Severe Pediatric COVID-19 and Multisystem Inflammatory Syndrome in Children From Wild-type to Population Immunity:A Prospective Multicenter Cohort Study With Real-time Reporting

BACKGROUND: SARS-CoV-2 variant evolution and increasing immunity altered the impact of pediatric SARS-CoV-2 infection. Public health decision-making relies on accurate and timely reporting of clinical data. METHODS: This international hospital-based multicenter, prospective cohort study with real-time reporting was active from March 2020 to December 2022. We evaluated longitudinal incident rates and risk factors for disease severity. RESULTS: We included 564 hospitalized children with acute COVID-19 (n = 375) or multisystem inflammatory syndrome in children (n = 189) from the Netherlands, Curaçao and Surinam. In COVID-19, 134/375 patients (36%) needed supplemental oxygen therapy and 35 (9.3%) required intensive care treatment. Age above 12 years and preexisting pulmonary conditions were predictors for severe COVID-19. During omicron, hospitalized children had milder disease. During population immunity, the incidence rate of pediatric COVID-19 infection declined for older children but was stable for children below 1 year. The incidence rate of multisystem inflammatory syndrome in children was highest during the delta wave and has decreased rapidly since omicron emerged. Real-time reporting of our data impacted national pediatric SARS-CoV-2 vaccination- and booster-policies. CONCLUSIONS: Our data supports the notion that similar to adults, prior immunity protects against severe sequelae of SARS-CoV-2 infections in children. Real-time reporting of accurate and high-quality data is feasible and impacts clinical and public health decision-making. The reporting framework of our consortium is readily accessible for future SARS-CoV-2 waves and other emerging infections.</p

Severe Pediatric COVID-19 and Multisystem Inflammatory Syndrome in Children From Wild-type to Population Immunity:A Prospective Multicenter Cohort Study With Real-time Reporting

BACKGROUND: SARS-CoV-2 variant evolution and increasing immunity altered the impact of pediatric SARS-CoV-2 infection. Public health decision-making relies on accurate and timely reporting of clinical data. METHODS: This international hospital-based multicenter, prospective cohort study with real-time reporting was active from March 2020 to December 2022. We evaluated longitudinal incident rates and risk factors for disease severity. RESULTS: We included 564 hospitalized children with acute COVID-19 (n = 375) or multisystem inflammatory syndrome in children (n = 189) from the Netherlands, Curaçao and Surinam. In COVID-19, 134/375 patients (36%) needed supplemental oxygen therapy and 35 (9.3%) required intensive care treatment. Age above 12 years and preexisting pulmonary conditions were predictors for severe COVID-19. During omicron, hospitalized children had milder disease. During population immunity, the incidence rate of pediatric COVID-19 infection declined for older children but was stable for children below 1 year. The incidence rate of multisystem inflammatory syndrome in children was highest during the delta wave and has decreased rapidly since omicron emerged. Real-time reporting of our data impacted national pediatric SARS-CoV-2 vaccination- and booster-policies. CONCLUSIONS: Our data supports the notion that similar to adults, prior immunity protects against severe sequelae of SARS-CoV-2 infections in children. Real-time reporting of accurate and high-quality data is feasible and impacts clinical and public health decision-making. The reporting framework of our consortium is readily accessible for future SARS-CoV-2 waves and other emerging infections.</p

Multidrug Resistant 2009 A/H1N1 Influenza Clinical Isolate with a Neuraminidase I223R Mutation Retains Its Virulence and Transmissibility in Ferrets

Only two classes of antiviral drugs, neuraminidase inhibitors and adamantanes, are approved for prophylaxis and therapy against influenza virus infections. A major concern is that influenza virus becomes resistant to these antiviral drugs and spreads in the human population. The 2009 pandemic A/H1N1 influenza virus is naturally resistant to adamantanes. Recently a novel neuraminidase I223R mutation was identified in an A/H1N1 virus showing cross-resistance to the neuraminidase inhibitors oseltamivir, zanamivir and peramivir. However, the ability of this virus to cause disease and spread in the human population is unknown. Therefore, this clinical isolate (NL/2631-R223) was compared with a well-characterized reference virus (NL/602). In vitro experiments showed that NL/2631-I223R replicated as well as NL/602 in MDCK cells. In a ferret pathogenesis model, body weight loss was similar in animals inoculated with NL/2631-R223 or NL/602. In addition, pulmonary lesions were similar at day 4 post inoculation. However, at day 7 post inoculation, NL/2631-R223 caused milder pulmonary lesions and degree of alveolitis than NL/602. This indicated that the mutant virus was less pathogenic. Both NL/2631-R223 and a recombinant virus with a single I223R change (recNL/602-I223R), transmitted among ferrets by aerosols, despite observed attenuation of recNL/602-I223R in vitro. In conclusion, the I223R mutated virus isolate has comparable replicative ability and transmissibility, but lower pathogenicity than the reference virus based on these in vivo studies. This implies that the 2009 pandemic influenza A/H1N1 virus subtype with an isoleucine to arginine change at position 223 in the neuraminidase has the potential to spread in the human population. It is important to be vigilant for this mutation in influenza surveillance and to continue efforts to increase the arsenal of antiviral drugs to combat influenza

