Zolpidem in Elderly Patients With Acute Treatment Resistant Catatonia: A Case Report.

Catatonia is defined by the Diagnostic and Statistical Manual of Mental Disorders-fifth Edition (DSM-5) as the presence of 3 or more of the following symptoms: catalepsy, waxy flexibility, stupor, agitation, mutism, negativism, posturing, mannerisms, stereotypies, grimacing, echolalia, and echopraxia. Screening instruments, such as the Bush Francis Catatonia Rating Scale (BFCRS) or the Northoff Catatonia Scale, are utilized to screen and track the severity and course of symptoms. The primary pharmacologic treatment for catatonia is the lorazepam challenge test. Second-line pharmacologic therapies are indicated when patients have an insufficient response to benzodiazepines and electroconvulsive therapy (ECT). In this case report, we report a case of a geriatric patient given high-dose zolpidem as an alternative agent with cardiac contraindications to ECT.

Pharmacotherapy considerations in pregnant patients on hemodialysis.

PURPOSE: Successful pregnancy rates on dialysis are increasing with the advent of intensive hemodialysis and advances in medical management. SUMMARY: Data support the use of intensive hemodialysis in pregnant women with end-stage kidney disease (ESKD). This paper provides an overview of common pharmacotherapeutic changes in management when caring for a pregnant woman receiving intensive hemodialysis. Pregnant patients on peritoneal dialysis were excluded from this analysis due to insufficient data. Topics covered include those related to anemia (iron and erythropoietin stimulating agents), blood pressure agents, monitoring of phosphorus, as well as nutrition and anticoagulation. CONCLUSION: When patients on hemodialysis become pregnant, medication adjustments are needed regarding antihypertensives, anemia management, and mineral-bone disease management as many agents require dose adjustment, switching agents due to teratogenicity, or cessation due to fetal complications. There are minimal data in this population; however, successful and healthy infants have been delivered in this patient population with the medication changes discussed.

N6-methyladenosine is required for efficient RNA synthesis of Ebola virus and other haemorrhagic fever viruses.

N6-methyladenosine (m6A) is one of the most abundant modifications of cellular RNA, where it serves various functions. m6A methylation of many viral RNA species has also been described; however, little is known about the m6A epitranscriptome of haemorrhagic fever-causing viruses like Ebola virus (EBOV). Here, we analysed the importance of the methyltransferase METTL3 for the life cycle of this virus. We found that METTL3 interacts with the EBOV nucleoprotein and the transcriptional activator VP30 to support viral RNA synthesis, and that METTL3 is recruited into EBOV inclusions bodies, where viral RNA synthesis occurs. Analysis of the m6A methylation pattern of EBOV mRNAs showed that they are methylated by METTL3. Further studies revealed that METTL3 interaction with the viral nucleoprotein, as well as its importance for RNA synthesis and protein expression, is also observed for other haemorrhagic fever viruses such as Junín virus (JUNV) and Crimean-Congo haemorrhagic fever virus (CCHFV). The negative effects on viral RNA synthesis due to loss of m6A methylation are independent of innate immune sensing, as METTL3 knockout did not affect type I interferon induction in response to viral RNA synthesis or infection. Our results suggest a novel function for m6A that is conserved among diverse haemorrhagic fever-causing viruses (i.e. EBOV, JUNV and CCHFV), making METTL3 a promising target for broadly-acting antivirals.

Ebola virus inclusion bodies are liquid organelles whose formation is facilitated by nucleoprotein oligomerization.

