Post-COVID syndrome is associated with capillary alterations, macrophage infiltration and distinct transcriptomic signatures in skeletal muscles (preprint)

The SARS-CoV-2 pandemic not only resulted in millions of acute infections worldwide, but also caused innumerable cases of post-infectious syndromes, colloquially referred to as long COVID. Due to the heterogeneous nature of symptoms and scarcity of available tissue samples, little is known about the underlying mechanisms. We present an in-depth analysis of skeletal muscle biopsies obtained from eleven patients suffering from enduring fatigue and post-exertional malaise after an infection with SARS-CoV-2. Compared to two independent historical control cohorts, patients with post-COVID exertion intolerance had fewer capillaries, thicker capillary basement membranes and increased numbers of CD169+ macrophages. SARS-CoV-2 RNA could not be detected in the muscle tissues, but transcriptomic analysis revealed distinct gene signatures compared to the two control cohorts, indicating immune dysregulations and altered metabolic pathways. We hypothesize that the initial viral infection may have caused immune-mediated structural changes of the microvasculature, potentially explaining the exercise-dependent fatigue and muscle pain.

The central nervous systems proteogenomic and spatial imprint upon systemic viral infections with SARS-CoV-2 (preprint)

In COVID-19 neurological alterations are noticed during the systemic viral infection. Various pathophysiological mechanisms on the central nervous system (CNS) have been suggested in the past two years, including the viral neurotropism hypothesis. Nevertheless, neurological complications can also occur independent of neurotropism and at different stages of the disease and may be persistent. Previous autopsy studies of the CNS from patients with severe COVID-19 show infiltration of macrophages and T lymphocytes, especially in the perivascular regions as well as pronounced microglial activation, but without signs of viral encephalitis. However, there is an ongoing debate about long-term changes and cytotoxic effects in the CNS due to the systemic inflammation. Here, we show the brain-specific host response during and after COVID-19. We profile single-nucleus transcriptomes and proteomes of brainstem tissue from deceased COVID-19 patients who underwent rapid autopsy. We detect a disease phase-dependent inflammatory type-I interferon response in acute COVID-19 cases. Integrating single-nucleus RNA sequencing and spatial transcriptomics, we could localize two patterns of reaction to severe systemic inflammation. One neuronal with direct focus on cranial nerve nuclei and one diffusely affecting the whole brainstem, the latter reflecting a bystander effect that spreads throughout the vascular unit and alters the transcriptional state of oligodendrocytes, microglia and astrocytes. Our results indicate that even without persistence of SARS-CoV-2 in the CNS, the tissue activates highly protective mechanisms, which also cause functional disturbances that may explain the neurological symptoms of COVID-19, triggered by strong systemic type-I IFN signatures in the periphery.

Gut microbiota dysbiosis is associated with altered tryptophan metabolism and dysregulated inflammatory response in severe COVID-19 (preprint)

The clinical course of the 2019 coronavirus disease (COVID-19) is variable and to a substantial degree still unpredictable, especially in persons who have neither been vaccinated nor recovered from previous infection. We hypothesized that disease progression and inflammatory responses were associated with alterations in the microbiome and metabolome. To test this, we integrated metagenome, metabolome, cytokine, and transcriptome profiles of longitudinally collected samples from hospitalized COVID-19 patients at the beginning of the pandemic (before vaccines or variants of concern) and non-infected controls, and leveraged detailed clinical information and post-hoc confounder analysis to identify robust within- and cross-omics associations. Severe COVID-19 was directly associated with a depletion of potentially beneficial intestinal microbes mainly belonging to Clostridiales, whereas oropharyngeal microbiota disturbance appeared to be mainly driven by antibiotic use. COVID-19 severity was also associated with enhanced plasma concentrations of kynurenine, and reduced levels of various other tryptophan metabolites, lysophosphatidylcholines, and secondary bile acids. Decreased abundance of Clostridiales potentially mediated the observed reduction in 5-hydroxytryptophan levels. Moreover, altered plasma levels of various tryptophan metabolites and lower abundances of Clostridiales explained significant increases in the production of IL-6, IFN{gamma} and/or TNF. Collectively, our study identifies correlated microbiome and metabolome alterations as a potential contributor to inflammatory dysregulation in severe COVID-19.

