Bat-borne H9N2 influenza virus evades MxA restriction and exhibits efficient replication and transmission in ferrets.

Influenza A viruses (IAVs) of subtype H9N2 have reached an endemic stage in poultry farms in the Middle East and Asia. As a result, human infections with avian H9N2 viruses have been increasingly reported. In 2017, an H9N2 virus was isolated for the first time from Egyptian fruit bats (Rousettus aegyptiacus). Phylogenetic analyses revealed that bat H9N2 is descended from a common ancestor dating back centuries ago. However, the H9 and N2 sequences appear to be genetically similar to current avian IAVs, suggesting recent reassortment events. These observations raise the question of the zoonotic potential of the mammal-adapted bat H9N2. Here, we investigate the infection and transmission potential of bat H9N2 in vitro and in vivo, the ability to overcome the antiviral activity of the human MxA protein, and the presence of N2-specific cross-reactive antibodies in human sera. We show that bat H9N2 has high replication and transmission potential in ferrets, efficiently infects human lung explant cultures, and is able to evade antiviral inhibition by MxA in transgenic B6 mice. Together with its low antigenic similarity to the N2 of seasonal human strains, bat H9N2 fulfils key criteria for pre-pandemic IAVs.

Atypical age distribution and high disease severity in children with RSV infections during two irregular epidemic seasons throughout the COVID-19 pandemic, Germany, 2021 to 2023.

BackgroundNon-pharmaceutical interventions (NPIs) during the COVID-19 pandemic affected respiratory syncytial virus (RSV) circulation worldwide.AimTo describe, for children aged < 5 years, the 2021 and 2022/23 RSV seasons in Germany.MethodsThrough data and 16,754 specimens from outpatient sentinel surveillance, we investigated RSV seasonality, circulating lineages, and affected children's age distributions in 2021 and 2022/23. Available information about disease severity from hospital surveillance was analysed for patients with RSV-specific diagnosis codes (n = 13,104). Differences between RSV seasons were assessed by chi-squared test and age distributions trends by Mann-Kendall test.ResultsRSV seasonality was irregular in 2021 (weeks 35-50) and 2022/23 (weeks 41-3) compared to pre-COVID-19 2011/12-2019/20 seasons (median weeks 51-12). RSV positivity rates (RSV-PR) were higher in 2021 (40% (522/1,291); p < 0.001) and 2022/23 (30% (299/990); p = 0.005) than in prior seasons (26% (1,430/5,511)). Known globally circulating RSV-A (lineages GA2.3.5 and GA2.3.6b) and RSV-B (lineage GB5.0.5a) strains, respectively, dominated in 2021 and 2022/23. In 2021, RSV-PRs were similar in 1 - < 2, 2 - < 3, 3 - < 4, and 4 - < 5-year-olds. RSV hospitalisation incidence in 2021 (1,114/100,000, p < 0.001) and in 2022/23 (1,034/100,000, p < 0.001) was approximately double that of previous seasons' average (2014/15-2019/20: 584/100,000). In 2022/23, proportions of RSV patients admitted to intensive care units rose (8.5% (206/2,413)) relative to pre-COVID-19 seasons (6.8% (551/8,114); p = 0.004), as did those needing ventilator support (6.1% (146/2,413) vs 3.8% (310/8,114); p < 0.001).ConclusionsHigh RSV-infection risk in 2-4-year-olds in 2021 and increased disease severity in 2022/23 possibly result from lower baseline population immunity, after NPIs diminished exposure to RSV.

A robust, scalable, and cost-efficient approach to whole genome sequencing of RSV directly from clinical samples.