Viral RNA synthesis of several non-segmented, negative-sense RNA viruses (NNSVs) takes place in inclusion bodies (IBs) that show properties of liquid organelles, which are formed by liquid-liquid phase separation of scaffold proteins. It is believed that this is driven by intrinsically disordered regions (IDRs) and/or multiple copies of interaction domains, which for NNSVs are usually located in their nucleo - and phosphoproteins. In contrast to other NNSVs, the Ebola virus (EBOV) nucleoprotein NP alone is sufficient to form IBs without the need for a phosphoprotein, and to facilitate the recruitment of other viral proteins into these structures. While it has been proposed that also EBOV IBs are liquid organelles, this has so far not been formally demonstrated. Here we used a combination of live cell microscopy, fluorescence recovery after photobleaching assays, and mutagenesis approaches together with reverse genetics-based generation of recombinant viruses to study the formation of EBOV IBs. Our results demonstrate that EBOV IBs are indeed liquid organelles, and that oligomerization but not IDRs of the EBOV nucleoprotein plays a key role in their formation. Additionally, VP35 (often considered the phosphoprotein-equivalent of EBOV) is not essential for IB formation, but alters their liquid behaviour. These findings define the molecular mechanism for the formation of EBOV IBs, which play a central role in the life cycle of this deadly virus.

Cryoelectron microscopic structure of the nucleoprotein-RNA complex of the European filovirus, Lloviu virus.

Lloviu virus (LLOV) is a novel filovirus detected in Schreiber's bats in Europe. The isolation of the infectious LLOV from bats has raised public health concerns. However, the virological and molecular characteristics of LLOV remain largely unknown. The nucleoprotein (NP) of LLOV encapsidates the viral genomic RNA to form a helical NP-RNA complex, which acts as a scaffold for nucleocapsid formation and de novo viral RNA synthesis. In this study, using single-particle cryoelectron microscopy, we determined two structures of the LLOV NP-RNA helical complex, comprising a full-length and a C-terminally truncated NP. The two helical structures were identical, demonstrating that the N-terminal region determines the helical arrangement of the NP. The LLOV NP-RNA protomers displayed a structure similar to that in the Ebola and Marburg virus, but the spatial arrangements in the helix differed. Structure-based mutational analysis identified amino acids involved in the helical assembly and viral RNA synthesis. These structures advance our understanding of the filovirus nucleocapsid formation and provide a structural basis for the development of antifiloviral therapeutics.

A dsRNA-binding mutant reveals only a minor role of exonuclease activity in interferon antagonism by the arenavirus nucleoprotein.

The arenavirus nucleoprotein (NP) plays an important role in the virus' ability to block interferon (IFN) production, and its exonuclease function appears to contribute to this activity. However, efforts to analyze this contribution are complicated by the functional overlap between the exonuclease active site and a neighboring region involved in IKKε-binding and subsequent inhibition of IRF3 activation, which also plays an important role in IFN production. To circumvent this issue, we mutated a residue located away from the active site that is involved in binding of the dsRNA substrate being targeted for exonuclease digestion, i.e. H426A. We found that expression of Tacaribe virus (TCRV) NP containing this RNA-binding H426A mutation was still able to efficiently block IFN-ß promoter activity in response to Sendai virus infection, despite being strongly impaired in its exonuclease activity. This was in contrast to a conventional exonuclease active site mutant (E388A), which was impaired with respect to both exonuclease activity and IFN antagonism. Importantly, growth of a recombinant virus encoding the RNA-binding mutation (rTCRV-H426A) was similar to wild-type in IFN-deficient cells, unlike the active site mutant (rTCRV-E388A), which was already markedly impaired in these cells. Further, in IFN-competent cells, the TCRV-H426A RNA-binding mutant showed more robust growth and delayed IFN-ß mRNA upregulation compared to the TCRV-E388A active site mutant. Taken together, this novel mutational approach, which allows us to now dissect the different contributions of the NP exonuclease activity and IKKε-binding/IRF3 inhibition to IFN antagonism, clearly suggests that conventional exonuclease mutants targeting the active site overestimate the contribution of the exonuclease function, and that rather other IFN antagonistic functions of NP play the dominant role in IFN-antagonism.

Evidence for Viral mRNA Export from Ebola Virus Inclusion Bodies by the Nuclear RNA Export Factor NXF1.