Local CCL18 and CCL21 expand lung fibrovascular niches and recruit lymphocytes, leading to tertiary lymphoid structure formation in prolonged COVID-19 (preprint)

Post-acute lung sequelae of COVID-19 are challenging many survivors across the world, yet the mechanisms behind are poorly understood. Our results delineate an inflammatory cascade of events occurring along disease progression within fibrovascular niches. It is initiated by endothelial dysfunction, followed by heme scavenging of CD163+ macrophages and production of CCL18. This chemokine synergizes with local CCL21 upregulation to influence the stromal composition favoring endothelial to mesenchymal transition. The local immune response is further modulated via recruitment of CCR7+ T cells into the expanding fibrovascular niche and imprinting an exhausted, T follicular helper like phenotype in these cells. Eventually, this culminates in the formation of tertiary lymphoid structures, further perpetuating chronic inflammation. Thus, our work presents misdirected immune-stromal interaction mechanisms promoting a self-sustained and non-resolving local immune response that extends beyond active viral infection and leads to profound tissue repurposing and chronic inflammation.

Increased neutralization and IgG epitope identification after MVA-MERS-S booster vaccination against Middle East respiratory syndrome (preprint)

Vaccine development is essential for pandemic preparedness. We previously conducted a Phase 1 clinical trial of the vector vaccine candidate MVA-MERS-S against the Middle East respiratory syndrome coronavirus (MERS-CoV), expressing its full spike glycoprotein (MERS-CoV-S), as a homologous two-dose regimen (Days 0 and 28). Here, we evaluate a third vaccination with MVA-MERS-S in a subgroup of trial participants one year after primary immunization. A booster vaccination with MVA-MERS-S is safe and well-tolerated. Both binding and neutralizing anti-MERS-CoV antibody titers increase substantially in all participants and exceed maximum titers observed after primary immunization more than 10-fold. We identify four immunogenic IgG epitopes, located in the receptor-binding domain (RBD, n=1) and the S2 subunit (n=3) of MERS-CoV-S. The level of baseline anti-human coronavirus antibody titers does not impact the generation of anti-MERS-CoV antibody responses. Our data support the rationale of a booster vaccination with MVA-MERS-S and encourage further investigation in larger trials. One Sentence Summary A late booster vaccination with the vector vaccine MVA-MERS-S against MERS-CoV is safe and significantly increases humoral immunogenicity including responses to four IgG epitopes.

Assessing and improving the validity of COVID-19 autopsy studies - a multi center approach to establish essential standards for immunohistochemical and ultrastructural analyses (preprint)

Background Autopsy studies have provided valuable insights into the pathophysiology of COVID-19. Controversies remain whether the clinical presentation is due to direct organ damage by SARS-CoV-2 or secondary effects, e.g. by an overshooting immune response. SARS-CoV-2 detection in tissues by RT-qPCR and immunohistochemistry (IHC) or electron microscopy (EM) can help answer these questions, but a comprehensive evaluation of these applications is missing. Methods We assessed publications using IHC and EM for SARS-CoV-2 detection in autopsy tissues. We systematically evaluated commercially available antibodies against the SARS-CoV-2 spike protein and nucleocapsid, dsRNA, and non-structural protein Nsp3 in cultured cell lines and COVID-19 autopsy tissues. In a multicenter study, we evaluated specificity, reproducibility, and inter-observer variability of SARS-CoV-2 nucleocapsid staining. We correlated RT-qPCR viral tissue loads with semiquantitative IHC scoring. We used qualitative and quantitative EM analyses to refine criteria for ultrastructural identification of SARS-CoV-2. Findings Publications show high variability in the detection and interpretation of SARS-CoV-2 abundance in autopsy tissues by IHC or EM. In our study, we show that IHC using antibodies against SARS-CoV-2 nucleocapsid yields the highest sensitivity and specificity. We found a positive correlation between presence of viral proteins by IHC and RT-qPCR-determined SARS-CoV-2 viral RNA load (r=-0.83, p-value <0.0001). For EM, we refined criteria for virus identification and also provide recommendations for optimized sampling and analysis. 116 of 122 publications misinterpret cellular structures as virus using EM or show only insufficient data. We provide publicly accessible digitized EM and IHC sections as a reference and for training purposes. Interpretation Since detection of SARS-CoV-2 in human autopsy tissues by IHC and EM is difficult and frequently incorrect, we propose criteria for a re-evaluation of available data and guidance for further investigations of direct organ effects by SARS-CoV-2.