Molecular characteristics of carbapenemase-producing Enterobacterales in the Netherlands; results of the 2014–2018 national laboratory surveillance

Objectives: Carbapenem resistance mediated by mobile genetic elements has emerged worldwide and has become a major public health threat. To gain insight into the molecular epidemiology of carbapenem resistance in The Netherlands, Dutch medical microbiology laboratories are requested to submit suspected carbapenemase-producing Enterobacterales (CPE) to the National Institute for Public Health and the Environment as part of a national surveillance system. Methods: Meropenem MICs and species identification were confirmed by E-test and MALDI-TOF and carbapenemase production was assessed by the Carbapenem Inactivation Method. Of all submitted CPE, one species/carbapenemase gene combination per person per year was subjected to next-generation sequencing (NGS). Results: In total, 1838 unique isolates were received between 2014 and 2018, of which 892 were unique CPE isolates with NGS data available. The predominant CPE species were Klebsiella pneumoniae (n = 388, 43%), Escherichia coli (n = 264, 30%) and Enterobacter cloacae complex (n = 116, 13%). Various carbapenemase alleles of the same carbapenemase gene resulted in different susceptibilities to meropenem and this effect varied between species. Analyses of NGS data showed variation of prevalence of carbapenemase alleles over time with blaOXA-48 being predominant (38%, 336/892), followed by blaNDM-1 (16%, 145/892). For the first time in the Netherlands, blaOXA-181, blaOXA-232 and blaVIM-4 were detected. The genetic background of K. pneumoniae and E. coli isolates was highly diverse. Conclusions: The CPE population in the Netherlands is diverse, suggesting multiple introductions. The predominant carbapenemase alleles are blaOXA-48 and blaNDM-1. There was a clear association between species, carbapenemase allele and susceptibility to meropenem