Many negative-sense RNA viruses, including the highly pathogenic Ebola virus (EBOV), use cytoplasmic inclusion bodies (IBs) for viral RNA synthesis. However, it remains unclear how viral mRNAs are exported from these IBs for subsequent translation. We recently demonstrated that the nuclear RNA export factor 1 (NXF1) is involved in a late step in viral protein expression, i.e., downstream of viral mRNA transcription, and proposed it to be involved in this mRNA export process. We now provide further evidence for this function by showing that NXF1 is not required for translation of viral mRNAs, thus pinpointing its function to a step between mRNA transcription and translation. We further show that RNA binding of both NXF1 and EBOV NP is necessary for export of NXF1 from IBs, supporting a model in which NP hands viral mRNA over to NXF1 for export. Mapping of NP-NXF1 interactions allowed refinement of this model, revealing two separate interaction sites, one of them directly involving the RNA binding cleft of NP, even though these interactions are RNA-independent. Immunofluorescence analyses demonstrated that individual NXF1 domains are sufficient for its recruitment into IBs, and complementation assays helped to define NXF1 domains important for its function in the EBOV life cycle. Finally, we show that NXF1 is also required for protein expression of other viruses that replicate in cytoplasmic IBs, including Lloviu and Junín virus. These data suggest a role for NXF1 in viral mRNA export from IBs for various viruses, making it a potential target for broadly active antivirals. IMPORTANCE Filoviruses such as the Ebola virus (EBOV) cause severe hemorrhagic fevers with high case fatality rates and limited treatment options. The identification of virus-host cell interactions shared among several viruses would represent promising targets for the development of broadly active antivirals. In this study, we reveal the mechanistic details of how EBOV usurps the nuclear RNA export factor 1 (NXF1) to export viral mRNAs from viral inclusion bodies (IBs). We further show that NXF1 is not only required for the EBOV life cycle but also necessary for other viruses known to replicate in cytoplasmic IBs, including the filovirus Lloviu virus and the highly pathogenic arenavirus Junín virus. This suggests NXF1 as a promising target for the development of broadly active antivirals.

A Genome-Wide CRISPR/Cas9 Screen Reveals the Requirement of Host Sphingomyelin Synthase 1 for Infection with Pseudorabies Virus Mutant gD-Pass.

Herpesviruses are large DNA viruses, which encode up to 300 different proteins including enzymes enabling efficient replication. Nevertheless, they depend on a multitude of host cell proteins for successful propagation. To uncover cellular host factors important for replication of pseudorabies virus (PrV), an alphaherpesvirus of swine, we performed an unbiased genome-wide CRISPR/Cas9 forward screen. To this end, a porcine CRISPR-knockout sgRNA library (SsCRISPRko.v1) targeting 20,598 genes was generated and used to transduce porcine kidney cells. Cells were then infected with either wildtype PrV (PrV-Ka) or a PrV mutant (PrV-gD-Pass) lacking the receptor-binding protein gD, which regained infectivity after serial passaging in cell culture. While no cells survived infection with PrV-Ka, resistant cell colonies were observed after infection with PrV-gD-Pass. In these cells, sphingomyelin synthase 1 (SMS1) was identified as the top hit candidate. Infection efficiency was reduced by up to 90% for PrV-gD-Pass in rabbit RK13-sgms1KO cells compared to wildtype cells accompanied by lower viral progeny titers. Exogenous expression of SMS1 partly reverted the entry defect of PrV-gD-Pass. In contrast, infectivity of PrV-Ka was reduced by 50% on the knockout cells, which could not be restored by exogenous expression of SMS1. These data suggest that SMS1 plays a pivotal role for PrV infection, when the gD-mediated entry pathway is blocked.

Requirements of a Group Intervention for Adolescents with Internet Gaming Disorder in a Clinical Setting: A Qualitative Interview Study.

