Thereafter, rats were single housed for 7 d before beginning self-administration training. Catheters were flushed daily with cefazolin  during this period to prevent infection and maintain catheter patency.All animal procedures were performed in accordance with the regulations of the animal care committees of Rosalind Franklin University and the Oregon Health & Science University, and with the National Institutes of Health (NIH) Cocaine was obtained from the National Institute on Drug Abuse and dissolved in 0.9% saline. Rats self-administered cocaine (0.5 mg/kg/infusion in a 100 μl/kg volume over 3 s) or saline, during 10 consecutive sessions (6 h/d starting at zeitgeber time 15), in a chamber equipped with two nose-poke holes. Active hole nose pokes resulted in intravenous delivery of the drug paired with a 20 s light cue (white light illuminating the active hole) on a fixed ratio 1 schedule. Each infusion was followed by a 20 s time-out period. Nose poking in the inactive hole had no consequences. After 10 d of training, rats underwent forced abstinence in home cages and were handled weekly until being used for experiments on or after WD40. This regimen reliably results in incubation of cocaine craving that is robustly expressed during this withdrawal period . Therefom HEPES, pH 7.4, 500 mm NaCl, 2 mm EDTA, 20 mm NaF, 1 mm PMSF, 0.1% NP-40 (v/v), 1 mm NaOV, 1× protease inhibitor cocktail Set 1 (Millipore), 1% Triton, and 0.5% deoxycholate. Three micrograms of DGL-α antibody , validated in previous studies (2O (dH2O) and blocked with 1% normal goat serum with 5% nonfat dry milk in 0.05% Tween-20 in TBS, pH 7.4, for 1 h at room temperature. Membranes were incubated overnight at 4°C with antibodies to mGlu5 , CaMKII , p-Thr286 CaMKII , p-Ser , Homer1b/c , or Homer2 . The mGlu5 dimer band (∼260 kDa) was analyzed because it represents the functional pool of these receptors  using a 1.5 mm biopsy punch (Thermo Fisher Scientific). NAc punches were homogenized in 500 μl of lysis buffer consisting of 25 m studies , was inceceptors . GAPDH wm HEPES, 0.5 mm EGTA, 1× Proteasome Inhibitor Cocktail Set 1 (Millipore)]. Homogenates were passed through a 100-μm-pore filter and then through a 5-μm-pore filter . After homogenates were passed through each filter, filters were washed with 50 μl of HB, and the washes were added to homogenates to maximize yield. Homogenates were then centrifuged at 14,000 × g for 20 min at 4°C. The pellet, which contains the synaptoneurosomes, was frozen on dry ice, stored at −80°C, and ultimately lysed in lysis buffer  for immunoblotting. NAc synaptoneurosomes prepared from individual rats (10 μg protein/lane) were mixed 1:1 with 2× sample treatment buffer  and analyzed by SDS-PAGE and immunoblotting. β-Tubulin was used as a loading control.Rats were decapitated and bilateral NAc tissue (primarily core) was rapidly dissected from a 2 mm coronal slice prepared with a brain matrix (ASI Instruments). Immediately following dissection, synaptoneurosomes were prepared according to published protocols . NAc punm HEPES, pH 7.0, 250 mm sucrose, 1 mm Roche PhosStop (Millipore Sigma), 0.5 mm tris (2-carboxyethyl)phosphine (TCEP), 0.01 mm leupeptin, 0.001 mm pepsin] was added to punched NAc tissue and macerated by a 27 gauge needle. The solution was homogenized with an electric pestle and then centrifuged for 30 min. Supernatant was removed from the pellet and discarded. The pellet was resuspended in 300 μl of membrane resuspension buffer (same as homogenization buffer but without sucrose), then vortexed and briefly sonicated on ice. Using a Bradford assay, protein concentration of the punched NAc tissue was determined. A 96-well plate was then prepared on ice with protein samples in triplicate at 40 μg total protein/well. As a negative control, tissue from cerebellum was run in the absence and presence of the DAGL inhibitor DO34 (2 μm). The FRET reporter compound MRJ20  to reach a final concentration of 2 μm. The assay buffer was then vortexed and sonicated on ice for 2 min. The plate was then taken to a prewarmed fluorescent plate reader , and the recently sonicated assay buffer was pipetted into the wells at 150 μl. The reader maintained a constant 37°C temperature and took fluorescent measurements every minute for 30 min. Samples were then analyzed for relative fluorescent units (RFU) over the 30 min, and a line of best fit for RFU per minute determined relative slopes (ΔRFU/minute) for each sample (GraphPad Prism).Rats were decapitated, and bilateral NAc was rapidly dissected as described above for synaptoneurosome preparation. Lipase activity was subsequently assayed using fluorescence resonance energy transfer (FRET) as adapted from a previous report . Brieflyt tests. Pearson correlation coefficients were used to assess the linear correlation between the magnitude of incubation of craving and DGL activity. Differences between experimental groups were considered statistically significant when p < 0.05.Data are expressed as the mean ± SEM. Groups were compared with unpaired (between group) or paired (within group) Three cohorts of rats underwent extended-access saline or cocaine self-administration , Bound (B) or immunoprecipitated fraction, and unbound (UB) fraction. mGlu5 is present in all fractions. Right, Results after immunoprecipitating mGlu5 from the same starting material and immunoblotting for DGL. Although DGL is more abundant in starting material and unbound fractions, it is detectable in the bound fraction. B, C, Coimmunoprecipitation experiments assessing the association between DGL and Homer1b/c. Although Homer1b/c is present in the starting material (B), it is not detected in the bound fraction after immunoprecipitation of DGL (C); the arrow in C shows the molecular weight at which the Homer1b/c band should have been observed. IB, Immunoblot. Download Figure 2-1, TIF file.Assessment of associations between DGL, mGlu5, and Homer1b/c in the NAc of drug-naive animals. t(14) = 2.59, p = 0.021), total CaMKIIβ (t(14) = 2.30, p = 0.037), phosphorylated (Thr286) CaMKIIα (t(14) = 2.14, p = 0.050), and phosphorylated CaMKIIβ (t(14) = 2.59, p = 0.014) were all increased in the cocaine group relative to saline controls (m NaF), our washing buffer for processing the bound fraction during the immunoprecipitation protocol did not, because at the time the tissue was processed, we did not intend to use it to detect protein phosphorylation. Therefore, DGL might have been dephosphorylated during the lengthy immunoprecipitation protocol .The loss of mGlu5-dependent synaptic depression after incubation of cocaine craving may be attributable to interactions that modulate DGL activity. For instance, CaMKIIα and CaMKIIβ bind DGL, and studies in dorsal striatum have found that CaMKII phosphorylation of DGL inhibits its activity and, conversely, that 2-AG-dependent depolarization-induced suppression of excitation is augmented by pharmacological inhibition of CaMKII . Furthercontrols . CaMKII controls . Therefocontrols . However10.1523/ENEURO.0220-21.2021.f3-1Figure 3-1t(13) = 2.54, p = 0.897). However, as noted in the main text, these data are not conclusive. Because we did not originally plan to assess the phosphorylation state of DGL using this tissue, some of the buffers used in the immunoprecipitation protocol did not contain phosphatase inhibitors. Thus, DGL may have been dephosphorylated during the lengthy immunoprecipitation protocol. IB, Immunoblot. Download Figure 3-1, TIF file.Assessment of DGL phosphorylation in the NAc after prolonged withdrawal from extended-access cocaine (Coc) self-administration. Rats underwent extended-access self-administration of cocaine or saline (6 h/d for 10 d), destined for preparation of NAc core homogenates on WD55 (t(16) = 3.748, p = 0.002; t(14) = 2.54, p = 0.024; p = 0.08; Based on earlier studies , the obsC with 5C and Extended Data We attempted to replicate this using an additional cohort of late withdrawal cocaine rats A for whiFig. 