mteb/CovidQA huggingface dataset:
The Katnal1 1H mutation was initially identified with a circadian deficit including a short free-running period and advanced activity onset. However subsequent ex vivo experiments using SCN slices of animals carrying the PER2::LUC reporter gene demonstrated no defects in SCN cellular rhythms, suggesting that the core circadian clock was unperturbed by the mutation. Phenotypes in circadian running wheel rhythms that are not associated with changes to the core clock mechanism have also been reported in mouse models of schizophrenia. 25 Here it has been suggested that the wheel running changes observed are the result in defects in output pathways from the SCN circadian clock. Similarly, in Katnal1 1H/1H mice we hypothesise that the defects we demonstrate in neuronal anatomy and neuronal morphology may disrupt output signals from the SCN. Alternatively given that various neuropeptides such as oxytocin are secreted in a circadian manner from ependymal cells lining the third ventricle of
the brain, 26 altered ventricular morphology and ciliary function in Katnal1 1H/1H mice may disrupt the circulation of factors secreted by the ciliated ventricular ependymal cells and contribute to the disruption of the behavioural rhythms observed.
The behavioural consequences of microtubule severing enzyme dysfunction in mouse models have been poorly characterised. Currently the phenotypes described are limited to motor dysfunction in mice lacking the Spg4 gene 27 and head shaking and circling in the Fign mutant. 7, 28, 29 In contrast, here we demonstrate that loss of function of Katnal1 is associated with a range of behavioural phenotypes, including changes in circadian activity, poor learning and memory, hyperactivity in a novel environment (the open field) and deficits in USVs. Notably the learning and memory, anxiety and vocalisation phenotypes reprise the clinical symptoms of ID, increased anxiety in novel situations and delays in language acquisition reported in human patients who carry microdeletions incorporating haploinsufficiency of KATNAL1. 8, 9 While it is also worth noting that mutant mice spend more time the centre of the open field than wild types (implying that Katnal1 1H/1H animals show reduced anxiety), we
suggest that this result is confounded by the hyperactivity in novel environments phenotype we also describe in mutant mice. This observation is backed up by the fact that mutant animals showed increased activity in all regions of the open field rather than just the anxiolytic periphery. Here we also highlight defects in Katnal1 1H/1H mice such as compromised neuronal migration and morphology which may underpin such phenotypes. In Drosophila, the homologue of Katnal1 (kat-60L1) has been demonstrated to play a critical role in neuronal morphology during development, 30 however the data that we present here is the first to demonstrate a similar phenotype in mammals and furthermore suggests how subtle perturbations to KATNAL1 function may contribute to specific neural and behavioural conditions. For example, defects in neuronal migration, synaptic spines and neuronal morphology such as those we have demonstrated here, have been suggested to underpin ID in conditions such as
lissencephaly, 18 Down's syndrome 31 and Rett syndrome. 32 While we are not suggesting that Katnal1 is causative for these conditions, similarities in symptoms and neuronal phenotypes between these conditions and those linked to Katnal1 dysfunction should be appreciated. Furthermore a rare mutation in KATNAL1 has been associated with schizophrenia 10 (http://atgu.mgh.harvard.edu/~spurcell/gene book/genebook.cgi?user = guest&cmd = verb-gene&tbox = KATNAL1) and KATNAL1 has been shown to interact with the schizophrenia associated gene DISC1. 33 In line with these observations we note that increases in ventricular volume and reductions in synaptic spines have been reported in schizophrenic patients 34, 35 and our data demonstrates the same phenotypes in Katnal1 1H/1H mice. Thus the range of phenotypes associated with defects in the function of Katnal1 strongly suggests that the gene should be considered in the pathology of disorders such as ID and schizophrenia.
We do note one key genetic difference between the human patients and Katnal1 1H/1H animals. While the human patients were all heterozygous for the Katnal1 deletion, we found no phenotype in heterozygous mutant mice (data not shown) suggesting that while haploinsufficiency is causative for phenotypes in humans, mice require complete loss of KATNAL1 function to show similar effects. A similar discrepancy between humans and mice has also been noted for the intellectual disability candidate gene CTNNB1. 17 While heterozygous loss of function mutations in CTNNB1 are causative for intellectual disability in humans, conditional knock outs for CTNNB1 have no reported behavioural or craniofacial phenotypes. 36, 37 These differences demonstrate that while mouse models of intellectual disability are of great use in our understanding of the causative mechanisms which underlie the condition, there are still genetic and neurodevelopmental differences between species which also must be taken into
account. We also note that while the Katnal1 1H mutation shows a loss of catalytic function in both HEK293 cells and Sertoli cells, 23 this loss of function has not been verified in neuronal cells. However, given that our data demonstrates that the Katnal1 1H mutation lies in an essential catalytic domain and that we show neuronal phenotypes in Katnal1 1H/1H mice, we would expect to see the same loss of catalytic function in neurons.