Transcriptomic responses of the human kidney to acute injury at single cell resolution (preprint)

Background: Acute kidney injury (AKI) occurs frequently in critically ill patients and is associated with adverse outcomes. Cellular mechanisms underlying AKI and kidney cell responses to injury remain incompletely understood. Methods: We performed single-nuclei transcriptomics, bulk transcriptomics, molecular imaging studies, and conventional histology on kidney tissues from 8 individuals with severe AKI (stage 2 or 3 according to Kidney Disease: Improving Global Outcomes (KDIGO) criteria). Specimens were obtained within 1-2 hours after individuals had succumbed to critical illness associated with respiratory infections, with 4 of 8 individuals diagnosed with COVID-19. Control kidney tissues were obtained post-mortem or after nephrectomy from individuals without AKI. Results: High-depth single cell-resolved gene expression data of human kidneys affected by AKI revealed enrichment of novel injury-associated cell states within the major cell types of the tubular epithelium, in particular in proximal tubules, thick ascending limbs and distal convoluted tubules. Four distinct, hierarchically interconnected injured cell states were distinguishable and characterized by transcriptome patterns associated with oxidative stress, hypoxia, interferon response, and epithelial-to-mesenchymal transition, respectively. Transcriptome differences between individuals with AKI were driven primarily by the cell type-specific abundance of these four injury subtypes rather than by private molecular responses. AKI-associated changes in gene expression between individuals with and without COVID-19 were similar. Conclusion: The study provides an extensive resource of the cell type-specific transcriptomic responses associated with critical illness-associated AKI in humans, highlighting recurrent disease-associated signatures and inter-individual heterogeneity. Personalized molecular disease assessment in human AKI may foster the development of tailored therapies.

SARS-CoV-2 Antibodies in Children: A One-Year Seroprevalence Study From June 2020 to May 2021 in Germany (preprint)

Background: Investigating the role of children in the COVID-19 pandemic is pivotal to prevent the virus spreading. In most cases, children infected with Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) develop non-specific symptoms or are asymptomatic. Therefore, the infection rate among this age group remains unclear. Seroprevalence studies, including clinical questionnaires, may contribute to our understanding of the time course and clinical manifestations of SARS-CoV-2 infections.Methods: SARS-CoV-2-KIDS is a longitudinal, hospital-based, multicentre study in Germany on the seroprevalence of anti-SARS-CoV-2 immunoglobulin G, as determined by an Enzyme-Linked Immunosorbent Assay in children (aged ≤17 years). A study-specific questionnaire provided additional information on clinical aspects.Findings: This analysis included 10,358 participants recruited from June 2020 to May 2021. The estimated anti-SARS-CoV-2 seroprevalence increased from 2·0% (95% confidence interval (95% CI) 1·6, 2·5) to 10·8% (95% CI 8·7, 12·9) in March 2021, without major change afterwards and was higher in children with migrant background (on average 6·6% vs. 2·8%). In the pandemic early stages, children under three years were 3·5 (95% CI 2·2, 5·6) times more likely to be seropositive than older children, with the levels equalising in later observations. History of self-reported respiratory tract infections or pneumonia was associated with seropositivity (OR 1·8 (95% CI 1·4, 2·3); 2·7 (95% CI 1·7, 4·1)).Interpretation: The majority of children in Germany do not have detectable SARS-CoV-2 IgG. To some extent, this may reflect the effect of differing containment measures implemented in the federal states. Detection levels might have been greater in certain age groups or migrant background. Lifting containment measurements is likely to cause a general increase in respiratory tract infections, which already pose a challenge to paediatric medical care during regular winter seasons. This challenge might become critical with additional infections caused by SARS-CoV-2.Funding: Funding Information: German Federal Ministry of Education and Research.Declaration of Interests: Authors have no conflicts of interest to declare.Ethics Approval Statement: Ethics committees of each study centre independently approved the study protocol. All parents/guardians gave written informed consent and children assented to the participation when appropriate for their age.