410.1523/ENEURO.0220-21.2021.f5-1Figure 5-1Figure 5-1, TIF file.DGL activity measurements in NAc core of cocaine rats and in two control groups. Rats self-administered cocaine A and recEnduring vulnerability to relapse is encoded by synaptic plasticity in the NAc . An impoHere, we assessed the postsynaptic mGlu5 signaling complex that mediates the generation of 2-AG  or input-specific adaptations in DGL activity. This could be one reason why we failed to identify a clear role for DGL adaptations in the incubation of craving. For example, one prominent incubation-related adaptation, synaptic insertion of CP-AMPARs, can occur on both D1 and D2 MSNs  but also participates in many other mechanisms regulating synaptic function , CaMKII may also contribute to homeostatic plasticity . Most noignaling . We haveignaling . Likewisignaling . The finignaling  combinedignaling .As DGL activity was reduced in rats that underwent incubation of cocaine craving compared with saline controls (above), we tested for a correlation between DGL activity and the magnitude of incubation of craving  for individual cocaine rats in two separate cohorts. Because the expression of incubation requires excitatory synaptic transmission onto NAc core MSN , and DGL1 receptor-expressing MSNs. These mice do not express mGlu5-dependent synaptic depression or demonstrate cue-induced reinstatement of cocaine or saccharin seeking; however, after 2-AG elevation with monoacylglycerol lipase (MAGL) inhibition, reinstatement of saccharin seeking is restored (However, a number of lines of evidence argue against this possibility. First, mGlu5/CB1R-dependent synaptic depression has been linked to the promotion of reward seeking in mice with constitutive knockdown of mGlu5 in Drestored . Furtherrestored . We noterestored , in contrestored .Additional evidence against a causal relationship between loss of mGlu5/CB1R-dependent synaptic depression and elevation of cue-induced drug craving comes from studies on the incubation of methamphetamine craving. Dihydroxyphenylglycine-induced, mGlu5-dependent synaptic depression in the NAc core was lost during the first week of abstinence from extended-access methamphetamine self-administration, which corresponds to the rising phase of incubation of methamphetamine craving, but at later withdrawal times this synaptic depression was restored whereas craving remained at high “incubated” levels . These rOverall, these results argue that synaptic depression can promote  or be diThe loss of mGlu5/CB1R-dependent synaptic depression in the NAc core after incubation of cocaine craving is likely attributable to multiple postsynaptic mechanisms, including reduced surface expression of mGlu5 and reduced mGlu5–Homer interactions  and, as


This study develops a theoretical model for the costs of an exam as a function of its duration. Two kind of costs are distinguished: (1) the costs of measurement errors and (2) the costs of the measurement. Both costs are expressed in time of the student. Based on a classical test theory model, enriched with assumptions on the context, the costs of the exam can be expressed as a function of various parameters, including the duration of the exam. It is shown that these costs can be minimized in time. Applied in a real example with reliability .80, the outcome is that the optimal exam time would be much shorter and would have reliability .675. The consequences of the model are investigated and discussed. One of the consequences is that optimal exam duration depends on the study load of the course, all other things being equal. It is argued that it is worthwhile to investigate empirically how much time students spend on preparing for resits. Six variants of the model are distinguished, which differ in their weights of the errors and in the way grades affect how much time students study for the resit. Exams at universities typically take between 0.5 and 5 hours, but what is the optimal duration? Recently, this was discussed by the faculty of the psychology department of the Radboud University, and several faculty members suggested that the courses with high study load require longer exams. The other side of this argument is that short exams, of say 1 hour, would suffice for small courses. Since I am Chairman of the Examination Board of the psychology program, my opinion on this matter was asked. My first thought on this was that a shorter exam will have less questions, and consequently a lower reliability, and that this might breach the common standards of reliability. For example, the Dutch Committee on Tests and Testing states that the reliability of a test being used for “important decisions at individual level” should be at least 0.80 , p. 34. The main problem of a short test is that the inevitable measurement errors will be relatively large. Consequently, there will be more students who fail the exam while they should have passed, and more students who pass the exam while they should have failed. But to how many students does this happen? In order to make an informed decision, we want to quantify this. The next section will develop a simple classical test theory model for this.The next question is what the costs of such incorrect decisions are. If the student fails, the student will retake the exam later, and for this the student needs additional study time. The present study will consider this time as the costs for the student. If the student passes undeservedly, it is harder to quantify the costs. In this case, there are usually no extra costs for the student, but there may be costs for the institution in terms of reputation damage if future employers or teachers note the lack of skills of some students who passed this exam. However, this kind of costs is much harder to quantify, and therefore it will be ignored in most of the article.Having a long exam, on the other hand, has also costs, because all students of the course will have to spend time on this. Based on these ingredients, the optimum exam duration may be defined as the exam duration for which the total costs in the student population are minimal, where the total costs are computed as (1) the hours extra study time for students who failed incorrectly plus (2) the hours spent by all students on the examination. This article will demonstrate that this optimum is easily computed if the relevant statistics are known from a previous administration.Obviously, there are in fact also other costs for the student, such as emotional stress. There will also be additional costs for the institution, such as the need of a bigger exam hall during the resit if more students fail. Furthermore, the costs of correcting the exam are ignored, which might be defensible for multiple choice exams that are automatically processed. Furthermore, one might argue that an item response theory model is needed instead of classical test theory. All these improvements will be ignored for the sake of simplicity of the model.The next section describes how the present article is related to articles with similar objectives. The following sections will explicate the test score model and explain how the probability of an incorrect fail on the exam can be derived from it, and include the two costs in the model and define the objective function. After illustrating this model with a real exam in discrete time, it will be shown that