The data we present here also demonstrate defects in motile cilia in Katnal1 1H/1H mice. Ciliary disruptions in humans (ciliopathies) include Bardet-Biedl and Joubert syndrome. 38 While there is currently limited data available regarding the behavioural phenotypes of mouse models of ciliopathies, we note that ciliary dysfunction in mice has been linked with learning and memory 39 and vocalisation phenotypes, 40 both of which were disturbed in the Katnal1 1H/1H mice described here. It is also notable that the neuronal migration and enlarged ventricle phenotypes that we describe in Katnal1 1H/1H mice recapitulate features associated with known ciliopathy gene mutations. [41] [42] [43] [44] Furthermore in Bardet-Biedl syndrome mouse models ciliary defects such as reduced CBF 45 and structural defects such as abnormal lengthening and swellings along their length 41 have been described, that are similar to those we describe in Katnal1 1H/1H mice. There is strong evidence that ciliopathy
associated genes play a number of roles in neuronal development by affecting processes such as progenitor proliferation or maintenance of the radial glia scaffold. 43 However it is also clear that defects in microtubule organisation also affect synaptic structure. 2 At present it is difficult to disentangle the relative contributions of defects in microtubule severing and ciliary abnormalities to the overall phenotypes we observe in Katnal1 1H/1H mice. Further investigations are required to clarify the impacts of these two processes. However it is notable that while defects in cilia structure may contribute to the phenotypes we describe in Katnal1 1H/1H mice, they are far less prominent in Katnal1 1H/1H mice than in other mouse ciliopathy models, 41 suggesting that the ciliary component of KATNAL1 dysfunction may be mild compared to other ciliopathies. Similarly while hydrocephalus has been suggested to be a component of some ciliopathy mouse models, 46 Katnal1 1H/1H mice showed only
increased ventricle size rather than an increased incidence of hydrocephalus, further suggesting the ciliary defects in these animals are mild compared to other ciliopathies.
In summary the data presented here clearly demonstrate that KATNAL1 plays an important role in a variety of neuronal processes including neuronal migration, neuronal morphology and ependymal ciliary function. The downstream effect of these defects leads in turn to a number of behavioural changes including in learning and memory, reaction to anxiogenic situations and circadian rhythms. These data therefore highlight how perturbations in KATNAL1 may play a role in neuronal dysfunction and demonstrates that the enzyme is a novel candidate in the study of behavioural and neurodevelopmental disorders.

The authors declare no conflict of interest.
Screening of FDA-Approved Drugs for Inhibitors of Japanese Encephalitis Virus Infection

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5640845/

SHA: 1bd2f6497996fc0fccd8dffd7f84846d3d36f964

Authors: Wang, Shaobo; Liu, Yang; Guo, Jiao; Wang, Peilin; Zhang, Leike; Xiao, Gengfu; Wang, Wei
Date: 2017-10-13
DOI: 10.1128/jvi.01055-17
License: cc-by
Abstract: Japanese encephalitis virus (JEV), an arthropod-borne flavivirus, is a major cause of acute viral encephalitis in humans. No approved drug is available for the specific treatment of JEV infections, and the available vaccines are not effective against all clinical JEV isolates. In the study described here, a high-throughput screening of an FDA-approved drug library for inhibitors of JEV was performed. Five hit drugs that inhibited JEV infection with a selective index of >10 were identified. The antiviral activities of these five hit drugs against other flavivirus, including Zika virus, were also validated. As three of the five hit drugs were calcium inhibitors, additional types of calcium inhibitors that confirmed that calcium is essential for JEV infection, most likely during viral replication, were utilized. Adaptive mutant analysis uncovered that replacement of Q130, located in transmembrane domain 3 of the nonstructural NS4B protein, which is relatively conserved in
flaviviruses, with R or K conferred JEV resistance to manidipine, a voltage-gated Ca(2+) channel (VGCC) inhibitor, without an apparent loss of the viral growth profile. Furthermore, manidipine was indicated to protect mice against JEV-induced lethality by decreasing the viral load in the brain, while it abrogated the histopathological changes associated with JEV infection. This study provides five antiflavivirus candidates and identifies cytoplasmic calcium to be a novel antiviral target for the treatment of JEV infection. The findings reported here provide therapeutic possibilities for combating infections caused by flaviviruses. IMPORTANCE No approved therapy for the treatment of Japanese encephalitis virus infection is currently available. Repurposing of approved drugs would accelerate the development of a therapeutic stratagem. In this study, we screened a library of FDA-approved drugs and identified five hit drugs, especially calcium inhibitors, exerting antiflavivirus activity
that blocked viral replication. The in vivo efficacy and toxicity of manidipine were investigated with a mouse model of JEV infection, and the viral target was identified by generating an adaptive mutant.