Respiratory syncytial virus (RSV) is a leading cause of acute lower respiratory tract infections causing significant morbidity and mortality among children and the elderly; two RSV vaccines and a monoclonal antibody have recently been approved. Thus, there is an increasing need for a detailed and continuous genomic surveillance of RSV circulating in resource-rich and resource-limited settings worldwide. However, robust, cost-effective methods for whole genome sequencing of RSV from clinical samples that are amenable to high-throughput are still scarce. We developed Next-RSV-SEQ, an experimental and computational pipeline to generate whole genome sequences of historic and current RSV genotypes by in-solution hybridization capture-based next generation sequencing. We optimized this workflow by automating library preparation and pooling libraries prior to enrichment in order to reduce hands-on time and cost, thereby augmenting scalability. Next-RSV-SEQ yielded near-complete to complete genome sequences for 98% of specimens with Cp values ≤31, at median on-target reads >93%, and mean coverage depths between ~1,000 and >5,000, depending on viral load. Whole genomes were successfully recovered from samples with viral loads as low as 230 copies per microliter RNA. We demonstrate that the method can be expanded to other respiratory viruses like parainfluenza virus and human metapneumovirus. Next-RSV-SEQ produces high-quality RSV genomes directly from culture isolates and, more importantly, clinical specimens of all genotypes in circulation. It is cost-efficient, scalable, and can be extended to other respiratory viruses, thereby opening new perspectives for a future effective and broad genomic surveillance of respiratory viruses. IMPORTANCE: Respiratory syncytial virus (RSV) is a leading cause of severe acute respiratory tract infections in children and the elderly, and its prevention has become an increasing priority. Recently, vaccines and a long-acting monoclonal antibody to protect effectively against severe disease have been approved for the first time. Hence, there is an urgent need for genomic surveillance of RSV at the global scale to monitor virus evolution, especially with an eye toward immune evasion. However, robust, cost-effective methods for RSV whole genome sequencing that are suitable for high-throughput of clinical samples are currently scarce. Therefore, we have developed Next-RSV-SEQ, an experimental and computational pipeline that produces reliably high-quality RSV genomes directly from clinical specimens and isolates.

The host RNA polymerase II C-terminal domain is the anchor for replication of the influenza virus genome.

The current model is that the influenza virus polymerase (FluPol) binds either to host RNA polymerase II (RNAP II) or to the acidic nuclear phosphoprotein 32 (ANP32), which drives its conformation and activity towards transcription or replication of the viral genome, respectively. Here, we provide evidence that the FluPol-RNAP II binding interface, beyond its well-acknowledged function in cap-snatching during transcription initiation, has also a pivotal role in replication of the viral genome. Using a combination of cell-based and in vitro approaches, we show that the RNAP II C-terminal-domain, jointly with ANP32, enhances FluPol replication activity. We observe successive conformational changes to switch from a transcriptase to a replicase conformation in the presence of the bound RNPAII C-terminal domain and propose a model in which the host RNAP II is the anchor for transcription and replication of the viral genome. Our data open new perspectives on the spatial coupling of viral transcription and replication and the coordinated balance between these two activities.

Different populations of A(H1N1)pdm09 viruses in a patient with hemolytic-uremic syndrome.

Respiratory viral infections may have different impacts ranging from infection without symptoms to severe disease or even death though the reasons are not well characterized. A patient (age group 5-15 years) displaying symptoms of hemolytic uremic syndrome died one day after hospitalization. qPCR, next generation sequencing, virus isolation, antigenic characterization, resistance analysis was performed and virus replication kinetics in well-differentiated airway cells were determined. Autopsy revealed hemorrhagic pneumonia as major pathological manifestation. Lung samples harbored a large population of A(H1N1)pdm09 viruses with the polymorphism H456H/Y in PB1 polymerase. The H456H/Y viruses replicated much faster to high viral titers than upper respiratory tract viruses in vitro. H456H/Y-infected air-liquid interface cultures of differentiated airway epithelial cells did reflect a more pronounced loss of ciliated cells. A different pattern of virus quasispecies was found in the upper airway samples where substitution S263S/F (HA1) was observed. The data support the notion that viral quasispecies had evolved locally in the lung to support high replicative fitness. This change may have initiated further pathogenic processes leading to rapid dissemination of inflammatory mediators followed by development of hemorrhagic lung lesions and fatal outcome.

Characteristics of two zoonotic swine influenza A(H1N1) viruses isolated in Germany from diseased patients.