with continuous time, the objective function is convex and has a unique minimum. The following section will investigate numerically how the minimum depends on the parameters. Several other versions of the model are discussed briefly.This section will sketch how the method developed in the present article differs from earlier approaches. Several authors have studied how one can optimize generalizability coefficients, reliability coefficients, or validity coefficients, or how one can minimize decision error rates. In many cases this was studied in the context of a budget constraint e.g.,  or fixedMany of the articles cited above use the Spearman–Brown formula, and this will be used in the present study too. Several articles assume continuous parameters, because this permits the use of derivatives. The present study will do that too.A different branch of psychometric optimization is the selection of items in Computerized Adaptive Testing e.g., , maximizAssuming that errors cost something is naturally done in decision problems in economics or industry. In educational testing, such assumptions are usually avoided because there is no obvious, generally agreed-upon method to quantify the costs of errors in pass/fail decisions. But is it possible to create defensible quantifications of such costs? Examiners will anyhow make exams of a certain length. Thus, at a certain point the examiner stops increasing the test length and implicitly accepts the corresponding error rate. Therefore, every finite exam contains an implicit assumption about how expensive it may be to avoid a decision error. It is worthwhile to make this assumption explicit and to investigate which information is needed for an evidence-based decision.This section will use a classical test theory model to derive a formula that shows how the exam duration affects the expected number of students who fail incorrectly. Assume that the test can be lengthened or shortened without systematically altering the content type or difficulty, thus leaving the true scores the same. That is, the examiner would add or delete test items and change the duration of the examination proportionally. Assume that the examiner estimated from this previous administration the values of  formula , p. 112:Assume that the cutoff for passing the test is some real number The correlation between Note that restudy fraction as This section will incorporate costs in the model. As explained in the introduction, the model will only consider the costs in terms student time. Suppose that the study load of a course is costs quotientTo find the optimum duration, we have to minimize this in If the restudy fraction In a multiple-choice exam of one of my courses, the nominal study load was 112 hours, the cutoff was If we consider An obvious weakness in this computation is the unknown value of This section will show that for continuous Let us first establish that Now consider the partial derivative which is negative for The second derivative with respect to For For the derivative of We can write the derivative in (2) asThe limit of this for In sum, exdur with the function minexamcosts that minimizes Because optimal reliability. Another name could be efficient reliability, similar to Thus, For the case discussed earlier, with In In Consider the possibility that the mean of the grades is equal to the cutoff: . ThusIf we write Although I do not know an analytical solution in Because of the simplicity of this case, it can be used a default model if one wants to avoid arbitrary assumptions on In the exam of the earlier example, we had As pointed out by a reviewer, it is possible that the amount of time that a student uses to prepare for the resit depends on the student’s score on the first attempt. This will now be modelled. Assume that, for students who fail the exam, the restudy fraction if For a student who incorrectly fails the exam, the expected restudy fraction would beThe conditional distribution of and the expected grade of students who failed incorrectly isHere, the first two terms do not depend on As a real data example, consider the case discussed earlier with The numerical example was based on empirical estimates of What is the best duration for an exam? How reliable should exam scores be? How is this affected by the study load of the course? In the construction of exams and tests it is often emphasized that they should be reliable. If this were the only criterion, exams should be very long, and in theory even infinitely long. The practical decision whether a certain test is long enough is inevitably partially based on intuitive reasoning and personal experience, since there are hitherto no formal models that justify a less than perfect reliability. This article shows that such models are possible and entail realistic outcomes.The model created here assumes a classical test theory model, with normally distributed true scores and error scores. From the mean, standard deviation, and reliability of the test, we can compute the probability of erroneous fail on the exam. To quantify the costs of such errors, it was assumed that the students who fail will lose a fraction of the nominal study load because they have to study for the resit. These hours were called the costs of errors. On the other hand, a long test will also consume time from students, and this was called cost of measurement. The compound costs were now defined as the costs of errors plus the costs of measurement. The compound costs were expressed as a function of the test length, and its minimum is easily obtained numerically.The models make clear that there are many factors contributing to the costs of the exam. If a course is repeated yearly, the examiner can presumably have a reasonable estimate of the values of What if there is no resit? In that case the model cannot be applied literally. However, one could argue that in this case a student who fails loses the time already spent in the course, which is probably proportional to the study load. Therefore, the same mathematical model would still be appropriate, albeit with a different interpretation of A limitation of the two models discussed is that students who incorrectly pass the exam , but even this condition is rarely satisfied. The Spearman–Brown formula has also been derived by The Spearman–Brown formula does not account for possible practice and fatigue effects that might influence the reliability if the test length is changed. I do not know of a model that describes the effect of this on the item parameters, but if these effects were strong then this would also invalidate the increasingly popular methods of computerized adaptive testing e.g., , which iThus, for the first time we have now a mathematical tool that allows us to estimate the optimal length of an exam from data of earlier similar exams. The tool is simple and easy to use, but obviously there are some limitations:The computation requires knowledge of the distribution of The computation ignored many other costs, such as extra financial costs if failing leads to dropping out of the program, the reputation damage when students with low true scores pass, emotional stress, costs of the school to make longer exams, and so on.The distribution of item characteristics should remain the same if the test is lengthened or shortened, such that the Spearman–Brown formula is valid. To avoid floor and ceiling effects, it may be better to work with an item response theory model instead of classical test theory.More detailed and realistic computations may be possible if all these components are added to the model, and the important conclusion from this article is that there is proof of concept.