Internet gaming disorder (IGD) has become an important health concern in a significant proportion of adolescents. Intervention studies in this age group are scarce, mostly follow quantitative designs, and rarely consider adolescents' experiences. This study aimed to evaluate the requirements for a group therapy program for adolescents with IGD. A qualitative interview study was conducted in a German clinic for addictive disorders in childhood and adolescence with nine participants (seven IGD patients (12-18 years, M = 15.86, SD = 1.95) and two psychotherapists). The semi-structured interviews addressed helpful contents, general conditions, and suggestions for alterations for an effective group intervention. Data were analyzed using content structuring qualitative analysis. Patient interview data resulted in 234 codings with eight main categories. Expert interview data yielded 151 codings with six main categories. The following treatment components were described as effective by the participants and experts: psychoeducation, emotion management, behavior analysis and modification, social skills training, parent participation, and relapse prevention. Additionally, adolescents emphasized the importance of group functionality for coherence, feedback and rewards, content presentation, physical activity and fun. The results are a valuable addition to findings from quantitative studies on IGD interventions and an interesting starting point for further representative studies.

The Great Gut Mimicker: A case report of MIS-C and appendicitis clinical presentation overlap in a teenage patient.

BACKGROUND: Abdominal pain and other gastrointestinal symptoms are common presenting features of multisystem inflammatory syndrome in children (MIS-C) and can overlap with infectious or inflammatory abdominal conditions, making accurate diagnosis challenging. CASE PRESENTATION: We describe the case of a 16-year-old female who presented with clinical symptoms suggestive of appendicitis and an abdominal computed tomography (CT) that revealed features concerning for appendicitis. After laparoscopic appendectomy, histopathology of the appendix demonstrated only mild serosal inflammation and was not consistent with acute appendicitis. Her overall clinical presentation was felt to be consistent with MIS-C and she subsequently improved with immunomodulatory and steroid treatment. CONCLUSIONS: We note that MIS-C can mimic acute appendicitis. This case highlights MIS-C as a cause of abdominal imaging with features concerning for appendicitis, and MIS-C should be considered in the differential for a patient with appendicitis-like symptoms and a positive COVID-19 IgG. Lab criteria, specifically low-normal white blood cell count and thrombocytopenia, appears to be of high relevance in differing MIS-C from acute appendicitis, even when appendix radiologically is dilated.

Remdesivir inhibits the polymerases of the novel filoviruses Lloviu and Bombali virus.

In recent years, a number of novel filoviruses (e.g. Lloviu virus (LLOV) and Bombali virus (BOMV)) have been discovered. While antibody-based therapeutics have recently been approved for treatment of infections with the filovirus Ebola virus (EBOV), no treatment options for novel filoviruses currently exist. Further, the development of antivirals against them is complicated by the fact that only sequence information, but no actual virus isolates, are available. To address this issue, we developed a reverse genetics-based minigenome system for BOMV, which allows us to assess the activity of the BOMV polymerase. Together with similar systems that we have developed for other filoviruses in the past (i.e. LLOV and Reston virus (RESTV)), we then assessed the efficiency of remdesivir, a known inhibitor of the EBOV polymerase that has recently been tested in a clinical trial for efficacy against Ebola disease. We show that remdesivir is indeed also active against the polymerases of BOMV, LLOV, and RESTV, with comparable IC50 values to its activity against EBOV. This suggests that treatment with remdesivir might represent a viable option in case of infections with novel filoviruses.

The Cellular Protein CAD is Recruited into Ebola Virus Inclusion Bodies by the Nucleoprotein NP to Facilitate Genome Replication and Transcription.