Novel enterovirus inhibitors; Viral and host factors

The genus Enterovirus of the family Picornaviridae includes many important human pathogens, such as poliovirus (PV), coxsackievirus, enterovirus 71 and rhinovirus. These viruses can cause a wide variety of diseases, ranging from mild to potentially fatal. Efficient vaccines against PV have almost succeeded to globally eradicate PV, but development of vaccines against all enteroviruses is not feasible due to the enormous amount of serotypes (almost 300 identified to date). At the moment, no antiviral drugs are available and treatment is limited to symptomatic and supportive care. For these reasons, antiviral drugs are urgently needed. In addition, antiviral drugs may be beneficial to contain any posteradication outbreaks of PV which may occur after stopping of the global vaccination program. Therefore, the aim of this thesis was to identify and characterize targets in the replication cycle of enteroviruses for antiviral therapy and compounds with antiviral activity against enteroviruses. These compounds may either exert their antiviral activity by targeting viral proteins or host factors that are essential for virus replication. Furthermore, by studying the mechanism of action of enterovirus inhibitors, we aim to increase our knowledge of the viral replication process and the requirements. This information may in turn benefit the search for new antiviral drugs, for example by identifying new drug targets. In CHAPTER 2, we have studied the effects of two putative inhibitors of the cellular protein GBF1 on enterovirus replication. The guanine nucleotide exchange factor GBF1 is an essential host factor for replication of enteroviruses. At the early secretory pathway, GBF1 activates the GTPase Arf1 by stimulating GTP exchange. Activated Arf1 then regulates membrane trafficking through the recruitment of effector proteins. Inhibition of GBF1 by the fungal metabolite Brefeldin A (BFA) strongly impairs enterovirus replication. The compound Golgicide A (GCA) was proposed to be an inhibitor of GBF1, with a similar mechanism of action as BFA, but with higher specificity. AG1478 was also reported to inhibit GBF1 activity, but in a different, probably indirect manner. Consequently, we investigated whether these compounds would share the antiviral effect of BFA. We found that GCA inhibited enterovirus replication, but this was not the case for AG1478, supporting the idea that this latter compound does not inhibit GBF1 in the same manner as the other compounds. A further major finding supporting this idea is that the effects of AG1478 on the function and integrity of the secretory pathway could be rescued by overexpression of Arf1, which was not the case for BFA and GCA. Both GCA, which unlike BFA does not target other large Arf activators, and AG1478, which disrupts secretory pathway integrity and function by a different mechanism than BFA and GCA, provide important new tools to further dissect the role of GBF1 in enterovirus RNA replication. CHAPTER 3 describes the identification and characterization of a newantiviral compound, TTP-8307. The spectrum of antiviral activity of TTP-8307 encompassed most, but not all, enteroviruses. To study the mechanism of action of the compound, TTP-8307-resistant coxsackievirus B3 (CVB3) mutants were selected by passaging 10 times in the presence of TTP-8307. These mutants harbored mutations in the viral protein 3A, a membrane protein implicated in the viral modification of secretory pathway membranes. Previously, a variety of mutations in 3A had been shown to confer resistance to enviroxime, an antiviral compound identified in the late 1970s of which the mechanism of action remained unknown. We showed that the TTP-8307-resistant mutants were cross-resistant to enviroxime, suggesting that these antiviral compounds have a similar or related mechanism of action. Subsequently, in CHAPTER 4 we showed that GW5074 also is an inhibitor of enterovirus replication, and that this kinase inhibitor, as well as enviroxime, act by inhibiting the activity of PI4KIIIß, a lipid kinase that had just been identified as an essential host factor for enterovirus replication3. We also showed that enviroxime- and GW5074-resistant viruses (i.e., the viruses with the single point mutations identified in CHAPTER 3) could replicate in the absence of high levels of PI4P, the product of PI4KIIIß. These results demonstrate that viruses are so versatile that they can even bypass the need for host factors for virus replication. In CHAPTER 5, NIH Clinical Collection compound library, a collection of small molecules that have a history of use in human clinical trials, was screened for inhibitors of CVB3. This screen revealed that itraconazole (ITZ), a compound which is in use as an antifungal drug, has antiviral activity. Known targets of ITZ, such as hCYP51, CYP3A4 or the hedgehog pathway were investigated if they mediated the antiviral activity of ITZ, but this appeared not to be the case. Interestingly, the antiviral activity of ITZ could be counteracted by the same mutations in 3A that provided protection against PI4KIIIß inhibitors, suggesting a similar mechanism of action. However, ITZ had no effect on PI4KIIIß activity. Also, unlike PI4KIIIß inhibitors, ITZ inhibited virus replication of cardioviruses, providing further evidence for a different target. CHAPTER 6 reports on the antiviral activity of fluoxetine, an anti-depressant (brand name Prozac) that was also identified in a screen of the NIH Clinical Collection. The antiviral spectrum of activity of fluoxetine encompassed some enteroviruses, but not all. The observation that mutations in the viral protein 2C of CVB3 offered protection to the compound, suggested that fluoxetine targets 2C. 2C has been implicated in many different aspects of virus replication, but its exact function is unknown. 2C is an ATPase, but fluoxetine had only a modest effect on 2C s ATPase activity. This finding suggests that fluoxetine targets another activity of 2C. In CHAPTER 7, we studied the antiviral activity of the small molecule GPC-N114. GPC-N114 inhibited virus replication of both enteroviruses and cardioviruses at the RNA replication stage. No resistant CVB3 variants were obtained despite many passages in the presence of GPC-N114. In the cardiovirus EMCV, mutations in the viral RNA-dependent RNA polymerase 3Dpol conferred resistance to the compound. Accordingly, GPC-N114 inhibited in vitro elongation activity of CVB3 and EMCV 3Dpol. Analysis of the three-dimensional structure of 3Dpol in complex with GPC-N114 revealed that the compound, a non-nucleoside analog, bound at the site where the templating nucleotide is located during the RNA replication process. This result suggests that GPC-N114 may interfere with template binding. This hypothesis was confirmed in functional studies. Collectively, these findings show that GPC-N114 inhibits picornavirus replication by affecting the binding of the RNA template to 3Dpol, making this the first picornavirus inhibitor with this mechanism of action. Finally, in CHAPTER 8 we describe the development of a cell-based assay for determining the capability of compounds to inhibit picornaviral protease activity. Inhibition of protease activity results in the induction of expression of a firefly luciferase reporter. A major advantage of this assay is that it is cell-based, in contrast to most protease assays available, allowing to select for non-toxic and cell-permeable compounds. We showed that the assay could accurately predict the potential of compounds to inhibit virus replication, with IC50 values obtained for several compounds in the same range as those determined with virus infections. In addition, the assay was versatile, applicable to CVB3 3Cpro and 2Apro and could easily be extended to 3Cpro of different genera. In combination with the excellent properties for use in a high-throughput setting, this assay could be a valuable tool for the discovery and validation of protease inhibitors.Chapter 1 General introduction and outline of this thesis Chapter 2 Mutations in the non-structural protein 3A confer resistance to the novel enterovirus replication inhibitor TTP-8307 Chapter 3 Differential effects of the putative GBF1 inhibitors golgicide A and AG1478 on enterovirus replication Chapter 4 Coxsackievirus mutants that can bypass host factor PI4KIIIβ and the need for high levels of PI4P lipids for replication Chapter 5 The antifungal itraconazole inhibits picornavirus RNA replication by targeting OSBP and ORP4 Chapter 6 Selective serotonin reuptake inhibitor fluoxetine inhibits replication of Human Enterovirus B and D by targeting viral protein 2C Chapter 7 A non-nucleoside inhibitor of the RNA-dependent RNA polymerase of enteroviruses and cardioviruses that affects binding of the RNA template Chapter 8 Application of a cell-based protease assay for testing inhibitors of picornavirus 3C proteases Chapter 9 Summary & General discussion Chapter 10 Nederlandse samenvatting Appendices Acknowledgments - Dankwoord; Curriculum vitae; Bibiographynrpages: 220status: publishe