Heterobasidion species are highly destructive basidiomycetous conifer pathogens of the Boreal forest region. Earlier studies have revealed dsRNA virus infections of families Curvulaviridae and Partitiviridae in Heterobasidion strains, and small RNA deep sequencing has also identified infections of Mitoviridae members in these fungi. In this study, the virome of Heterobasidion parviporum was examined for the first time by RNA-Seq using total RNA depleted of rRNA. This method successfully revealed new viruses representing two established (+)ssRNA virus families not found earlier in Heterobasidion: Narnaviridae and Botourmiaviridae. In addition, we identified the presence of a recently described virus group tentatively named “ambiviruses” in H. parviporum. The H. parviporum isolates included in the study originated from experimental forest sites located within 0.7 km range from each other, and a population analysis including 43 isolates was conducted at one of the experimental plots to establish the prevalence of the newly identified viruses in clonally spreading H. parviporum individuals. Our results indicate that viral infections are considerably more diverse and common among Heterobasidion isolates than known earlier and include ssRNA viruses with high prevalence and interspecies variation. Agaricus bisporus. Fungal viruses have since then been described in diverse fungal taxa, including Ascomycota and Basidiomycota as well as early diverging fungal lineages such as Chytridiomycota, Blastocladiomycota, Neocallimastigomycota, Zoopagomycota, and Mucoromycota , and the re-classification of many fungal viruses, for example, the division of family Narnaviridae into Mitoviridae and Narnaviridae.High-throughput sequencing (HTS) studies have recently revolutionized our understanding on the virosphere and revealed numerous novel virus clades that await classification by the International Committee on Taxonomy of Viruses (ICTV). Among these are many new fungal narna-like viruses  and the Heterobasidion species complex includes several highly destructive conifer pathogens of the Boreal forest region (via root contacts. Heterobasidion parviporum prefers Norway spruce (Picea abies) as its host tree and is widely distributed in the Northern hemisphere following the distribution of its primary host (The t region . These rary host .Heterobasidion strains (Heterobasidion spp. (including H. parviporum) is Heterobasidion RNA virus 6, a bisegmented virus in family Curvulaviridae  virus infections in ca. 15–17% of  strains . The mosaviridae , 2015a. basidion . The alppression , and anolitation .Heterobasidion strains have been investigated by HTS, and both small RNA deep sequencing and transcriptomics analysis have successfully revealed the presence of mitoviruses not detectable as dsRNA in H. parviporum and H. annosum  virus taxa that may be more difficult to detect as dsRNA) could be discovered in H. parviporum using this methodology. The investigated isolates originated from experimental forest sites located within 0.7 km range from each other, one of which has been extensively screened for the presence and distribution of dsRNA viruses earlier  are adjacent Heterobasidion clones infected with identical ssRNA virus variants suggesting lateral virus transmission; (ii) are ssRNA virus infections temporally stable within Heterobasidion clones; (iii) do ssRNA viruses accumulate in Heterobasidion clones through time, leading to increase in viral co-infections in single isolates.Thus far, only a few  annosum . However earlier . The earH. parviporum strains included in the RNA-Seq analyses were isolated from the Ruotsinkylä research forest in Tuusula, situated approximately 30 km north of Helsinki. They originated from five different experimental plots , all of which were located within ca. 700 m distance from each other. More detailed site characteristics for RKU are described in Heterobasidion isolates subjected to RNA-Seq (except RKU172) contained dsRNA (data not shown) as revealed by cellulose chromatography analysis  by laboratory pairing experiments as described by LAP3.3.2H. parviporum population utilized in the RT-PCR screening of mycoviruses newly discovered in the present study originated from a naturally regenerated Norway spruce stand 7R of 875 m2 located in Ruotsinkylä , southern Finland (see H. parviporum strains took place in 2005 (H. parviporum isolates had been determined previously using vegetative incompatibility reactions and microsatellite markers (N = 43) are presented in The  in 2005  and 2012 in 2005 , and the markers . The isoH. parviporum strains utilized in the study were stored for long-term in agar slides  in cryotubes and kept in a cold room (4°C). The long-term preservations were renewed (by cultivating the strain on fresh MEA plates for ∼2 weeks) every time the particular strain was accessed. The estimated cultivation count is three for the oldest strains (isolated in 2003) and three or less for the H. parviporum strains isolated after 2003.The H. parviporum isolates was examined using RNA sequencing (RNA-Seq). The cDNA library named 7Het contained eight H. parviporum isolates , one H. annosum isolate, and one Oidiodendron maius isolate. Here, we report the results for the H. parviporum strains.The presence of mycoviruses in H. parviporum isolates were cultivated for 6–9 days in 1.5% malt extract on an orbital shaker (120 rpm) at 23°C. The collected mycelia was freeze-dried for 3 days and kept in −80°C until the total RNA was isolated using Spectrum Plant Total RNA Kit (Sigma-Aldrich) as described by The A pooled RNA sample, composed of 0.5 μg total RNA of each fungal isolate, was sent to Macrogen Korea for library preparation and HTS. The library was generated using the TruSeq Stranded Total RNA Library prep kit with Human/Mouse/Rat Gold for rRNA removal (Illumina), and Novaseq (Illumina) was utilized in the production of 101 bp pair-end reads (100 M reads/sample).de novo assembly in R  using a database (db) generated by combining translated CDS of hosts  detection. For SNP determination, the mapping of reads to virus strains and variants or contigs was conducted with Geneious RNA assembler with the loose mapping criteria . Variant call parameters for virus sequences are presented in in silico contig assembly. All the Sanger sequencing was conducted at Macrogen Europe, and Geneious 10.2.6  was utilized in the examination of sequences and assembly of mycovirus sequences.In order to determine the sequence differences between virus variants hosted by different H. parviporum isolate, its complete genome or complete CDS was determined by Sanger sequencing at least from one isolate. The RT-PCRs were conducted with Phusion High-Fidelity DNA Polymerase (Thermo Fisher Scientific) and virus contig specific primer pairs (If a novel virus was hosted by more than one er pairs . All seqThe cellulose chromatography method  was utilThe nucleic acid isolation and reverse transcription was performed as described previously. The RT-PCRs were conducted with Phusion High-Fidelity DNA Polymerase (Thermo Fisher Scientific) and virus contig specific primer pairs producing partial CDS regions . All mycPhylogenetic trees were constructed