Ebola virus (EBOV) is a zoonotic pathogen causing severe hemorrhagic fevers in humans and non-human primates with high case fatality rates. In recent years, the number and extent of outbreaks has increased, highlighting the importance of better understanding the molecular aspects of EBOV infection and host cell interactions to control this virus more efficiently. Many viruses, including EBOV, have been shown to recruit host proteins for different viral processes. Based on a genome-wide siRNA screen, we recently identified the cellular host factor carbamoyl-phosphate synthetase 2, aspartate transcarbamylase, and dihydroorotase (CAD) as being involved in EBOV RNA synthesis. However, mechanistic details of how this host factor plays a role in the EBOV life cycle remain elusive. In this study, we analyzed the functional and molecular interactions between EBOV and CAD. To this end, we used siRNA knockdowns in combination with various reverse genetics-based life cycle modelling systems and additionally performed co-immunoprecipitation and co-immunofluorescence assays to investigate the influence of CAD on individual aspects of the EBOV life cycle and to characterize the interactions of CAD with viral proteins. Following this approach, we could demonstrate that CAD directly interacts with the EBOV nucleoprotein NP, and that NP is sufficient to recruit CAD into inclusion bodies dependent on the glutaminase (GLN) domain of CAD. Further, siRNA knockdown experiments indicated that CAD is important for both viral genome replication and transcription, while substrate rescue experiments showed that the function of CAD in pyrimidine synthesis is indeed required for those processes. Together, this suggests that NP recruits CAD into inclusion bodies via its GLN domain in order to provide pyrimidines for EBOV genome replication and transcription. These results define a novel mechanism by which EBOV hijacks host cell pathways in order to facilitate genome replication and transcription and provide a further basis for the development of host-directed broad-spectrum antivirals.

The Ebola Virus Nucleoprotein Recruits the Nuclear RNA Export Factor NXF1 into Inclusion Bodies to Facilitate Viral Protein Expression.

Ebola virus (EBOV) causes severe outbreaks of viral hemorrhagic fever in humans. While virus-host interactions are promising targets for antivirals, there is only limited knowledge regarding the interactions of EBOV with cellular host factors. Recently, we performed a genome-wide siRNA screen that identified the nuclear RNA export factor 1 (NXF1) as an important host factor for the EBOV life cycle. NXF1 is a major component of the nuclear mRNA export pathway that is usurped by many viruses whose life cycles include nuclear stages. However, the role of NXF1 in the life cycle of EBOV, a virus replicating in cytoplasmic inclusion bodies, remains unknown. In order to better understand the role of NXF1 in the EBOV life cycle, we performed a combination of co-immunoprecipitation and double immunofluorescence assays to characterize the interactions of NXF1 with viral proteins and RNAs. Additionally, using siRNA-mediated knockdown of NXF1 together with functional assays, we analyzed the role of NXF1 in individual aspects of the virus life cycle. With this approach we identified the EBOV nucleoprotein (NP) as a viral interaction partner of NXF1. Further studies revealed that NP interacts with the RNA-binding domain of NXF1 and competes with RNA for this interaction. Co-localization studies showed that RNA binding-deficient, but not wildtype NXF1, accumulates in NP-derived inclusion bodies, and knockdown experiments demonstrated that NXF1 is necessary for viral protein expression, but not for viral RNA synthesis. Finally, our results showed that NXF1 interacts with viral mRNAs, but not with viral genomic RNAs. Based on these results we suggest a model whereby NXF1 is recruited into inclusion bodies to promote the export of viral mRNA:NXF1 complexes from these sites. This would represent a novel function for NXF1 in the life cycle of cytoplasmically replicating viruses, and may provide a basis for new therapeutic approaches against EBOV, and possibly other emerging viruses.

High-throughput screening for negative-stranded hemorrhagic fever viruses using reverse genetics.

Viral hemorrhagic fevers (VHFs) cause thousands of fatalities every year, but the treatment options for their management remain very limited. In particular, the development of therapeutic interventions is restricted by the lack of commercial viability of drugs targeting individual VHF agents. This makes approaches like drug repurposing and/or the identification of broad range therapies (i.e. those directed at host responses or common proviral factors) highly attractive. However, the identification of candidates for such antiviral repurposing or of host factors/pathways important for the virus life cycle is reliant on high-throughput screening (HTS). Recently, such screening work has been increasingly facilitated by the availability of reverse genetics-based approaches, including tools such as full-length clone (FLC) systems to generate reporter-expressing viruses or various life cycle modelling (LCM) systems, many of which have been developed and/or greatly improved during the last years. In particular, since LCM systems are capable of modelling specific steps in the life cycle, they are a valuable tool for both targeted screening (i.e. for inhibitors of a specific pathway) and mechanism of action studies. This review seeks to summarize the currently available reverse genetics systems for negative-sense VHF causing viruses (i.e. arenaviruses, bunyaviruses and filoviruses), and to highlight the recent advancements made in applying these systems for HTS to identify either antivirals or new virus-host interactions that might hold promise for the development of future treatments for the infections caused by these deadly but neglected virus groups.