Interspecies transmission of influenza A viruses (IAV) from pigs to humans is a concerning event as porcine IAV represent a reservoir of potentially pandemic IAV. We conducted a comprehensive analysis of two porcine A(H1N1)v viruses isolated from human cases by evaluating their genetic, antigenic and virological characteristics. The HA genes of those human isolates belonged to clades 1C.2.1 and 1C.2.2, respectively, of the A(H1N1) Eurasian avian-like swine influenza lineage. Antigenic profiling revealed substantial cross-reactivity between the two zoonotic H1N1 viruses and human A(H1N1)pdm09 virus and some swine viruses, but did not reveal cross-reactivity to H1N2 and earlier human seasonal A(H1N1) viruses. The solid-phase direct receptor binding assay analysis of both A(H1N1)v showed a predominant binding to α2-6-sialylated glycans similar to human-adapted IAV. Investigation of the replicative potential revealed that both A(H1N1)v viruses grow in human bronchial epithelial cells to similar high titers as the human A(H1N1)pdm09 virus. Cytokine induction was studied in human alveolar epithelial cells A549 and showed that both swine viruses isolated from human cases induced higher amounts of type I and type III IFN, as well as IL6 compared to a seasonal A(H1N1) or a A(H1N1)pdm09 virus. In summary, we demonstrate a remarkable adaptation of both zoonotic viruses to propagate in human cells. Our data emphasize the needs for continuous monitoring of people and regions at increased risk of such trans-species transmissions, as well as systematic studies to quantify the frequency of these events and to identify viral molecular determinants enhancing the zoonotic potential of porcine IAV.

Relevance of Host Cell Surface Glycan Structure for Cell Specificity of Influenza A Viruses.

Influenza A viruses (IAVs) initiate infection via binding of the viral hemagglutinin (HA) to sialylated glycans on host cells. HA's receptor specificity towards individual glycans is well studied and clearly critical for virus infection, but the contribution of the highly heterogeneous and complex glycocalyx to virus-cell adhesion remains elusive. Here, we use two complementary methods, glycan arrays and single-virus force spectroscopy (SVFS), to compare influenza virus receptor specificity with virus binding to live cells. Unexpectedly, we found that HA's receptor binding preference does not necessarily reflect virus-cell specificity. We propose SVFS as a tool to elucidate the cell binding preference of IAVs, thereby including the complex environment of sialylated receptors within the plasma membrane of living cells.

An appeal for strengthening genomic pathogen surveillance to improve pandemic preparedness and infection prevention: the German perspective.

The SARS-CoV-2 pandemic has highlighted the importance of viable infection surveillance and the relevant infrastructure. From a German perspective, an integral part of this infrastructure, genomic pathogen sequencing, was at best fragmentary and stretched to its limits due to the lack or inefficient use of equipment, human resources, data management and coordination. The experience in other countries has shown that the rate of sequenced positive samples and linkage of genomic and epidemiological data (person, place, time) represent important factors for a successful application of genomic pathogen surveillance. Planning, establishing and consistently supporting adequate structures for genomic pathogen surveillance will be crucial to identify and combat future pandemics as well as other challenges in infectious diseases such as multi-drug resistant bacteria and healthcare-associated infections. Therefore, the authors propose a multifaceted and coordinated process for the definition of procedural, legal and technical standards for comprehensive genomic pathogen surveillance in Germany, covering the areas of genomic sequencing, data collection and data linkage, as well as target pathogens. A comparative analysis of the structures established in Germany and in other countries is applied. This proposal aims to better tackle epi- and pandemics to come and take action from the "lessons learned" from the SARS-CoV-2 pandemic.

Dual-Action Heteromultivalent Glycopolymers Stringently Block and Arrest Influenza A Virus Infection In Vitro and Ex Vivo.

Here, we demonstrate the concerted inhibition of different influenza A virus (IAV) strains using a low-molecular-weight dual-action linear polymer. The 6'-sialyllactose and zanamivir conjugates of linear polyglycerol are optimized for simultaneous targeting of hemagglutinin and neuraminidase on the IAV surface. Independent of IAV subtypes, hemagglutination inhibition data suggest better adsorption of the heteromultivalent polymer than homomultivalent analogs onto the virus surface. Cryo-TEM images imply heteromultivalent compound-mediated virus aggregation. The optimized polymeric nanomaterial inhibits >99.9% propagation of various IAV strains 24 h postinfection in vitro at low nM concentrations and is up to 10000× more effective than the commercial zanamivir drug. In a human lung ex vivo multicyclic infection setup, the heteromultivalent polymer outperforms the commercial drug zanamivir and homomultivalent analogs or their physical mixtures. This study authenticates the translational potential of the dual-action targeting approach using small polymers for broad and high antiviral efficacy.