using predicted RNA-dependent RNA polymerases (RdRPs) of mycoviruses. The RdRPs of related viruses were retrieved from the GenBank and aligned using MAFFT v7.450 with Blosum45 substitution matrix. The evolutionary history was inferred by using the Maximum Likelihood method in MEGA X . DetaileHeterobasidion parviporum strains isolated in 2005 and 2012 (H. parviporum clones were examined for the presence of novel viruses identified in the present study by HTSs. The virus specific primer pairs (and 2012  represener pairs  were useH. parviporum RNA-Seq library 7Het was composed of ∼118 million paired-end reads (SRA ID: SRR14842285). Using a bioinformatics pipeline based on Trinity de novo assembly , we were able to identify a new narna-like virus and a new ourmia-like virus, which were the first putative members of families Botourmiaviridae and Narnaviridae described in species of Heterobasidion  with the RdRP of Plasmopara viticola lesion associated orfanplasmovirus 1 (Sclerotinia sclerotiorum (Mr = 140.491 kDa) with no conserved domains identified based on the NCBI conserved domain search but sharing similarity with predicted proteins of narna-like viruses (Mr = 134.058 kDa). Based on BlastX, the insect narna-like virus 1  showed highest similarity with RNA2 of HetNlV1 (Plasmopara viticola lesions and thrips (A Trinity contig (DN23600) of 7Het library showed the highest similarity  hosted by 3R4  shared 9H. parviporum isolates in the RNA-Seq library (Mr = 72.491 kDa). No conserved domains were detected based on the NCBI conserved domain search, but the putative RdRP shared conserved amino acids with other RdRPs of ourmia-like viruses. Based on phylogenetic analysis, HetOlV1 is included in a highly supported cluster with classified members of genus Magoulivirus (A Trinity contig (DN24714) showing similarity with ourmia-like viruses and having the mean coverage of more than 3,640 reads (with loose mapping criteria) was detected by RT-PCR in six  library . The seq library . The San library ; Table 1 library , a magouulivirus .H. parviporum isolates included in the RNA-Seq library, one of them represented by three different variants. The studied sequences included altogether 58 SNP sites, all of which were detected by variant calling of mapped reads , whereas HetAlV2 and HetAlV3 had multiple hosts  have been described at the Ruotsinkylä research forest (plot 7R) earlier . These v 7R plot , sharing 7R plot . Therefo 7R plot .Interestingly, read mapping revealed a very high read depth for HetMV3-pa1-4 and HetMV3-pa4 but the presence of HetMV3-pa5 could not be confirmed. Thus, ca. 0.38 and 0.21 million reads mapped to 1,452 nt long Sanger confirmed sequences of HetMV3-pa1-4 and pa4, respectively, when no mismatches were allowed in the mapping. In contrast, the HetMV3-pa5 was only partially covered, and moreover, less than 15 raw reads were aligned to the sites containing unique SNPs of the strain. For the variant calling, mapping was conducted with loose parameters and all but one SNPs of HetMV3-pa1-4 and pa4 were detected , but theH. parviporum isolates included in the RNA-Seq analysis were infected with HetRV6 based on RT-PCR (Eight different strains of the orthocurvulavirus Heterobasidion RNA virus 6 (HetRV6) had been detected at the Ruotsinkylä research forest (plot 7R) earlier . In thisn RT-PCR . The Hetn RT-PCR  shared 9n RT-PCR  and was n RT-PCR .A total of 10,635 reads mapped to dsRNA1 of HetRV6-pa33 and all 138 SNP identified in Sanger confirmed complete ORFs of the seven HetRV6 strains and variants listed above were detected by variant calling . Using c∗5 multivirus isolate . The dsRNA2 of HetPV21 encoding a putative CP  shows highest similarity to Rosellinia necatrix partitivirus 13 , and its complete genome sequence with 5′ and 3′ UTRs was determined . The dsRvirus 13 ; Table 1itivirus . The firitivirus .We also completed the genome sequence of HetPV4-pa1 by determining its dsRNA2 sequence from its original host isolate RT3.49C from plot 4R as described earlier for dsRNA1 . The twoThe read coverage was also determined for HetPV13-an1, which has been completely sequenced earlier . The reaH. parviporum isolates examined by RNA-Seq were infected altogether by 11 different viruses, of which 6 infected more than one isolate , the artificially infected fungal strain had been cured of two of the four partitivirus infections  and HetRV6 since it was generated in 2013  collected in 2005 and 2012 from the 7R study plot  were confirmed to occur at 7R study plot in fungal strains isolated in 2005 (H. parviporum isolate (242-05). The sequence identity of HetAlV4-pa2 was 95.6% with the HetAlV4-pa1 hosted by RKU3.1.25 included in the RNA-Seq. This particular clone was found in the forest plot only in 2005.All  in 2005 ; HetAlV1 in 2005 . The fun in 2005 . In contThe novel ssRNA viruses discovered here, HetAlV3, HetOlV1, and HetAlV2, had infected 60, 56, and 42% of the 7R fungal isolates screened in this study, respectively. This presents a significant increase in the viral content of the isolates when compared to our earlier dsRNA-based analysis , where tHeterobasidion species: Partitiviridae , Mitoviridae (HetMV3 represented by two new strains and one variant), and Curvulaviridae  but also viruses representing groups not detected in isolates of Heterobasidion earlier: a putative narna-like virus, an ourmia-like virus, and four ambi-like viruses. Therefore, only half of the virus families or unclassified family level groups detected in this study had been discovered in Heterobasidion spp. prior to the employment of RNA-Seq, and only two of them had been discovered based on dsRNA screening. A similar situation has been revealed in several other fungal plant pathogens recently subjected to HTS analysis despite extensive screening for viruses based on traditional methods earlier . The raw read coverages of all HetAlV3 variants were very low and, thus, the sequencing coverage was not optimal for this particular virus. moisture . Yet, thNarnaviridae was recently divided into two families when mitoviruses were split into a new family Mitoviridae (ratified by the ICTV Executive Committee in 2019).1 Currently, there are only two classified species in family Narnaviridae. However, in addition to numerous typical narnaviruses, several new groups of narna-like viruses have been recently discovered in fungi and oomycetes. This includes bisegmented narnaviruses with a split polymerase palm domain  resembles viruses in the genus Magoulivirus. Based on ICTV criteria for the classification of botourmiaviruses , whereas an isolate of the 7R forest site was infected with all four ambi-like viruses (HetAlV1–4), supporting the assumption (based on the sequence identities) that all four represent distinct virus species, including HetAlV3 with low read depth in the RNA-Seq. Furthermore, PCR analysis of DNA templates as well as new infections by HetAlV3-pa1-e in a Heterobasidion clone at our study site during the 7-year sampling period indicates that HetAlV3 is not integrated to the host DNA.