Text: F laviviruses are taxonomically classified in the genus Flavivirus and family Flaviviridae.

These viruses comprise over 70 different pathogens, such as Japanese encephalitis virus (JEV), Zika virus (ZIKV), dengue virus (DENV), West Nile virus (WNV), and yellow fever virus (YFV). Most flaviviruses are arthropod borne and cause public health problems worldwide (1) . The development and usage of vaccines against some flaviviruses, such as JEV, YFV, and tick-borne encephalitis virus (TBEV), have decreased the rates of morbidity and mortality from infections caused by these viruses (2) ; however, flavivirus-induced diseases are still pandemic, and few therapies beyond intensive supportive care are currently available.
Flaviviruses have an approximately 11-kb positive-stranded RNA genome containing a single open reading frame (ORF) flanked by untranslated regions (UTRs) at both termini. The ORF encodes three structural proteins, including the capsid (C), membrane (premembrane [prM] and membrane [M] ), and envelope (E), and seven nonstructural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) (3) . These seven nonstructural proteins participate in viral replication, virion assembly, and virus escape from immune surveillance.
To date, no specific antivirals with activity against flaviviruses are available. To address this, we conducted a screen of a library of 1,018 FDA-approved drugs. Since flaviviruses are similar in structure and pathogenesis, we first utilized JEV as the prototype to screen the drug library and subsequently validated the antiviral activities with ZIKV, WNV, and DENV type 2 (DENV-2). The hit drugs identified in this study offer potential new therapies for the treatment of flavivirus infection and disease.
Screening of an FDA-approved drug library for inhibitors of JEV infection. Recombinant viral particles (RVPs) with the luciferase-reporting replicon enveloped by the JEV structural proteins were used to select inhibitors, with a focus on those that inhibit virus entry and replication, by a high-throughput screening (HTS) assay (4, 5) . The number of genomic RNA copies of RVP was determined to be 8.4 ϫ 10 6 copies/ml by using a standard curve generated with plasmids carrying the infectious clone. The HTS assay conditions, including the seeding cell density and RVP dose, were optimized to be 10,000 cells per 96-well plate and 20 l (16 copies/cell) RVP for the infective dose, respectively. Under the optimized conditions, the signal-to-basal (S/B) ratio, coefficient of variation (CV), and Z= factor were 38,374, 2.8%, and 0.89, respectively, which demonstrated that the assay was robust and suitable for the large-scale screening of compounds.
A schematic of the HTS assay is depicted in Fig. 1B . After three rounds of screening, five hits with a selective index (SI; which is equal to the 50% cytotoxic concentration [CC 50 [/50% inhibitory concentration [IC 50 ]) of Ͼ10 were selected. The CC 50 values of the hit drugs exhibited in Fig. 1B were similar to those previously published for diverse cell systems but determined using different toxicity assays (6) (7) (8) (9) (10) (11) (12) (13) . Three of the hit drugs, manidipine, cilnidipine, and benidipine hydrochloride, were dihydropyridine (DHP) voltage-gated Ca 2ϩ channel (VGCC) antagonists, while pimecrolimus is an inhibitor of inflammatory cytokine secretion and nelfinavir mesylate is an HIV-1 protease blocker. All five drugs exhibited a dose-dependent inhibition of JEV RVP infection (Fig. 1C) . To validate the antiviral effect, hit drugs were purchased from other commercial sources and tested. In the reconfirmation screen, all hit drugs showed antiviral and cytotoxic
effects similar to those found in the primary screen.