Serum but not mucosal antibody responses are associated with pre-existing SARS-CoV-2 spike cross-reactive CD4+ T cells following BNT162b2 vaccination in the elderly (preprint)

Advanced age is a main risk factor for severe COVID-19. However, low vaccination efficacy and accelerated waning immunity have been reported in this age group. To elucidate age-related differences in immunogenicity, we analysed human cellular, serological and salivary SARS-CoV-2 spike glycoprotein-specific immune responses to BNT162b2 COVID-19 vaccine in old (69-92 years) and middle-aged (24-57 years) vaccinees compared to natural infection (COVID-19 convalescents, 21-55 years). Serological humoral responses to vaccination exceeded those of convalescents but salivary anti-spike subunit 1 (S1) IgA and neutralizing capacity were less durable in vaccinees. In old vaccinees, we observed that pre-existing spike-specific CD4 + T cells are associated with efficient induction of anti-S1 IgG and neutralizing capacity in serum but not saliva. Our results suggest pre-existing SARS-CoV-2 cross-reactive CD4 + T cells as predictor of an efficient COVID-19 vaccine-induced humoral immune response in old individuals.

Complement Activation Induces Excessive T Cell Cytotoxicity in Severe COVID-19 (preprint)

Severe COVID-19 is linked to both dysfunctional immune response and unrestrained immunopathogenesis, and it remains unclear if T cells contribute to disease pathology. Here, we combined single-cell transcriptomics and proteomics with mechanistic studies assessing pathogenic T cell functions and inducing signals. We identified activated, CD16+ T cells with increased cytotoxic functions in severe COVID-19. CD16 expression enabled immune complex-mediated, T cell receptor-independent degranulation and cytotoxicity not found in other diseases. CD16+ T cells from COVID-19 patients promoted microvascular endothelial cell injury and release of neutrophil and monocyte chemoattractants. CD16+ T cell clones persisted beyond acute disease maintaining their cytotoxic phenotype. Age-dependent generation of C3a in severe COVID-19 induced activated CD16 + cytotoxic T cells. The proportion of activated CD16+ T cells and plasma levels of complement proteins upstream of C3a correlated with clinical outcome, supporting a pathological role of exacerbated cytotoxicity and complement activation in COVID-19.Funding: This work was supported by the German Research Foundation (DFG): SA1383/3-1 to B.S.; SFB-TR84 114933180 to L.E.S., S.B., P.G., S.H. and W.M.K. INST 37/1049-1, INST 216/981- 1, INST 257/605-1, INST 269/768-1, INST 217/988-1, INST 217/577-1, and EXC2151- 390873048 to J.L.S.; GRK 2168 – 272482170, ERA CVD (00160389 to J.L.S.; SFB 1454 – 432325352 to A.C.A. and J.L.S.; SFB TR57 and SPP1937 to J.N.; GRK2157 to A.-E.S.; and ME 3644/5-1 to H.E.M.; RTG2424 to N.B.; SFB-TRR219 322900939, BO3755/13-1 Project- ID 454024652 to P.B.; the Berlin University Alliance (BUA) (PreEP-Corona grant to L.E.S. and V.M.C.); the Berlin Institute of Health (BIH) (to L.E.S., V.M.C.,B.S. and W.M.K.); Helmholtz- Gemeinschaft Deutscher Forschungszentren, Germany (sparse2big to J.L.S.), EU projects SYSCID (733100 to J.L.S.); European Research Council Horizon 2020 (grant agreement No 101001791 to P.B.); the DZIF, Germany (TTU 04.816 and 04.817 to J.N.); the Hector Foundation (M89 to J.N.); the EU projects ONE STUDY (260687), BIO-DrIM (305147) and INsTRuCT (860003) to B.S.); German Registry of COVID-19 Autopsies through Federal Ministry of Health (ZMVI1-2520COR201 to P.B.); Federal Ministry of Education and Research (DEFEAT PANDEMICs, 01KX2021 and STOP-FSGS-01GM1901A to P.B.); the Berlin Senate to German Rheumatism Research Centre (DRFZ); the Berlin Brandenburg School for regenerative Therapies (BSRT) to C.B.; the German Federal Ministry of Education and Research (BMBF) projects RECAST (01KI20337) to B.S., V.M.C., L.E.S and M.R.; VARIPath (01KI2021) to V.M.C.; NUM COVIM (01KX2021) to L.E.S., V.M.C., F.K., J.L.S., J.N. and B.S.; RAPID to and S.H.,; SYMPATH to N.S. and W.M.K.; PROVID to S.H. and W.M.K.; ZissTrans (02NUK047E) to N.B; National Research Node ‘Mass spectrometry in Systems Medicine (MSCoresys) (031L0220A) to M.R. and N.B.; Diet–Body–Brain (DietBB) (01EA1809A) to J.L.S.; the UKRI/NIHR through the UK Coronavirus Immunology Consortium (UK-CIC), the Francis Crick Institute through the Cancer Research UK (FC001134), the UK Medical Research Council (FC001134), the Wellcome Trust (FC001134 and IA 200829/Z/16/Z) to M.R.; a Charité 3R project (to B.S., S.H., W.M.K.); and an intramural grant from the Department of Genomics & Immunoregulation at the LIMES Institute to A.C.A. We are grateful to the patients and donors volunteering to participate in this study making this research possible in the first place and wish for a speedy and full recovery.Conflict of Interest: V.M.C. is named together with Euroimmun GmbH on a patent application filed recently regarding SARS-CoV-2 diagnostics via antibody testing. A.R.S. and H.E.M. are listed asinventors on a patent application by the DRFZ Berlin in the field of mass cytometry.Ethical Approval: The study was approved by the Institutional Review board of Charité(EA2/066/20).