The RNA Template Channel of the RNA-Dependent RNA Polymerase as a Target for Development of Antiviral Therapy of Multiple Genera within a Virus Family

Lonneke van der Linden et al.The genus Enterovirus of the family Picornaviridae contains many important human pathogens (e.g., poliovirus, coxsackievirus, rhinovirus, and enterovirus 71) for which no antiviral drugs are available. The viral RNA-dependent RNA polymerase is an attractive target for antiviral therapy. Nucleoside-based inhibitors have broad-spectrum activity but often exhibit off-target effects. Most non-nucleoside inhibitors (NNIs) target surface cavities, which are structurally more flexible than the nucleotide-binding pocket, and hence have a more narrow spectrum of activity and are more prone to resistance development. Here, we report a novel NNI, GPC-N114 (2,2'-[(4-chloro-1,2-phenylene)bis(oxy)]bis(5-nitro-benzonitrile)) with broad-spectrum activity against enteroviruses and cardioviruses (another genus in the picornavirus family). Surprisingly, coxsackievirus B3 (CVB3) and poliovirus displayed a high genetic barrier to resistance against GPC-N114. By contrast, EMCV, a cardiovirus, rapidly acquired resistance due to mutations in 3Dpol. In vitro polymerase activity assays showed that GPC-N114 i) inhibited the elongation activity of recombinant CVB3 and EMCV 3Dpol, (ii) had reduced activity against EMCV 3Dpol with the resistance mutations, and (iii) was most efficient in inhibiting 3Dpol when added before the RNA template-primer duplex. Elucidation of a crystal structure of the inhibitor bound to CVB3 3Dpol confirmed the RNA-binding channel as the target for GPC-N114. Docking studies of the compound into the crystal structures of the compound-resistant EMCV 3Dpol mutants suggested that the resistant phenotype is due to subtle changes that interfere with the binding of GPC-N114 but not of the RNA template-primer. In conclusion, this study presents the first NNI that targets the RNA template channel of the picornavirus polymerase and identifies a new pocket that can be used for the design of broad-spectrum inhibitors. Moreover, this study provides important new insight into the plasticity of picornavirus polymerases at the template binding site. © 2015 van der Linden et al.This work was supported by research grants from the SILVER Large Scale Collaborative Project (grant agreement number 260644) project of the European Union 7th Framework, the European Virus Archive (EVA) project (European FP7 Capacities Project number 153 228292), the “Convenant K.U. Leuven-Radboud University Nijmegen” framework, GOA/10/014 (for work performed at KULeuven), NWO-VICI grant no. 91812628 (FJMvK), FWO Krediet aan Navorsers no. 1.5.206.11 (AMDP), the “Agency for Innovation by Science and Technology in Flanders (IWT)” (CL)Peer Reviewe