[Establishment of genomic pathogen surveillance to strengthen pandemic preparedness and infection prevention in Germany]. / Etablierung der Genomischen Erreger-Surveillance zur Stärkung des Pandemie- und Infektionsschutzes in Deutschland.

The SARS-CoV­2 pandemic has shown a deficit of essential epidemiological infrastructure, especially with regard to genomic pathogen surveillance in Germany. In order to prepare for future pandemics, the authors consider it urgently necessary to remedy this existing deficit by establishing an efficient infrastructure for genomic pathogen surveillance. Such a network can build on structures, processes, and interactions that have already been initiated regionally and further optimize them. It will be able to respond to current and future challenges with a high degree of adaptability.The aim of this paper is to address the urgency and to outline proposed measures for establishing an efficient, adaptable, and responsive genomic pathogen surveillance network, taking into account external framework conditions and internal standards. The proposed measures are based on global and country-specific best practices and strategy papers. Specific next steps to achieve an integrated genomic pathogen surveillance include linking epidemiological data with pathogen genomic data; sharing and coordinating existing resources; making surveillance data available to relevant decision-makers, the public health service, and the scientific community; and engaging all stakeholders. The establishment of a genomic pathogen surveillance network is essential for the continuous, stable, active surveillance of the infection situation in Germany, both during pandemic phases and beyond.

Inhibition of p38 signaling curtails the SARS-CoV-2 induced inflammatory response but retains the IFN-dependent antiviral defense of the lung epithelial barrier.

SARS-CoV-2 is the causative agent of the immune response-driven disease COVID-19 for which new antiviral and anti-inflammatory treatments are urgently needed to reduce recovery time, risk of death and long COVID development. Here, we demonstrate that the immunoregulatory kinase p38 MAPK is activated during viral entry, mediated by the viral spike protein, and drives the harmful virus-induced inflammatory responses. Using primary human lung explants and lung epithelial organoids, we demonstrate that targeting p38 signal transduction with the selective and clinically pre-evaluated inhibitors PH-797804 and VX-702 markedly reduced the expression of the pro-inflammatory cytokines IL6, CXCL8, CXCL10 and TNF-α during infection, while viral replication and the interferon-mediated antiviral response of the lung epithelial barrier were largely maintained. Furthermore, our results reveal a high level of drug synergism of both p38 inhibitors in co-treatments with the nucleoside analogs Remdesivir and Molnupiravir to suppress viral replication of the SARS-CoV-2 variants of concern, revealing an exciting and novel mode of synergistic action of p38 inhibition. These results open new avenues for the improvement of the current treatment strategies for COVID-19.

SARS-CoV-2 variant Alpha has a spike-dependent replication advantage over the ancestral B.1 strain in human cells with low ACE2 expression.

Epidemiological data demonstrate that Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) variants of concern (VOCs) Alpha and Delta are more transmissible, infectious, and pathogenic than previous variants. Phenotypic properties of VOC remain understudied. Here, we provide an extensive functional study of VOC Alpha replication and cell entry phenotypes assisted by reverse genetics, mutational mapping of spike in lentiviral pseudotypes, viral and cellular gene expression studies, and infectivity stability assays in an enhanced range of cell and epithelial culture models. In almost all models, VOC Alpha spread less or equally efficiently as ancestral (B.1) SARS-CoV-2. B.1. and VOC Alpha shared similar susceptibility to serum neutralization. Despite increased relative abundance of specific sgRNAs in the context of VOC Alpha infection, immune gene expression in infected cells did not differ between VOC Alpha and B.1. However, inferior spreading and entry efficiencies of VOC Alpha corresponded to lower abundance of proteolytically cleaved spike products presumably linked to the T716I mutation. In addition, we identified a bronchial cell line, NCI-H1299, which supported 24-fold increased growth of VOC Alpha and is to our knowledge the only cell line to recapitulate the fitness advantage of VOC Alpha compared to B.1. Interestingly, also VOC Delta showed a strong (595-fold) fitness advantage over B.1 in these cells. Comparative analysis of chimeric viruses expressing VOC Alpha spike in the backbone of B.1, and vice versa, showed that the specific replication phenotype of VOC Alpha in NCI-H1299 cells is largely determined by its spike protein. Despite undetectable ACE2 protein expression in NCI-H1299 cells, CRISPR/Cas9 knock-out and antibody-mediated blocking experiments revealed that multicycle spread of B.1 and VOC Alpha required ACE2 expression. Interestingly, entry of VOC Alpha, as opposed to B.1 virions, was largely unaffected by treatment with exogenous trypsin or saliva prior to infection, suggesting enhanced resistance of VOC Alpha spike to premature proteolytic cleavage in the extracellular environment of the human respiratory tract. This property may result in delayed degradation of VOC Alpha particle infectivity in conditions typical of mucosal fluids of the upper respiratory tract that may be recapitulated in NCI-H1299 cells closer than in highly ACE2-expressing cell lines and models. Our study highlights the importance of cell model evaluation and comparison for in-depth characterization of virus variant-specific phenotypes and uncovers a fine-tuned interrelationship between VOC Alpha- and host cell-specific determinants that may underlie the increased and prolonged virus shedding detected in patients infected with VOC Alpha.

