Prediction of the Spatial Origin of Puumala Virus Infections Using L Segment Sequences Derived from a Generic Screening PCR

To screen diagnostic specimens for the presence of hantavirus genomes or to identify new hantaviruses in nature, the pan-hanta L-PCR assay, a broadly reactive nested reverse transcription polymerase chain reaction (RT-PCR) assay targeting the L segment, is highly preferred over other assays because of its universality and high sensitivity. In contrast, the geographic allocation of Puumala virus strains to defined outbreak regions in Germany was previously done based on S segment sequences. We show that the routinely generated partial L segment sequences resulting from the pan-hanta L-PCR assay provide sufficient phylogenetic signal to inform the molecular epidemiology of the Puumala virus. Consequently, an additional S segment analysis seems no longer necessary for the identification of the spatial origin of a virus strain

Cluster of human Puumala orthohantavirus infections due to indoor exposure?—An interdisciplinary outbreak investigation

Puumala orthohantavirus (PUUV) is the most important hantavirus species in Europe, causing the majority of human hantavirus disease cases. In central and western Europe, the occurrence of human infections is mainly driven by bank vole population dynamics influenced by beech mast. In Germany, hantavirus epidemic years are observed in 2- to 5-year intervals. Many of the human infections are recorded in summer and early autumn, coinciding with peaks in bank vole populations. Here, we describe a molecular epidemiological investigation in a small company with eight employees of whom five contracted hantavirus infections in late 2017. Standardized interviews with employees were conducted to assess the circumstances under which the disease cluster occurred, how the employees were exposed and which counteractive measures were taken. Initially, two employees were admitted to hospital and serologically diagnosed with hantavirus infection. Subsequently, further investigations were conducted. By means of a self-administered questionnaire, three additional symptomatic cases could be identified. The hospital patients' sera were investigated and revealed in one patient a partial PUUV L segment sequence, which was identical to PUUV sequences from several bank voles collected in close proximity to company buildings. This investigation highlights the importance of a One Health approach that combines efforts from human and veterinary medicine, ecology and public health to reveal the origin of hantavirus disease clusters.Peer Reviewe

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

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.Peer Reviewe

Prediction of the Spatial Origin of Puumala Virus Infections Using L Segment Sequences Derived from a Generic Screening PCR

To screen diagnostic specimens for the presence of hantavirus genomes or to identify new hantaviruses in nature, the pan-hanta L-PCR assay, a broadly reactive nested reverse transcription polymerase chain reaction (RT-PCR) assay targeting the L segment, is highly preferred over other assays because of its universality and high sensitivity. In contrast, the geographic allocation of Puumala virus strains to defined outbreak regions in Germany was previously done based on S segment sequences. We show that the routinely generated partial L segment sequences resulting from the pan-hanta L-PCR assay provide sufficient phylogenetic signal to inform the molecular epidemiology of the Puumala virus. Consequently, an additional S segment analysis seems no longer necessary for the identification of the spatial origin of a virus strain

Absence of detectable fitness advantages of VOC Alpha in primary human respiratory cells, organoids, and hamsters.

(A) Virus growth kinetics were performed in infected hNAECs (MOI 0.1). Samples were collected from the apical and basal side at indicated time points and titrated by plaque assay. n = 3 biological replicates. (B) Virus growth kinetics was conducted in infected bronchial AEC (MOI 0.5). Samples were collected from the apical side and titrated by plaque assay. Data are derived from 1 experiment conducted in triplicates. (C) Intestinal organoids were infected (MOI 0.05) and viral load in supernatant (left) and organoid lysates (right) was quantified at indicated time points by E-gene-specific quantitative RT-PCR. Data are derived from 4 independent experiments. (D) Virus replication was monitored in infected lung organoids (MOI 1). Samples harvested at indicated time points were titrated by plaque assay. Data are derived from 3 independent experiments. (E) Dwarf hamsters were intranasally infected (100,000 PFU) and infectious virus particles from lung homogenates were quantified using plaque assay (left). Donor hamsters were cohoused with naive animals and transmission efficiency was determined from lung homogenates at the indicated time points (right). n = 1–3 animals per experimental condition. Dotted horizontal lines indicate the lower detection limit of the plaque assays. AEC, airway epithelial culture; GE, genome equivalents; hNAEC, human nasal airway epithelial culture; MOI, multiplicity of infection; n.d., not detected; PFU, plaque-forming units; RT-PCR, real-time PCR; VOC, variant of concern. See S1 Data.</p

Inoculum data from all experiments conducted in this study.