“Ambiviruses” are bicistronic viruses with a ∼5 kb linear genome containing two ORFs in ambisense orientation and, which thus far, have been found by means of HTS in lla spp. . They arIV and V . In the Heterobasidion remains undetected by traditional methods, we investigated samples from an extensively studied forest plot for the occurrence of novel ssRNA viruses discovered in the present study. Previously known Heterobasidion viruses from the Ruotsinkylä site (two study plots) were the alphapartitiviruses HetPV4 and HetPV9, the betapartitiviruses HetPV2 and HetPV7, the orthocurvulavirus HetRV6 (eight distinct strains), and the mitovirus HetMV3 . During the 7-year study period, we also detected within-clone spread of HetOlV1 and HetAlV1–3. Moreover, we were able to confirm that HetOlV1 and HetAlV1–3 infections persisted at the study site during the study period of 7 years, indicating that infections by these ssRNA viruses are mostly stable. Their prevalence was also higher than that of partitiviruses and similar to that of orthocurvulaviruses at the site (As our RNA-Seq analysis suggested that a major part of viral diversity in s HetMV3 . By examt clones , suggestthe site . In the the site .H. parviporum and H. annosum (Heterobasidion, irregularity in the virus distribution within fungal clones has been reported for C. parasitica (Helicobasidium mompa (R. necatrix (Lactarius rufus and Lactarius tabidus (H. parviporum isolates harbored more viral infections in 2012 than in 2005, although the cultivation counts of hosts isolated year 2005 and 2012 may differ and thus contribute to the results obtained.The ssRNA virus community showed similar features as detected earlier for dsRNA viruses in  annosum , that israsitica , Helicobum mompa , R. necanecatrix , as well tabidus , for ins tabidus . As the  tabidus , we hypoS. sclerotiorum infecting annual crops (The highly stable and gradually expanding virus community at the site seems to be related to the host biology, i.e., presence of long-living clonal host individuals, and is very different from the annually variable virus community seen in the plant pathogen al crops . The detMZ502381-90 and the RNA-Seq reads deposited in the SRA and can be accessed through PRJNA738388.The sequences of virus genomes are available in the GenBank database under the accession numbers EV: conceptualization, project administration, and funding acquisition. EV, SS, and TP: formal analysis and investigation. EV and SS: writing original draft preparation. All authors have read and agreed to the published version of the manuscript.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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


A recent study reported that an aerosolized virus (COVID-19) can survive in the air for a few hours. It is highly possible that people get infected with the disease by breathing and contact with items contaminated by the aerosolized virus. However, the aerosolized virus transmission and trajectories in various meteorological environments remain unclear. This paper has investigated the movement of aerosolized viruses from a high concentration source across a dense urban area. The case study looks at the highly air polluted areas of London: University College Hospital (UCH) and King's Cross and St Pancras International Station (KCSPI). We explored the spread and decay of COVID-19 released from the hospital and railway stations with the prescribed meteorological conditions. The study has three key findings: the primary result is that the concentration of viruses decreases rapidly by a factor of 2–3 near the sources although the virus may travel from meters up to hundreds of meters from the source location for certain meteorological conditions. The secondary finding shows viruses released into the atmosphere from entry and exit points at KCSPI remain trapped within a small radial distance of < 50 m. This strengthens the case for the use of face coverings to reduce the infection rate. The final finding shows that there are different levels of risk at various door locations for UCH; depending on which door is used there can be a higher concentration of COVID-19. Although our results are based on London, since the fundamental knowledge processes are the same, our study can be further extended to other locations  in the world. I.https://www.worldometers.info/coronavirus/).The earliest confirmed case of COVID-19 was in December 2019, and the disease has since become a global pandemic with over 125 711 889 confirmed cases and 2 760 406 deaths worldwide as of March 25, 2021 . Social distancing in the range 1–2.5 m has been recommended to mitigate this situation . This study investigates the effect of the exponential decay of the virus and the complexity of the spreading phenomenon: how long can the virus spread for given certain meteorological conditions? We will explore these in two different locations in London: University College Hospital (UCH); King's Cross Station and St Pancras International (KCSPI). This study will explore the spread of this virus from hospitals and railway stations, and the impact of meteorological conditions on this virus. Our aim is to explore the impact of the aerosol transport of the virus under specific meteorological conditions and highlight the importance of treating the pandemic seriously. Although our study is based on London, it can be extended to other locations  in the world since all the fundamental knowledge principles are the same.The aim of this computational study is to tackle the aforementioned issues with the computational fluid dynamics (CFD) model Fluidity, developed by Imperial College. Fluidity is an open source large eddy simulation (LES) model with an advanced adaptive mesh capability  density of the atmosphere, The three-dimensional (3D) Navier–Stokes equations and generic atmospheric transport (advection-diffusion) equation are utilized for COVID-19 spreading simulations. The 3D Navier–Stokes equations are written asC is the mass concentration of the virus, S represents the source term of virus, and D is the decay term of virus. In this study, the aerosol physical interaction between particles of different diameters is not considered. The emitted virus concentration around the source locations is positively correlated with the around aerosol concentration. This article investigates the potential pathways routes for COVID-19-saturated aerosol flow from local sources.The COVID-19 virus transport equation isλ denotes the decay rate. The decay term in Eq. The virus exponential decay formulation  and King's Cross and St Pancras International Station (KCSPI) on the Euston road in the center of London are close to significant traffic along Euston road, resulting in high pollution levels around that area. The computational area is a rectangular 2 × 1.5 km2 domain centered on UCH and 1000 m high to capture the dynamic part of the atmospheric boundary layer. As shown in •Wind field: In March, the wind direction is mostly from the southwest and a typical mean wind speed of approximately 5.23 m/s at 10 m above the ground (see https://weatherspark.com/m/45061/3/Average-Weather-in-March-in-City-of-London-United-Kingdom).COVID-19 virus lifetime: Given the range of the lifetime is from 1 to 4 h for COVID-19(2). Within such a short period (10 min), the effect of the decay rate can be ignored. Compared to the role of the wind speed on the transmission and diffusion process of the virus concentration, the effect of the decay rate is relatively small in outdoor simulations.