Transcriptomic comparison of primary human lung cells with lung tissue samples and the human A549 lung cell line highlights cell type specific responses during infections with influenza A virus.

Influenza A virus (IAV) causes pandemics and annual epidemics of severe respiratory infections. A better understanding of the molecular regulation in tissue and cells upon IAV infection is needed to thoroughly understand pathogenesis. We analyzed IAV replication and gene expression induced by IAV strain H3N2 Panama in isolated primary human alveolar epithelial type II cells (AECIIs), the permanent A549 adenocarcinoma cell line, alveolar macrophages (AMs) and explanted human lung tissue by bulk RNA sequencing. Primary AECII exhibit in comparison to AM a broad set of strongly induced genes related to RIG-I and interferon (IFN) signaling. The response of AECII was partly mirrored in A549 cells. In human lung tissue, we observed induction of genes unlike in isolated cells. Viral RNA was used to correlate host cell gene expression changes with viral burden. While relative induction of key genes was similar, gene abundance was highest in AECII cells and AM, while weaker in the human lung (due to less IAV replication) and A549 cells (pointing to their limited suitability as a model). Correlation of host gene induction with viral burden allows a better understanding of the cell-type specific induction of pathways and a possible role of cellular crosstalk requiring intact tissue.

SARS-CoV-2 Omicron variant is attenuated for replication in a polarized human lung epithelial cell model.

SARS-CoV-2 and its emerging variants of concern remain a major threat for global health. Here we introduce an infection model based upon polarized human Alveolar Epithelial Lentivirus immortalized (hAELVi) cells grown at the air-liquid interface to estimate replication and epidemic potential of respiratory viruses in the human lower respiratory tract. hAELVI cultures are highly permissive for different human coronaviruses and seasonal influenza A virus and upregulate various mediators following virus infection. Our analysis revealed a significantly reduced capacity of SARS-CoV-2 Omicron BA.1 and BA.2 variants to propagate in this human model compared to earlier D614G and Delta variants, which extends early risk assessments from epidemiological and animal studies suggesting a reduced pathogenicity of Omicron.

Molecular characterization and evolution dynamics of influenza B viruses circulating in Germany from season 1996/1997 to 2019/2020.