Assessment of the function and intergenus-compatibility of Ebola and Lloviu virus proteins.

Sequences for Lloviu virus (LLOV), a putative novel filovirus, were first identified in Miniopterus schreibersii bats in Spain following a massive bat die-off in 2002, and also recently found in bats in Hungary. However, until now it is unclear if these sequences correspond to a fully functional, infectious virus, and whether it will show a pathogenic phenotype like African filoviruses, such as ebola- and marburgviruses, or be apathogenic for humans, like the Asian filovirus Reston virus. Since no infectious virus has been recovered, the only opportunity to study infectious LLOV is to use a reverse genetics-based full-length clone system to de novo generate LLOV. As a first step in this process, and to investigate whether the identified sequences indeed correspond to functional viral proteins, we have developed life cycle modelling systems for LLOV, which allow us to study genome replication and transcription as well as entry of this virus. We show that all LLOV proteins fulfill their canonical role in the virus life cycle as expected based on the well-studied related filovirus Ebola virus. Further, we have analysed the intergenus-compatibility of proteins that have to act in concert to facilitate the virus life cycle. We show that some but not all proteins from LLOV and Ebola virus are compatible with each other, emphasizing the close relationship of these viruses, and informing future studies of filovirus biology with respect to the generation of genus-chimeric proteins in order to probe virus protein-protein interactions on a functional level.

A genome-wide siRNA screen identifies a druggable host pathway essential for the Ebola virus life cycle.

BACKGROUND: The 2014-2016 Ebola virus (EBOV) outbreak in West Africa highlighted the need for improved therapeutic options against this virus. Approaches targeting host factors/pathways essential for the virus are advantageous because they can potentially target a wide range of viruses, including newly emerging ones and because the development of resistance is less likely than when targeting the virus directly. However, systematic approaches for screening host factors important for EBOV have been hampered by the necessity to work with this virus at biosafety level 4 (BSL4). METHODS: In order to identify host factors involved in the EBOV life cycle, we performed a genome-wide siRNA screen comprising 64,755 individual siRNAs against 21,566 human genes to assess their activity in EBOV genome replication and transcription. As a screening platform, we used reverse genetics-based life cycle modelling systems that recapitulate these processes without the need for a BSL4 laboratory. RESULTS: Among others, we identified the de novo pyrimidine synthesis pathway as an essential host pathway for EBOV genome replication and transcription, and confirmed this using infectious EBOV under BSL4 conditions. An FDA-approved drug targeting this pathway showed antiviral activity against infectious EBOV, as well as other non-segmented negative-sense RNA viruses. CONCLUSIONS: This study provides a minable data set for every human gene regarding its role in EBOV genome replication and transcription, shows that an FDA-approved drug targeting one of the identified pathways is highly efficacious in vitro, and demonstrates the power of life cycle modelling systems for conducting genome-wide host factor screens for BSL4 viruses.

Analysis of a Putative Late Domain Using an Ebola Virus Transcription and Replication-Competent Virus-Like Particle System.

The Ebola virus (EBOV) matrix protein VP40 drives budding of virions and encodes 2 overlapping late domain motifs at amino acid positions 7-13 (PTAPPEY). However, these motifs are not absolutely essential for replication in cell culture, and recently a potential third late domain motif (YPx(6)I) was proposed at amino acid positions 18-26 of VP40. To analyze the importance of this motif in viral budding, we used a transcription and replication-competent virus-like particle system. Using this system, we show that this motif does not contribute to EBOV budding or particle propagation.