(XLSX)</p

Enhanced cell–cell fusion and reduced virus particle entry by VOC Alpha SARS-CoV-2 spike.

(A) For Tat-mediated cell–cell fusion assay, CHO cells were cotransfected with plasmids expressing indicated spike-HA and HIV-1 Tat. LTR-luciferase-expressing target TZM-bl cells were transfected with plasmids encoding human ACE2 and TMPRSS2. Transfected cells were cocultured for 8 hours and luciferase expression resulting from intercellular Tat transfer was quantified luminometrically. All values were normalized to B.1 spike (indicated by a dotted line). Shown are results from 3–6 biological replicates, each performed in triplicates. (B) Calu-3 cells were transduced with lentiviral pseudoparticles expressing luciferase and decorated with indicated spike-HA. Transduction efficiency was quantified luminometrically. Dotted line indicates background levels of luciferase nontransduced cultures. Shown are results from 6 independent biological replicates (using independent lentivirus particle preparations), each performed in triplicates, indicated by symbols. (C) Indicated A549 cells were transduced with increasing quantities (0.5 μl, 5 μl, and 50 μl) of lentiviral, luciferase-expressing particles pseudotyped with B.1- or VOC Alpha-spike. Transduction efficiency was determined luminometrically. Dotted line indicates luciferase background level of luciferase detected in nontransduced cells. Symbols represent individual values of 3 biological replicates, each performed in triplicates. (D, E) Indicated A549 (D) and Calu-3 (E) cells were infected at 4°C with B.1 or VOC Alpha isolates (MOI 1) to allow synchronized infection. Total cellular RNA was isolated at the indicated time points and nucleocapsid-encoding sgRNA was quantified by Q-RT-PCR. N = mean of 3–4 biological replicates, indicated by symbols. (F) Synchronized infection of Calu-3 cells was performed with B.1 and VOC Alpha virions that were pretreated with trypsin for 1 hour at 37°C. Data were normalized to the respective log10 relative N sgRNA level of the untreated (0 μg/ml) 4 hours postinfection sample (dotted line). Means +/− SD of 6 independently performed experiments are shown. (G) Synchronized infection of Calu-3 cells was performed with B.1 and VOC Alpha virions that were pretreated with saliva (pooled saliva from 3 healthy donors) for 1 hour at 37°C. Data were normalized to the respective log10 relative sgRNA N level of the untreated (-) 4 hours postinfection sample (dotted line). Results show means of 4 independently performed experiments, which were each performed in triplicates. Del, deletion; MOI, multiplicity of infection; Q-RT-PCR, quantitative real-time PCR; RLU, relative light units; SARS-CoV-2, Severe Acute Respiratory Syndrome Coronavirus 2; sgRNA N, subgenomic nucleocapsid RNA; TMPRSS2, transmembrane protease serine subtype 2; VOC, variant of concern. See S1 Data.</p

Competition assay, additional targets.

Calu-3 cells were infected with a mixture of B.1 and VOC Alpha at indicated ratios (B.1:VOC Alpha/v1 ratio of 1:1, 9:1, and 1:9) with a total infectious dose of 10,000 PFU (corresponding to an MOI of 0.04). After serial passaging, viral RNA from the supernatant was isolated, sequenced, and the relative proportion of B.1- and VOC Alpha-corresponding sequences, discriminated by mutations in NSP3 (A), Spike amino acid positions 501 (B) and 681 (C) was plotted. Data show individual values of triplicates of 1 experiment. MOI, multiplicity of infection; PFU, plaque-forming units; p0-p5, passage 0–passage 5; VOC, variant of concern. See S1 Data. (TIF)</p

Raw numeric values corresponding to plotted data in corresponding figures.

(XLSX)</p