Comparative sensitivity evaluation for 122 CE-marked SARS-CoV-2 antigen rapid tests (preprint)

Abstract Objective Independent evaluation of the sensitivity of CE-marked SARS-CoV-2 antigen rapid diagnostic tests (Ag RDT) offered in Germany. Method The sensitivity of 122 Ag RDT was adressed using a common evaluation panel. Minimum sensitivity of 75% for panel members with CT<25 was used for differentiation of devices eligible for reimbursement in in the German healthcare system. Results The sensitivity of different SARS-CoV-2 Ag RDT varied over a wide range. The sensitivity limit of 75% for panel members with CT <25 was met by 96 of the 122 tests evaluated; 26 tests exhibited lower sensitivity, few of which were completely failing. Some devices exhibited high sensitivity, e.g. 100% for CT<30. Conclusion This comparative evaluation succeeded to distinguish less sensitive from better performing Ag RDT. Most of the Ag RDT evaluated appear to be suitable for fast identification of acute infections associated with high viral loads. Market access of SARS-CoV-2 Ag RDT should be based on minimal requirements for sensitivity and specificity.

Limited specificity of SARS-CoV-2 antigen-detecting rapid diagnostic tests at low temperatures (preprint)

SARS-CoV-2 antigen-detecting rapid diagnostic tests (Ag-RDTs) are available within and outside of health care settings to enable increased access to COVID-19 diagnosis. These environments include provisional testing facilities, lacking temperature control; as outside temperatures fall, recommended testing temperatures cannot be guaranteed. We report impaired specificity in two out of six Ag-RDTs when used at 2-4°C, indicating that testing in cold settings might cause false-positive results potentially entailing unwarranted quarantine assignments and incorrect incidence estimates.

