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ObjectivesThe first German SARS-CoV-2 outbreak was a superspreading event in Gangelt, North Rhine-Westphalia, during indoor carnival festivities called ‘Kappensitzung’ (15 February 2020). We determined SARS-CoV-2 RT-PCR positivity rate, SARS-CoV-2-specific antibodies, and analysed the conditions and dynamics of superspreading, including ventilation, setting dimensions, distance from infected persons and behavioural patterns.DesignIn a cross-sectional epidemiological study (51 days postevent), participants were asked to give blood, pharyngeal swabs and complete self-administered questionnaires.SettingThe SARS-CoV-2 superspreading event took place during festivities in the small community of Gangelt in February 2020. This 5-hour event included 450 people (6–79 years of age) in a building of 27 m × 13.20 m × 4.20 m.ParticipantsOut of 450 event participants, 411 volunteered to participate in this study.Primary and secondary outcome measuresPrimary outcome: infection status (determined by IgG ELISA). Secondary outcome: symptoms (determined by questionnaire).ResultsOverall, 46% (n=186/404) of participants had been infected, and their spatial distribution was associated with proximity to the ventilation system (OR 1.39, 95% CI 0.86 to 2.25). Risk of infection was highly associated with age: children (OR 0.33, 95% CI 0.267 to 0.414) and young adults (age 18–25 years) had a lower risk of infection than older participants (average risk increase of 28% per 10 years). Behavioural differences were also risk associated including time spent outside (OR 0.55, (95% CI 0.33 to 0.91) or smoking (OR 0.32, 95% CI 0.124 to 0.81).ConclusionsOur findings underline the importance of proper indoor ventilation for future events. Lower susceptibility of children/young adults indicates their limited involvement in superspreading.

BackgroundThe beginning of the COVID-19 pandemic was shaped by superspreading events including large-scale outbreaks. In Germany the first SARS-CoV-2 outbreak was a superspreading event in a rural area during indoor carnival festivities in February 2020.Methods51 days after the event all known participants were asked to give blood samples, pharyngeal swabs and answer a self-administered questionnaire. Metric room coordinates for all tables, seats, and ventilation-points were assessed.FindingsWe analyzed infection rates among all 411 participants, and the risk of infection in relation to various factors including age, alcohol consumption, and ventilation system. Overall, 46% (n=186/404) of the participants had been infected. We demonstrate that the spatial distribution of infected participants was associated with proximity to the ventilation system (represented as inverse distance, with Odds Ratio OR 1.39, 95% KI [0.86; 2.25]). Interestingly, the risk of infection was highly associated with age, whereby children (OR: 0.33 [0.267; 0.414]) and young adults (age 18-25) had a lower risk of infection than older participants resulting in an average infection risk increase of 28% per 10 years age difference. Behavioral differences also impacted the risk of infection including time spent outside (OR: 0.55 [0.33; 0.91]) or smoking (OR: 0.32 [0.124; 0.81]).InterpretationOur findings underline the importance of proper indoor ventilation for events in the future. The lower susceptibility for children and young adults indicates their limited involvement in superspreading events.FundingThe government of North Rhine-Westphalia (Germany) supported the study with 65,000 Euro.Research in contextEvidence before this studyThe scientific literature was searched for the term “superspreading event AND Covid-19 OR Sars Cov 2” and identified published papers from China, South Korea, Europe, and North America. Most researchers analyzed superspreading events within a health care setting e.g. in hospitals or nursing homes, or described the general impact of superspreading events on the global pandemic. Only a few metanalyses of transmission clusters analyzed party occasions (e.g. a nightclub in Berlin, Germany) as superspreading events. These reports describe less than 100 infections and are very limited due to missing data or reporting biases. Therefore, the ability to draw scientific conclusions is also limited. Additionally, to our knowledge, there are no studies, which investigated individual behavior, the location, and role of children during a superspreading event. The research for the study started April 2020 and was concluded in June 2021.Added value of this studyOur report analyzes the first COVID-19 superspreading event in Germany in detail, which was not only a unique setting but also included children and adults in the same room. We demonstrate that nearly half of the participants were infected with SARS-CoV-2 and that the proximity of the seating to the ventilation system was an important risk factor for infection. The data showed that low physical distance including singing and duration of attendance at this event increased the risk of infection, while regular smoking and spending the break of the event outside lowered the risk of infection. This underlines the benefit of airing to lower the amount of both droplets and aerosols. Furthermore, we found lower infection in children than adults despite being in the same room suggesting differences in infectability in children. Indeed, we observed that an additional 10 years of age is on average associated with 28% increased risk of infection.Implications of all the available evidenceTaken together, the results demonstrate the importance of the ventilation system during superspreading events. In particular children and young adults had a lower risk of infection during the event indicating that they have a limited role during this pandemic. Overall, our data demonstrate in detail age-dependent infectability as well as highlights to understand transmission dynamics in order to improve comprehensive public health preparedness measures.

