Search

Acknowledgements: Anna Stoliaroff-Pépin for help with taking blood samples; Viktoria Streib and Hanna Töpfer for administrative assistance; Magdalena Simstich and Corinna Fruth for entering data; Romy Hildebrandt (Kreisverwaltung Mainz-Bingen, Amt für Gesundheitswesen), Florian Burckhardt (State Health Department Rhineland-Palatinate) as well as Manfred Vogt, Monika Cremer-Kahn and Benedikt Frei (Landesuntersuchungsamt, Institut für Hygiene und Infektionsschutz, Koblenz) for investigating and providing specimens from the party outbreak, Julia Lange, Hansjörg Rotheudt, Lukas Schumann from the Hermann-Rietschel-Institute (for help in particle measurements), Eugen Lichtner, Kevin Lausch from the Hermann-Rietschel-Institute (for setting up the models for the seating order), Clemens Bodenstein, Caroline Eberle, Juliane Fraissinet, Melanie Hoffmeister, Bianca Hube, Silvia Muschter, Therese Muzeniek, Stefanie Schürer, Franziska Schwarz, Anette Teichmann and Julia Tesch (Robert Koch Institute) for support with serological testing, Steven Uddin and Ute Kramer (Robert Koch Institute) for sequencing and Jan Walter (Robert Koch Institute) for valuable comments on the manuscript., BACKGROUND: Superspreading events are important drivers of the SARS-CoV-2 pandemic and long-range (LR) transmission is believed to play a major role. We investigated two choir outbreaks with different attack rates (AR) to analyze the contribution of LR transmission and highlight important measures for prevention. METHODS: We conducted two retrospective cohort studies and obtained demographic, clinical, laboratory and contact data, performed SARS-CoV-2 serology, whole genome sequencing (WGS), calculated LR transmission probabilities, measured particle emissions of selected choir members, and calculated particle air concentrations and inhalation doses. RESULTS: We included 65 (84%) and 42 (100%) members of choirs 1 and 2, respectively, of whom 58 (89%) and 10 (24%) became cases. WGS confirmed strain identity in both choirs. Both primary cases transmitted presymptomatically. Particle emission rate when singing was 7 times higher compared to talking. In choir 1, the median concentration of primary cases' emitted particles in the room was estimated to be 8 times higher, exposure at least 30 minutes longer and room volume smaller than in choir 2, resulting in markedly different estimated probabilities for LR transmission (mode: 90% vs. 16%, 95% CI: 80-95% vs. 6-36%). According to a risk model, the first transmission in choir 1 occurred likely after 8 minutes of singing. CONCLUSIONS: The attack rate of the two choirs differed significantly reflecting the differences in LR transmission risks. The pooled proportion of cases due to LR transmission was substantial (81%; 55/68 cases) and was facilitated by likely highly infectious primary cases, high particle emission rates, and indoor rehearsing for an extended time. Even in large rooms, singing of an infectious person may lead to secondary infections through LR exposure within minutes. In the context of indoor gatherings without mask-wearing and waning or insufficient immunity, these results highlight the ongoing importance of non-pharmaceutical interventions wherever aerosols can accumulate.

Additional file 1. 1. Laboratory procedures. 2. Figure. Four-tiered testing strategy for the detection of SARS-CoV-2 antibodies. 3. Table. Stratified seroprevalence sensitivity analysis. 4. Table. Results of SARS-CoV-2 seropositivity compared to reported past PCR positivity in each survey sample. 5. Table. COVID-19-like symptoms, December 2020. 6. Table. SARS-CoV-2 Seroprevalence by characteristics of HCW participants in each survey sample. 7. Table. Self-reported COVID-19 exposures and infection prevention and control (IPC) measures, May 2020. 8. References.

Background: Superspreading events are important drivers of the SARS-CoV-2 pandemic. By analyzing two outbreaks associated with choir rehearsals in March 2020, we demonstrate the risk of indoor, long-range (LR) transmission and singing, to help prevent similar outbreaks. Methods: We conducted two retrospective cohort studies and obtained demographic, clinical, laboratory and contact data, performed SARS-CoV-2 serology, whole genome sequencing (WGS), calculated LR transmission probabilities, measured particle emissions of selected choir members, and calculated particle air concentrations and inhalation doses.Results: We included 65 (84%) and 42 (100%) members of choirs 1 and 2, respectively. WGS confirmed strain identity in both choirs and the primary case of choir 1 (transmitting presymptomatically). Particle emission rate when singing was 7 times higher compared to talking. In choir 1, the median concentration of primary cases’ emitted particles was 8 times higher, exposure at least 30 minutes longer and room volume smaller than in choir 2, resulting in markedly different estimated probabilities for LR transmission (median: 89% vs. 18%, 95%CI: 80-95% vs. 6-36%). Observed AR in choir 1 (89%) was significantly higher than in choir 2 (24%). According to a risk model, first transmission in choir 1 occurred likely after 7 minutes of singing. The number of inhaled particles emitted by an infectious case, sufficient to infect 50% of exposed, was calculated to 1039-2883 particles (95%CI).Conclusions: Even in large rooms, singing of an infectious person may lead to secondary infections through LR exposure within minutes. Given the potential for presymptomatic infectiousness, greatest caution is required wherever aerosols can accumulate.

Additional file 1: Figure S1. Correlation of the CT values obtained for 407 specimens positive for the E-Gene PCR with the orf1ab PCR. Shown is the correlation over all specimens (blue line, R2 = 0.94), for specimens with E-Gene PCR CT values below CT = 33 (red line, R2 = 0.95) and for specimens with E-Gene PCR CT values above CT = 33 (green line, R2 = 0.33).