Human Lungs Show Limited Permissiveness for SARS-CoV-2 Due to Scarce ACE2 Levels But Strong Virus-Induced Immune Activation in Alveolar Macrophages (preprint)

SARS-CoV-2 utilizes the ACE2 transmembrane peptidase as essential cellular entry receptor. Several studies have suggested abundant ACE2 expression in the human lung, inferring strong permissiveness to SARS-CoV-2 infection with resultant alveolar damage and lung injury. Against this expectation, we provide evidence that ACE2 expression must be considered scarce, thereby limiting SARS-CoV-2 propagation in the human alveolus. Instead, spectral imaging of ex vivo infected human lungs and COVID-19 autopsy samples depicted that alveolar macrophages were frequently positive for SARS-CoV-2, indicating viral phagocytosis. Single-cell transcriptomics of SARS-CoV-2 infected human lung tissue further revealed strong inflammatory and anti-viral activation responses in macrophages and monocytes, comparable to those induced by MERS-CoV, but with virus-specific gene expression profiles. Collectively, our findings indicate that severe lung injury in COVID-19 likely results from an overwhelming immune activation rather than direct viral damage of the alveolar compartment.Funding: ACH, LES, SH were supported by Berlin University Alliance GC2 Global Health (Corona Virus Pre-Exploration Project). ACH, SH, TW and CD were supported by BMBF (RAPID) and ACH, SH by BMBF (alvBarriereCOVID-19). KH, LB, SL, SH, CD, TW, ACH were funded by BMBF (NFN-COVID 19, Organo-Strat). KH, NS, LES, MW, SH, ADG, CD, TW and ACH were supported by DFG (SFB-TR 84). ACH was supported by BIH, Charite 3R, and Charité-Zeiss MultiDim. KH was supported by BMBF (Camo-COVID-19). MW, NS and SH was supported by BMBF (PROVID). MW and NS was supported by BIH and BMBF (SYMPATH, CAPSyS, NAPKON). BO and DB were funded through the BIH Clinical Single Cell Bioinformatics Pipeline. LB was supported by the BMBF (CoIMMUNE), the DFG (KFO 342) and the IZKF of the Medical Faculty of the WWU. Conflict of Interest: The authors declare no competing interests.Ethical Approval: The study was approved by the ethics committee at the Charité clinic (projects EA2/079/13) and Ärztekammer Westfalen-Lippe and of the Westfälischen Wilhelms-Universität (AZ: 2016-265-f-S). Written informed consent was obtained from all patients.

A Sars-Cov-2 Neutralizing Antibody Protects from Lung Pathology in a Covid-19 Hamster Model (preprint)

The emergence of SARS-CoV-2 led to pandemic spread of coronavirus disease 2019 (COVID-19), manifesting with respiratory symptoms and multi-organ dysfunction. Detailed characterization of virus-neutralizing antibodies and target epitopes is needed to understand COVID-19 pathophysiology and guide immunization strategies. Among 598 human monoclonal antibodies (mAbs) from ten COVID-19 patients, we identified 40 strongly neutralizing mAbs. The most potent mAb CV07-209 neutralized authentic SARS-CoV-2 with IC 50 of 3.1 ng/ml. Crystal structures of two mAbs in complex with the SARS-CoV-2 receptor-binding domain at 2.55 and 2.70 Å revealed a direct block of ACE2 attachment. Interestingly, some of the near-germline SARS-CoV-2 neutralizing mAbs reacted with mammalian self-antigens. Prophylactic and therapeutic application of CV07-209 protected hamsters from SARS-CoV-2 infection, weight loss and lung pathology. Our results show that non-self-reactive virus-neutralizing mAbs elicited during SARS-CoV-2 infection are a promising therapeutic strategy.