Validation of hit drugs. To verify the results obtained by the luciferase reporter assays, we also investigated the antiviral effect of the five hit drugs on wild-type JEV strain AT31. As expected from the HTS assay, all five drugs robustly inhibited virus production, with a reduction of approximately 4 to 5 log units at the highest concentration and an approximately 1-log-unit decrease with 2.5 M the drugs (Fig. 2B) . A sharp decrease in JEV RNA levels was also detected (Fig. 2C) . The attenuated RNA levels in the high-dose, middle-dose, and low-dose groups were all above 40%. In particular, in the manidipine-treated group, the inhibitory effect was at least 80% compared to that for the control, which showed a strong inhibition of viral replication. Consistent with the inhibition of virus replication and production, expression of the viral structural protein prM was hardly detectable following treatment with the drugs at the high concentration (Fig. 2D) . Overall, the results in Fig.
2 confirmed that the five hit drugs inhibited JEV infection in a dose-dependent manner in vitro.
Drugs inhibit JEV infection during viral RNA synthesis. Because RVPs, which have a natural virus-like envelope on the outside and a replicon on the inside, permitted the quantification of JEV productive entry and replication, a time-of-addition experiment was performed to investigate whether the hit drugs blocked the entry step or the replication step. As shown in Fig. 3B , no suppression of luciferase activity by any of the hit drugs was observed when they were used as treatments before infection or during infection or as a virucide, suggesting that these drugs do not inhibit JEV infection either by inactivating the virus directly or by blocking JEV entry. However, these drugs exerted fully inhibitory effects when they were added at 1 h postinfection, suggesting that viral replication was the stage at which these drugs showed inhibitory activity.
To confirm this suggestion, we investigated the inhibitory effects of these drugs on the JEV replicon. The highest concentration of manidipine and nelfinavir mesylate tested in baby hamster kidney (BHK-21) cells was adjusted to 5 M and 10 M, respectively. It was shown that all five drugs inhibited JEV RNA synthesis in a dosedependent manner, while neither drug inhibited the initial translation of replicon RNA (5, 14) (Fig. 3C) , confirming that these drugs inhibited JEV infection at the stage of replication.
Hit drugs exhibit broad-spectrum antiflavivirus activity. In order to determine whether the antiviral activity of the five hit drugs extended to other flaviviruses, we explored their antiviral effect against ZIKV. Similar to the findings for JEV, the ZIKV titer was decreased by multiple log units when ZIKV was treated with a high concentration of each of the drugs (Fig. 4A) . Moreover, ZIKV exhibited a higher sensitivity to the two calcium channels inhibitors manidipine and cilnidipine than JEV, with no plaque formation being observed at 10 M. Consistent with this result, sharp decreases in the level of replication of ZIKV RNA and the level of expression of viral protein were also detected (Fig. 4A) . Notably, treatment with 5 M manidipine produced a 95% inhibition of viral replication, translation, and viral yields. Taken together, these results indicate that the hit drugs could effectively inhibit ZIKV infection.
Since these drugs exhibited their anti-JEV effects at the stage of viral replication, we further tested the effects against WNV and DENV-2 by using WNV and DENV-2 replicons. Similar to the results for JEV, a dose-dependent reduction in the level of WNV replication was observed with the drug treatments. The same phenotype was observed for DENV-2 for all drugs except nelfinavir mesylate, which showed no effect at the concentrations tested ( Fig. 4B and C). Together, these results indicate that the five hit drugs are excellent candidates for broad-spectrum antiflavivirus treatment. Antiviral effect of calcium inhibitors. Since three hit drugs, manidipine, cilnidipine, and benidipine hydrochloride, were DHP VGCC inhibitors, we asked whether other calcium antagonists could block JEV infection. To address this question, we employed four different classes of inhibitors. Verapamil, a prototype phenylalkylamine (PAA) VGCC inhibitor (15) , exhibited a dose-dependent inhibition of JEV on both
African Green monkey kidney (Vero) and human hepatocellular carcinoma (Huh-7) cells (Fig. 5) , which was consistent with the inhibitory effects of the DHP inhibitors, suggesting that calcium channels play an important role in JEV infection. Cyclosporine and 2-aminobiphenyl borate (2-APB), which inhibit the efflux of Ca 2ϩ from the mitochondrial and endoplasmic reticulum (ER) pool, respectively (16) (17) (18) (19) , were also found to block JEV infection effectively. Similarly, treatment with the cell-permeant Ca 2ϩ chelator 1,2-bis-(o-aminophenoxy)-ethane-N,N,N=,N=-tetraacetic acid, tetraacetoxymethyl ester (BAPTA-AM), could also suppress JEV infection. Taken together, we concluded that intracellular Ca 2ϩ is essential for JEV infection and cytoplasmic calcium is a potent target for antiflavivirus treatment.