Adaptive changes of viruses enable the virus-host coevolution and infection of new host species. While viruses with an RNA genome adapt primarily by genomic variabilities, adaptation mechanisms of viruses with a DNA genome have remained largely elusive. Since genomes of DNA viruses are more stable compared to genomes of RNA viruses, it is assumed that mechanisms other than genomic changes underlie the adaptation of DNA viruses. However, these mechanisms have hardly been investigated so far. Since isolated virus particles are capable of crossing the species barrier, it is assumed that adaptive changes can be identified in the virus particles themselves. Therefore, this study aimed to elucidate adaptation mechanisms of DNA virus particles by proteomics and genomics technologies. While next-generation sequencing technologies are frequently used to analyze genomic changes of viruses, adaptive changes of virus particles by mass spectrometry-based proteomics have not been analyzed yet. In the present study, cowpox virus (CPXV) was used as a model virus and cell culture as a model system to study adaptive changes of DNA virus particles. CPXV is a member of the genus Orthopoxvirus (OPV) and is able infect a remarkably broad range of host species, e.g. rats, cats, elephants and humans. Increasing numbers of CPXV infections in Europe underline the need for a comprehensive understanding of OPV adaptation mechanisms. CPXV particles were isolated from a rat, which is a natural host of these viruses, and serially passaged five times in a rat and a human cell line. During passaging, an increase in viral fitness was observed exclusively in human cells, suggesting an adaptation of virus particles. Strikingly, proteome analysis revealed that the composition of virus particles changed in a cell line-specific manner, while the viral genome remained overall stable during passaging in both cell lines. Because several ubiquitination sites in virus proteins were identified, the role of ubiquitin for CPXV infection was analyzed. It was shown by the first global ubiquitination site analysis of virus particles that ubiquitin is a major conserved CPXV modification. Additionally, the dependence of CPXV replication on this protein modification was verified, making ubiquitination changes an attractive hypothesis of OPV adaptation. Furthermore, it was shown that CPXV particles incorporate intact transcripts, which presumably enable the rapid expression of viral immunomodulatory proteins upon infection. Summarized, the results of the present study lead to new findings about OPV adaptation mechanisms in vitro. These mechanisms may also apply to in vivo adaptation of DNA viruses and may enable, for example, a crossing of the species barrier. The methods established in this study enable the further characterization of OPV adaptation and, moreover, can be applied to elucidate adaptation mechanisms of viruses belonging to other families., Adaptive Veränderungen von Viren ermöglichen eine Virus-Wirt Koevolution und die Infektion neuer Spezies. Während der Adaption von Viren mit RNA Genom primär eine genomische Variabilität zugrunde liegt, sind Adaptationsmechanismen von Viren mit DNA Genom bislang nur wenig erforscht. Da das Genom von DNA-Viren stabiler ist als das von RNA-Viren, wird vermutet, dass der Adaptation von DNA-Viren andere Mechanismen zugrunde liegen als rein genomische Veränderungen. Weiterhin ist anzunehmen, dass adaptive Veränderungen im Viruspartikel selbst zu identifizieren sind, da isolierte Viruspartikel in der Lage sind die Speziesbarriere zu überwinden. Das Ziel der hier vorliegenden Studie war es daher Adaptationsmechanismen von DNA-Viren mittels Proteomik und Genomik aufzuklären. Während genomische Veränderungen von Viren häufig mittels Next Generation Sequencing analysiert werden, wurden proteomische Veränderungen viraler Partikel bislang noch nicht mit Massenspektrometrie-basierten Methoden analysiert. Als Modell-DNA-Virus für die hier vorliegende Studie wurde das Kuhpockenvirus (CPXV) gewählt und Zellkultur als System um adaptive Veränderungen zu untersuchen. CPXV gehören zum Genus der Orthopockenviren (OPV) und können eine Vielzahl von Spezies infizieren, z.B. Ratten, Katzen, Elefanten und Menschen. Die Anzahl an CPXV-Infektionen in Europa nimmt stetig zu, weshalb es notwendig ist Adaptionsmechanismen von OPV aufzudecken. CPXV Partikel wurden aus einer Ratte isoliert und für fünf Passagen in einer humanen und in einer Ratten Zelllinie vermehrt. Dabei konnte ausschließlich in humanen Zellen eine Steigerung der viralen Fitness beobachtet werden, was auf eine Adaptation der Viren hindeutet. Die Proteomanalyse zeigte, dass sich die Viruspartikel während des Passagierens Zelllinien-spezifisch veränderten, während das virale Genom jedoch insgesamt stabil blieb. Da mehrere Ubiquitinierungsstellen in viralen Proteinen detektiert wurden, wurde die Rolle von Ubiquitin für die CPXV Infektion untersucht. Mittels der ersten globalen Ubiquitinomanalyse von Viruspartikeln konnte gezeigt werden, dass Ubiquitin eine konservierte Modifikation in CPXV Proteinen ist, dessen biologische Relevanz in weiteren Experimenten bestätigt werden konnte. Veränderungen der Ubiquitinierung stellen somit eine attraktive Hypothese zur Adaptation von OPV dar. Darüber hinaus konnte gezeigt werden, dass CPXV Partikel Transkripte inkorporieren, die vermutlich eine schnelle Expression viraler Immunmodulatoren ermöglichen. Die Ergebnisse der vorliegenden Studie führen zu neuen Erkenntnissen über die Adaptationsmechanismen von OPV in vitro, die möglicherweise auch für die Adaption von DNA-Viren in vivo gelten. Die im Rahmen dieser Arbeit etablierten Methoden können weiterhin dazu genutzt werden, um die Adaptation von OPV näher zu charakterisieren und um die Adaptation von Viren anderer Familien zu untersuchen.