Differential Effects of the Putative GBF1 Inhibitors Golgicide A and AG1478 on Enterovirus Replication▿

The genus Enterovirus, belonging to the family Picornaviridae, includes well-known pathogens, such as poliovirus, coxsackievirus, and rhinovirus. Brefeldin A (BFA) impedes replication of several enteroviruses through inhibition of Golgi-specific BFA resistance factor 1 (GBF1), a regulator of secretory pathway integrity and transport. GBF1 mediates the GTP exchange of Arf1, which in activated form recruits coatomer protein complex I (COP-I) to Golgi vesicles, a process important in transport between the endoplasmic reticulum and Golgi vesicles. Recently, the drugs AG1478 and Golgicide A (GCA) were put forward as new inhibitors of GBF1. In this study, we investigated the effects of these putative GBF1 inhibitors on secretory pathway function and enterovirus replication. We show that both drugs induced fragmentation of the Golgi vesicles and caused dissociation of Arf1 and COP-I from Golgi membranes, yet they differed in their effect on GBF1 localization. The effects of AG1478, but not those of GCA, could be countered by overexpression of Arf1, indicating a difference in their molecular mechanism of action. Consistent with this idea, we observed that GCA drastically reduced replication of coxsackievirus B3 (CVB3) and other human enterovirus species, whereas AG1478 had no effect at all on enterovirus replication. Time-of-addition studies and analysis of RNA replication using a subgenomic replicon both showed that GCA suppresses RNA replication of CVB3, which could be countered by overexpression of GBF1. These results indicate that, in contrast to AG1478, GCA inhibits CVB3 RNA replication by targeting GBF1. AG1478 and GCA may be valuable tools to further dissect enterovirus replication

Strain-dependent neutralization reveals antigenic variation of human parechovirus 3

Human parechovirus 3 (HPeV3), a member of the Picornavirus family, is frequently detected worldwide. However, the observed seropositivity rates for HPeV3 neutralizing antibodies (nAbs) vary from high in Japan to low in the Netherlands and Finland. To study if this can be explained by technical differences or antigenic diversity among HPeV3 strains included in the serological studies, we determined the neutralizing activity of Japanese and Dutch intravenous immunoglobulin batches (IVIG), a rabbit HPeV3 hyperimmune polyclonal serum, and a human HPeV3-specific monoclonal antibody (mAb) AT12-015, against the HPeV3 A308/99 prototype strain and clinical isolates from Japan, the Netherlands and Australia, collected between 1989 and 2015. The rabbit antiserum neutralized all HPeV3 isolates whereas the neutralization capacity of the IVIG batches varied, and the mAb exclusively neutralized the A308/99 strain. Mapping of the amino acid variation among a subset of the HPeV3 strains on an HPeV3 capsid structure revealed that the majority of the surface-exposed amino acid variation was located in the VP1. Furthermore, amino acid mutations in a mAb AT12-015-resistant HPeV3 A308/99 variant indicated the location for potential antigenic determinants. Virus aggregation and the observed antigenic diversity in HPeV3 can explain the varying levels of nAb seropositivity reported in previous studies.Peer reviewe

Broad-range inhibition of enterovirus replication by OSW-1, a natural compound targeting OSBP

Enteroviruses, e.g., polio-, coxsackie- and rhinoviruses, constitute a large genus within the Picornaviridae family of positive-strand RNA viruses and include many important pathogens linked to a variety of acute and chronic diseases. Despite their huge medical and economic impact, no approved antiviral therapy is yet available. Recently, the oxysterol-binding protein (OSBP) was implicated as a host factor for enterovirus replication. Here, we investigated the antiviral activity of the natural compound OSW-1, a ligand of OSBP that is under investigation as an anti-cancer drug. OSW-1 potently inhibited the replication of all enteroviruses tested, with IC50 values in the low nanomolar range, acted at the genome replication stage and was effective in all tested cell types of three different species. Importantly, OSBP overexpression rescued viral replication, demonstrating that the antiviral effect of OSW-1 is due to targeting OSBP. Together, we here report the anti-enterovirus activity of the natural anti-cancer compound OSW-1.publisher: Elsevier articletitle: Broad-range inhibition of enterovirus replication by OSW-1, a natural compound targeting OSBP journaltitle: Antiviral Research articlelink: http://dx.doi.org/10.1016/j.antiviral.2015.02.013 content_type: article copyright: Copyright © 2015 Elsevier B.V. All rights reserved.status: publishe