Functional comparison of MERS-coronavirus lineages reveals increased zoonotic potential of the recombinant lineage 5 (preprint)

Middle East respiratory syndrome coronavirus (MERS-CoV) can cause severe pneumonia in humans. The virus is enzootic in dromedary camels across the Middle East and Africa. It is acquired through animal contact and undergoes limited onward transmission particularly in hospitals. Because of this initial potential for human-to-human transmission, we monitor the virus for phenotypic changes related to its pandemic potential. Potential phenotypic changes have been suspected since the year 2015, when a novel recombinant clade (MERS-CoV lineage 5) caused large nosocomial outbreaks in Saudi Arabia and South Korea that effectively swept other, hitherto co-circulating viral lineages. To this day, lineage 5 remains the only circulating MERS-CoV lineage on the Arabian Peninsula. In spite of available sequence data, no studies of viral phenotype have been carried out to date. Here we performed a comprehensive in-vitro and ex-vivo comparison of live virus isolates taken in Saudi Arabia immediately before and after the shift toward lineage 5. We characterized seven isolates representing the recombination-parental lineage 3, eight isolates representing parental lineage 4, as well as eight isolates representing lineage 5. Replication of lineage 5 viruses is significantly increased over isolates from parental lineages in cell culture and ex-vivo lung models. Transcriptional profiling by real-time RT-PCR shows that several key immune genes (IFNb1, CCL5, IFNL1) are significantly less induced in lung cells infected with lineage 5 MERS-CoV compared to parental strains. In IFN receptor knock out cells, as well as under chemical inhibition of IFN signalling, the differences in replication level between lineage 5 and parental lineages are reduced, suggesting that phenotypic differences may be determined by IFN antagonism. Concordantly, lineage 5 shows increased resilience against interferon (IFN) pre-treatment of Calu-3 cells and maintains a 10-fold higher replication level under low and high concentrations of IFN. Reduced immune activation combined with enhanced virus replication and IFN resilience may explain the dominance of lineage 5 on the Arabian Peninsula. This phenotypic difference is highly relevant with regard to pandemic potential, and has remained undiscovered in spite of viral sequence surveillance.

Outbreak of COVID-19 in Germany Resulting from a Single Travel-Associated Primary Case (preprint)

Background: In December 2019, a newly identified coronavirus (SARS-CoV-2) emerged in Wuhan, China, causing respiratory disease (COVID-19) presenting with fever, cough and frequently pneumonia. WHO has set the strategic objective to interrupt virus spread of SARS-CoV-2 worldwide. An outbreak in Bavaria, Germany, starting end of January 2020, gave the opportunity to study transmission events, incubation period, and attack rates.Methods: A case was defined as a person with SARS-CoV-2-infection confirmed by PCR. Case interviews were conducted to i) describe timing of onset and nature of symptoms, ii) identify and classify contacts. High-risk contacts were actively followed and monitored for symptoms, low-risk contacts were tested upon self-reporting of symptoms. Whole genome sequencing was used to confirm epidemiological links and clarify transmission events where contact histories were ambiguous; integration with epidemiological data enabled precise reconstruction of exposure events and incubation periods.Results: Case #0 was a Chinese person who visited Germany for professional reasons. Sixteen subsequent cases emerged in four transmission generations. Signature mutations occurred upon foundation of generation 2, as well as in one patient pertaining to generation 4. Median incubation period and serial interval were 4.0 days, respectively. Transmissions occurred frequently pre-symptomatic, at day of symptom onset and during prodromal phase (symptoms other than fever and cough for ≥1 day at beginning of illness phase). Attack rates were 75% among members of a household cluster in common isolation, 10% among household contacts only together until isolation of case, and 5% among non-household high-risk contacts.Conclusions: While our cases present with predominately mild, non-specific symptoms, infectiousness before or on the day of symptom onset or during prodromal phase is substantial. Additionally, the incubation period is often very short, false-negative tests may occur. Although the outbreak was apparently controlled, successful long-term and global containment of COVID-19 may be difficult to achieve.Funding Statement: Contributions by C. D. and V. M. C. were funded by the German Ministry of Health (Konsiliarlabor für Coronaviren), as well as the German Center for Infection Research. S.B., T.W., K.P., N.M, and T.S.B. are fellows of the ECDC Fellowship Programme, supported financially by the European Centre for Disease Prevention and Control (ECDC).Declaration of Interests: The authors declared no competing interest. Ethics Approval Statement: The outbreak investigation was conducted as part of the authoritative, official tasks of the county health departments as well as the state health department of the Bavarian Health and Food Safety Authority, supported by the Robert Koch Institute. As conducted in response to a public health emergency, this study was exempt from institutional review board approval.