Selection and characterization of manidipine-resistant JEV. To identify the viral target of the calcium channel inhibitor, we selected a manidipine-resistant virus by serially passaging JEV in the presence of manidipine. Viruses from passage 20 (P20) showed robust resistance compared with the wild type (WT) (Fig. 6A ). When JEV from P20 was treated with 5 M or 10 M manidipine, the viral titer was about 10-and 100-fold higher than that of the WT, respectively. Individual virus clones were isolated, and two isolates were randomly selected and amplified. An amino acid substitution was observed in two isolated clones, resulting in a glutamine (Q)-to-arginine (R) switch at amino acid position 130 in transmembrane domain 3 (TMD3) of NS4B, i.e., position 2401 of the translated polyprotein in the JEV infectious cDNA clone (Fig. 6B ). Sequence alignment of NS4B indicated that Q130 was conserved in all flaviviruses except YFV, which possessed a lysine at that position (Fig.  6B) . The conserved
Q130 of NS4B may account for the sensitivity of JEV, ZIKV, WNV, and DENV-2 to manidipine, as described above (Fig. 4) , while YFV showed resistance to the drug (data not shown).
To confirm that the Q130R mutation did confer manidipine resistance and to investigate the role of Q130 in NS4B function, we produced JEV clones with the Q130R, Q130K, Q130E, or Q130A mutation by introducing the desired mutations into the infectious cDNA clone and rescuing the mutant viruses. To investigate the biological properties of the mutant viruses, we first examined the growth kinetics of the rescued viruses. As shown in Fig. 6C , all mutant viruses had an accumulation of infectious virions and reached the highest titer at 60 h postinfection. Infection of the Q130R and Q130K mutant viruses resulted in growth curves similar to the growth curve for the WT (Fig.  6C) , while the Q130E and Q130A mutants produced smaller amounts of viruses between 24 and 60 h. Analysis of the plaque morphology revealed that the plaques of the Q130R, Q130K, and Q130E mutants were similar to the plaques of the WT, whereas the plaques of the Q130A mutant were smaller than those of the WT.
We next investigated the sensitivity of the four mutant viruses to manidipine. As shown in Fig. 6D , the Q130R and Q130K mutant viruses were resistant to manidipine. At a 10 M concentration, manidipine efficiently inhibited WT JEV infection and reduced the viral yields by approximately 4 log units, while the Q130R and Q130K mutant viruses were resistant to manidipine and the viral titer decreased less than 2 log units. The Q130A mutant virus demonstrated moderate resistance and a slightly higher Taken together, it could be concluded that Q130 not only is critical for conferring manidipine sensitivity but also is important for JEV replication. The replacement of glutamine with basic amino acids conferred resistance to manidipine without an apparent loss of growth.
In vivo efficacy of manidipine. As manidipine exhibited the strongest inhibitory activities on JEV replication as well as ZIKV infection when its activities were compared with those of the five hit drugs (Fig. 2 and 4A) , we further examined the protective effect of manidipine against JEV-induced lethality in a mouse model. As anticipated, mice in the JEV-infected vehicle-treated group started to show symptoms, including limb paralysis, restriction of movement, piloerection, body stiffening, and whole-body tremor, from day 5 postinfection. Within 21 days postinfection, most mice in the JEV-infected group succumbed to the infection, with the mortality rate being 73% (4 out of 15 animals survived). Manidipine treatment following JEV infection reduced the mortality rate to 20% (12 out of 15 animals survived) (Fig. 7A ). Mice treated with manidipine alone or treated with manidipine and infected with JEV showed little abnormal behavior, similar to the findings for the mice in the
vehicle-treated group. These results suggest that manidipine provided effective protection against JEVinduced mortality.