Influenza B viruses are responsible for significant disease burden caused by viruses of both the Yamagata- and Victoria-lineage. Since the circulating patterns of influenza B viruses in different countries vary we investigated molecular properties and evolution dynamics of influenza B viruses circulating in Germany between 1996 and 2020. A change of the dominant lineage occurred in Germany in seven seasons in over past 25 years. A total of 676 sequences of hemagglutinin coding domain 1 (HA1) and 516 sequences of neuraminidase (NA) genes of Yamagata- and Victoria-lineage viruses were analyzed using time-scaled phylogenetic tree. Phylogenetic analysis demonstrated that Yamagata-lineage viruses are more diverse than the Victoria-lineage viruses and could be divided into nine genetic groups whereas Victoria-lineage viruses presented six genetic groups. Comparative phylogenetic analyses of both the HA and NA segments together revealed a number of inter-lineage as well as inter- and intra-clade reassortants. We identified key amino acid substitutions in major HA epitopes such as in four antigenic sites and receptor-binding sites (RBS) and in the regions close to them, with most substitutions in the 120-loop of both lineage viruses. Altogether, seventeen substitutions were fixed over time within the Yamagata-lineage with twelve of them in the antigenic sites. Thirteen substitutions were identified within the Victoria-lineage, with eleven of them in the antigenic sites. Moreover, all Victoria-lineage viruses of the 2017/2018 season were characterized by a deletion of two amino acids at the position 162-163 in the antigenic site of HA1. The viruses with triple deletion Δ162-164 were found in Germany since season 2018/2019. We highlighted the interplay between substitutions in the glycosylation sites and RBS and antigenic epitope during HA evolution. The results obtained underscore the need for continuous monitoring of circulating influenza B viruses. Early detection of strains with genetic and antigenic variation is essential to predict the circulation patterns for the following season. Such information is important for the development of optimal vaccines and strategies for prevention and control of influenza.

Advancing Precision Vaccinology by Molecular and Genomic Surveillance of Severe Acute Respiratory Syndrome Coronavirus 2 in Germany, 2021.

BACKGROUND: Comprehensive pathogen genomic surveillance represents a powerful tool to complement and advance precision vaccinology. The emergence of the Alpha variant in December 2020 and the resulting efforts to track the spread of this and other severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern led to an expansion of genomic sequencing activities in Germany. METHODS: At Robert Koch Institute (RKI), the German National Institute of Public Health, we established the Integrated Molecular Surveillance for SARS-CoV-2 (IMS-SC2) network to perform SARS-CoV-2 genomic surveillance at the national scale, SARS-CoV-2-positive samples from laboratories distributed across Germany regularly undergo whole-genome sequencing at RKI. RESULTS: We report analyses of 3623 SARS-CoV-2 genomes collected between December 2020 and December 2021, of which 3282 were randomly sampled. All variants of concern were identified in the sequenced sample set, at ratios equivalent to those in the 100-fold larger German GISAID sequence dataset from the same time period. Phylogenetic analysis confirmed variant assignments. Multiple mutations of concern emerged during the observation period. To model vaccine effectiveness in vitro, we employed authentic-virus neutralization assays, confirming that both the Beta and Zeta variants are capable of immune evasion. The IMS-SC2 sequence dataset facilitated an estimate of the SARS-CoV-2 incidence based on genetic evolution rates. Together with modeled vaccine efficacies, Delta-specific incidence estimation indicated that the German vaccination campaign contributed substantially to a deceleration of the nascent German Delta wave. CONCLUSIONS: SARS-CoV-2 molecular and genomic surveillance may inform public health policies including vaccination strategies and enable a proactive approach to controlling coronavirus disease 2019 spread as the virus evolves.

Archival influenza virus genomes from Europe reveal genomic variability during the 1918 pandemic.

The 1918 influenza pandemic was the deadliest respiratory pandemic of the 20th century and determined the genomic make-up of subsequent human influenza A viruses (IAV). Here, we analyze both the first 1918 IAV genomes from Europe and the first from samples prior to the autumn peak. 1918 IAV genomic diversity is consistent with a combination of local transmission and long-distance dispersal events. Comparison of genomes before and during the pandemic peak shows variation at two sites in the nucleoprotein gene associated with resistance to host antiviral response, pointing at a possible adaptation of 1918 IAV to humans. Finally, local molecular clock modeling suggests a pure pandemic descent of seasonal H1N1 IAV as an alternative to the hypothesis of origination through an intrasubtype reassortment.

Determination of respiratory syncytial virus epidemic seasons by using 95% confidence interval of positivity rates, 2011-2021, Germany.

Based on our national outpatient sentinel surveillance, we have developed a novel approach to determine respiratory syncytial virus (RSV) epidemic seasons in Germany by using RSV positivity rate and its lower limit of 95% confidence interval. This method was evaluated retrospectively on nine RSV seasons, and it is also well-suited to describe off-season circulation of RSV in near real time as observed for seasons 2020/21 and 2021/22 during the COVID-19 pandemic. Prospective application is of crucial importance to enable timely actions for health service delivery and prevention.