•Source locations and concentrations: The locations of virus sources at UCH are chosen at the entries in Doors 1–4, shown in 3  mesh size is set to be 3 m (100 m). The maximum number of nodes is set to be 600 000, which is large enough to ensure the a priori error to be achieved. To avoid spurious dilution of virus emission, high-resolution meshes are located around the sources [see rces see .•Boundary and initial conditions: At the inlet boundary, the velocity profile is given by Eq. B.In this discussion, we will focus on (1) the spatial distribution of viruses released from UCH and KCSPI for given wind field; (2) the release locations of viruses in hospital; and (3) the distance of virus transmission.Wind field and spatial distribution of viruses: The wind velocity solution from the CFD simulations is shown in μg/m3) (see the https://www.londonair.org.uk), and the aerosol present there could transmit the disease. The increased wind speed can carry the aerosolized virus along East Euston road and North Gower street, reaching Euston Square Gardens up to KCSPI. This demonstrates how the viruses emitted from UCH can infect neighboring communities—the area around University College London (UCL)—and the commuters passing through Euston road and Gower street, to list a few.Spatial distribution of viruses: We have plotted the virus contour maps in 3) can be found around UCH, Gower road, A400, Gower P1, and all the way up to Euston Square Gardens. Further downwind past the Euston Square Gardens—places like the British library, St Pancras International, and King's Cross stations—the concentration is much lower, in the range of 0.1–0.2 copies/m3, roughly 0.1% of the concentration found in the immediate vicinity of UCH. From the contour maps, we can observe that the majority of the viruses remain trapped within a short radial distance of under 50 m along St. Pancras Road, past that, the concentration rapidly diluted to under 0.1 copies/m3. Concentrations around 0.1 copies/m3 are found within 250 m away from UCH. This can be attributed to the weaker wind field of around 0.5 m/s around the railway stations compared to the stronger wind field near UCH at 0.5–3 m/s.3) of the virus emitted at Door 2 is larger than those of other doors. The virus released from Door 1 is directly transported to Euston road while those released from Door 2 are blown down Grafton way, splits between going down Huntley street and staying in Grafton way, some of the latter merges with the viruses from Door 4 and with Door 3's at A400, from which it moves continuously to Euston road. The infectious area of the virus emitted from Doors 1 and 2 mostly focuses on Wolfson Institute on Grafton way, UCL along A400, while the virus emitted from Doors 1 and 3 mainly infects the commuters passing through Euston road and Euston square station to list a few.Source location of viruses: We have experimented with test cases that have many different release locations . We also notice that the infectious area from the virus released from Door 2 in UCH is larger than the other doors. This suggests that Door 2 in UCH is not a good location for A & E; the entry location in hospital must be chosen carefully. Our study found that the majority of the viruses released from the St Pancras International and King's Cross stations remain trapped within a short radical distance of less than 50 m and will not affect the people living nearby. In summary, it is suggested that a face cover is needed for personal protection if people travel to public dense places . Finally, the impact of urban green  environment on reducing the virus spreading will be further investigated in our future work.Our findings suggest that the aerosolized virus particles can be transmitted a long distance (hundreds of meters) due to the fully developed turbulent flows around the source locations. For example, around the UCH, there is a strong wind field (∼2.5 m/s) at the height of 5 m and viruses with the concentration of >0.2 copies/m


Both CFTR and SLC26A9 contribute to basal chloride currents in non-stimulated BCi-NS1 airway epithelia, with CFTR being the dominating Cl− conductance. In wtCFTR-expressing CFBE human airway epithelial cells, SLC26A9 was partially located in the plasma membrane, whereas CFBE cells expressing F508del-CFTR showed exclusive cytosolic localization of SLC26A9. Membrane localization of SLC26A9 and basal chloride currents were augmented by interleukin 13 in wild-type CFTR-expressing cells, but not in cells expressing the most common disease-causing mutant F508del-CFTR. The data suggest an upregulation of SLC26A9-dependent chloride secretion in asthma, but not in the presence of F508del-CFTR.SLC26A9 is an epithelial anion transporter with a poorly defined function in airways. It is assumed to contribute to airway chloride secretion and airway surface hydration. However, immunohistochemistry showing precise localization of SLC26A9 in airways is missing. Some studies report localization near tight junctions, which is difficult to reconcile with a chloride secretory function of SLC26A9. We therefore performed immunocytochemistry of SLC26A9 in sections of human and porcine lungs. Obvious apical localization of SLC26A9 was detected in human and porcine superficial airway epithelia, whereas submucosal glands did not express SLC26A9. The anion transporter was located exclusively in ciliated epithelial cells. Highly differentiated BCi-NS1 human airway epithelial cells grown on permeable supports also expressed SLC26A9 in the apical membrane of ciliated epithelial cells. BCi-NS1 cells expressed the major Cl SLC26A9 was suggested to have a role in inflammatory airway diseases, such as cystic fibrosis (CF) and asthma [− transport rates and substantial anion currents [The epithelial anion transporter SLC26A9 (solute carrier 26 family member A9) possibly contributes to airway surface hydration by operating as an uncoupled Clnsporter ,2. SLC26d asthma ,4,5. SLCd asthma ,8,9. Altcurrents ,14,15,16− secretory function. Moreover, studies in renal and gastrointestinal mucosa suggested an apical membrane location of SLC26A9 [In order to fulfil a chloride secretory function in airways, localization of SLC26A9 in the apical membrane is required. Data on the expression of TMEM16A in human airways are still scarce. Surprisingly, immunofluorescence stainings in well-differentiated airway epithelial cells revealed expression of SLC26A9 near tight junctions . A recen SLC26A9 ,19,20. H SLC26A9 , which s SLC26A9 . The preWe analysed expression of SLC26A9 in human airways. Immunohistochemistry of SLC26A9 (green fluorescence) shows expression in the apical pole of the superficial epithelium in normal non-CF airways A. In conLow levels of SLC26A9 are detected in the cytosol of CF-airways. This corresponds to several earlier studies indicating a lack of proper biosynthesis and apical expression of SLC26A9 in cultured CF airway epithelial cells ,22,23,24A more detailed analysis indicates expression of SLC26A9 in the apical membrane of ciliated epithelial cells. Thus, SLC26A9 is coexpressed together with CFTR in ciliated cells, whereas expression of SLC26A9 was not detected in non-ciliated cells . NotablyIn order to provide further evidence for an apical localization of SLC26A9 in airways, we analysed the expression of SLC26A9 in the porcine airway epithelium. To this end, we performed co-staining of SLC26A9 and acetylated tubulin in the superficial airway epithelium of wt-piglets (CFTR+/+) and littermate CFTR-knockout piglets (CFTR−/−). Identically to human airways, superficial airways of CFTR+/+ piglets also demonstrated clear apical expression of SLC26A9 in ciliated epithelial cells A. Remark− conductance. To further examine the role of SLC26A9 in human airway epithelial cells, we made use of the BCi-NS1 basal cell line which maintains multipotent differentiation capacity [+ absorption, which is inhibited by amiloride, the inhibitor of epithelial Na+ channels (ENaC). Steady-state and transient short-circuit currents (Isc) were activated by an increase in intracellular cAMP (using IBMX and forskolin) and by the purinergic agonist ATP, respectively  Isc, although the epithelium was not previously stimulated by IBMX/forskolin. A transient Isc was activated by stimulation with ATP . Moreover, exposure to IL-13 further enhanced constitutive whole-cell currents and augmented 5Cl−-induced current inhibition  BCi-NS1 and CFBE cells but found very little SLC26A9 in the biotinylated fraction (data not shown). Moreover, SLC26A9 expressed in HEK293 cells shows almost exclusively cytosolic expression , similarLocalization of SLC26A9 near tight junctions was reported previously ,18. AlthExpression of SLC26A9 was essentially absent in human F508del-CFTR/F508del-CFTR airways. Moreover, SLC26A9 present in non-polarized CFBE cells expressing F508del-CFTR was detected exclusively in the cytosol. Both findings highlight the pronounced abrogating effect of F508del-CFTR on SLC26A9 ,23. In c− secretion in airways of wild-type but not Slc26a9-deficient mice. Thus, an upregulation of SLC26A9-dependent Cl− secretion may help to prevent mucus obstruction in asthma [− conductance, suggesting enhanced SLC26A9 conductance in asthmatic airways. It should be mentioned that IL-13 (and IL-4) also strongly upregulated airway expression of the Ca2+-activated Cl− channel TMEM16A in vivo and in vitro [− and fluid secretion and antagonizes mucus plugging, as proposed earlier [It was found that IL-13 treatment increases Cln asthma . In airwin vitro . It was in vitro ,33,34,35in vitro ,33,34. T− secretion. This may therefore suggest an additional role of SLC26A9, maybe for bicarbonate transport. Plasma membrane location of SLC26A9 was essentially unchanged in lungs from CFTR-knockout piglets. This may explain why we did not find evidence for acidification of the airway surface liquid (ASL) in lungs of CFTR-knockout piglets in our previous study [A limitation of the present study is the limited number of available lung sections. Thus, subsequent studies should analyse expression of SLC26A9 in CF lungs from patients with different class 1–7 mutations, as well as the effect of CFTR correctors on correcting the plasma membrane location of SLC26A9. Moreover, quantitative comparison of SLC26A9 membrane expression in normal and asthmatic airways would be helpful to clarify whether asthmatic airways express more apical SLC26A9. Nevertheless, the results suggest a limited contribution of SLC26A9 to airway Clus study . Finally®, Serumwerk Bernburg, Bernburg Germany) and Azaperone  anaesthesia by intracardiac injection of T61® . At least three human and piglet samples  were analysed. The human lung sections were kindly provided by Prof. Eric Verbeken and Prof. Kris De Boeck .Airway sections from CFTR+/+ and CFTR−/− littermate piglets were generously provided by Prof. Dr. Nick Klymiuk  CO2. For the IL-13 experiment, cells were treated with 20 ng/mL IL-13  for 72 h in OptiMEM reduced serum medium ; IL-13 was refreshed every day. Cell membrane surface biotinylation was performed as described earlier [Human cystic fibrosis bronchial epithelial cell lines (CFBE) stably expressing wtCFTR or F508del-CFTR  were a g earlier . SLC26A9For RT-PCR, total RNA from BCi-NS1 and CFBE human airway epithelial cells was isolated using NucleoSpin RNA II columns . Total RNA (0.5 µg/25 µL reaction) was reverse-transcribed using random primer  and M-MLV reverse transcriptase RNase H minus . Each RT-PCR reaction contained sense (0.5 µM) and antisense primer (0.5 µM) , 0.5 µL Protein was isolated from filter-grown BCi-NS1 cells using a lysis buffer containing 25 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM EDTA, 5% glycerol, 0.43% Nonidet P-40, 100 mM dithiothreitol  and 1× protease inhibitor mixture . Proteins were then separated by 8.5% SDS-PAGE and transferred to a PVDF membrane . Membranes were incubated overnight at 4 °C with rabbit primary antibodies against TMEM16A  and α-SLC26A9  and a mouse primary antibody against CFTR . Rabbit anti-actin  was used as a loading control. Membranes were then incubated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit/sheep anti-mouse secondary antibodies at room temperature for 2 h, and immunoreactive signals were visualized using a SuperSignal HRP chemiluminescence substrate-detection kit .For staining, cells were fixed for 10 min with methanol and acetone (5:1) mixing solution at −20 °C. Paraffin-embedded lung sections and monolayer CFBE sections (5 µm) were deparaffinized with xylene and rehydrated through a series of ethanol. The standard protocol for immunofluorescence was performed. In brief, after blocking with 5% bovine serum albumin in PBS, sections or cells were incubated in rabbit anti-SCL26A9 antibody  or rabbit anti-SCL26A9 antibody  overnight at 4 °C. Antigen retrieval was performed by Davids Biotechnologie, Regensburg, Germany in pre-heated Tris-EDTA buffer (pH 9.0) for 15 min, using a microwave before blocking . Cells wTransepithelial Using chamber recordings: BCi-NS1 cells polarized on permeable supports were measured under short-circuit conditions in non-perfused chambers with bicarbonate-Ringer solution . Comprehensive methods have been detailed in previous reports [ reports ,40.Patch Clamp: Cells were patch-clamped when grown on coated glass coverslips, and experiments were performed at 37 °C. Patch pipettes were filled with a cytosolic-like solution containing (in mM): KCl 30, K-Gluconate 95, NaH2PO4 1.2, Na2HPO4 4.8, EGTA 1, Ca-Gluconate 0.758, MgCl2 1.03, D-Glucose 5, ATP 3; pH 7.2. The intracellular (pipette) Ca2+ activity was 0.1 µM. The bath was perfused continuously at a rate of 4–6 mL/min with Ringer’s solution (in mM): NaCl 145, KH2PO4 0.4, K2HPO4 1.6, Glucose 5, MgCl2 1, Ca-Gluconate 1.3. The substitution of extracellular Cl− (5 mM) was replaced by gluconate. Patch pipettes had an input resistance of 2–5 MΩ, and whole-cell currents were corrected for serial resistance. The current–voltage (I–V) relationship was determined by pulsing from the holding potential of −100 mV to test potentials between −100 and +100 mV increasing in 20 mV increments. Currents were recorded using an EPC-9 computer-controlled amplifier and PULSE software , as well as Chart software .