The world faces an unprecedented SARS-CoV2 pandemic where many critical factors still remain unknown. The case fatality rates (CFR) reported in the context of the SARS-CoV-2 pandemic substantially differ between countries. For SARS-CoV-2 infection with its broad clinical spectrum from asymptomatic to severe disease courses, the infection fatality rate (IFR) is the more reliable parameter to predict the consequences of the pandemic. Here we combined virus RT-PCR testing and assessment for SARS-CoV2 antibodies to determine the total number of individuals with SARS-CoV-2 infections in a given population.MethodsA sero-epidemiological GCP- and GEP-compliant study was performed in a small German town which was exposed to a super-spreading event (carnival festivities) followed by strict social distancing measures causing a transient wave of infections. Questionnaire-based information and biomaterials were collected from a random, household-based study population within a seven-day period, six weeks after the outbreak. The number of present and past infections was determined by integrating results from anti-SARS-CoV-2 IgG analyses in blood, PCR testing for viral RNA in pharyngeal swabs and reported previous positive PCR tests.ResultsOf the 919 individuals with evaluable infection status (out of 1,007; 405 households) 15.5% (95% CI: [12.3%; 19.0%]) were infected. This is 5-fold higher than the number of officially reported cases for this community (3.1%). Infection was associated with characteristic symptoms such as loss of smell and taste. 22.2% of all infected individuals were asymptomatic. With the seven SARS-CoV-2-associated reported deaths the estimated IFR was 0.36% [0.29%; 0.45%]. Age and sex were not found to be associated with the infection rate. Participation in carnival festivities increased both the infection rate (21.3% vs. 9.5%, pConclusionsWhile the number of infections in this high prevalence community is not representative for other parts of the world, the IFR calculated on the basis of the infection rate in this community can be utilized to estimate the percentage of infected based on the number of reported fatalities in other places with similar population characteristics. Whether the specific circumstances of a super-spreading event not only have an impact on the infection rate and number of symptoms but also on the IFR requires further investigation. The unexpectedly low secondary infection risk among persons living in the same household has important implications for measures installed to contain the SARS-CoV-2 virus pandemic.

Diversity of prion strains was attributed to an elusive nucleic acid, yet a search spanning nearly two decades has failed to identify a prion-specific polynucleotide. In our search for a prion-specific nucleic acid, we analyzed nucleic acids in purified fractions from the brains of Syrian hamsters infected with Sc237 prions. Purification of Sc237 prions removed nucleic acids larger than 50 nucleotides as measured by return refocusing electrophoresis (RRGE). To determine the size of the largest polynucleotide present in purified fractions at an abundance of one molecule per infectious (ID 50 ) unit, we measured prions present after inoculation. In order to account for the rapid clearance of prions after intracerebral inoculation, we determined the number of PrP Sc molecules and ID 50 units of prions that were retained in brain. Factoring in clearance after inoculation, we estimate that the largest polynucleotide present in our purified fractions at one molecule per ID 50 unit is ≈25 nucleotides in length. In the same fractions, there were ≈3,000 protease-resistant PrP Sc molecules per ID 50 unit after accounting for clearance of PrP Sc following inoculation. We compared the resistance of Sc237 and 139H prions to inactivation by UV irradiation at 254 nm. Irradiation of homogenates and microsomes diminished prion infectivity by a factor of ≈1,000 but did not alter the strain-specified properties of the Sc237 and 139H prions. The data reported here combined with the production of synthetic prions argue that the 25-mer polynucleotides found in purified prion preparations are likely to be host encoded and of variable sequence; additionally, these 25-mers are unlikely to be prion specific.

As recently published (Kellings et al. J. gen Vir. 73, 1025-1029 (1992)), the analysis of purified scrapie prions by return refocusing gel electrophoresis revealed remaining nucleic acids in the size range up to 1100 nucleotides. The results defined the possible characteristics of a hypothetical scrapie-specific nucleic acid. If homogeneous in size, such a molecule would be less than 80 nucleotides in length at a particle-toinfectivity ratio (p: i) near unity; if heterogeneous, scrapie-specific nucleic acids would have to include molecules smaller than 240 nucleotides. To decrease the amount of nucleic acids, several modifications of the PrP Sc purification scheme were introduced. Instead of sucrose gradient, ultrafiltration was applied as a purification step and nucleic acids were degraded by BenzonasetM after ultrafiltration, but significant reduction of the p: i ratio could not be achieved. To prevent trapping of nucleic acids in prion rods, nuclease (Benzonase™ ) was added into the tissue homogenate and incubated at 37°C, overnight. The Benzonase treatment revealed no loss of infectivity, but the whole procedure of nucleic acid analysis did not lead to a reduction of the p :i ratio. In another approach the number of nucleic acid degradations steps was reduced to essentially two steps: Zn 2+ hydrolysis and Benzonase digestion. Higher Zn 2+ concentrations and prolonged incubation times resulted in a more efficient nucleic acid degradation. The bioassays yielded complete recovery of infectivity. Large-scale preparations for determining the p: i ratio are still underway

Although increasingly unlikely, the possibility of a scrapie-specific nucleic acid carried by infectious prion particles is still unresolved. Return refocusing gel electrophoresis was developed to detect homogeneous and heterogeneous nucleic acids extracted from highly purified scrapie prion preparations. This method was improved with respect to the size range from 13 to 1100 nucleotides (nt) over which analyses could be performed. The yield of nucleic acid, particularly of small DNA oligonucleotides and polyadenylated RNA, was determined after deproteinization and two-phase extraction. Despite extensive nuclease digestions some small polynucleotides remained. Although a scrapie-specific nucleic acid cannot be excluded, the results further define the possible characteristics of a hypothetical molecule. If homogeneous in size, such a molecule would be less than 80 nt in length at a particle-to-infectivity ratio near unity, if heterogeneous, scrapie-specific nucleic acids would have to include molecules smaller than 240 nt.

Prions are composed largely, if not entirely, of an abnormal isoform of the prion protein (PrP) designated PrPSc. A protease resistant polypeptide, PrP 27–30, can be derived from PrPSc by limited proteolysis with retention of infectivity. Both PrPSc and the cellular isoform PrPC are encoded by a chromosomal gene; PrPSc is produced from the cellular isoform by a posttranslational process (for review see Prusiner, 1991).

An abnormal isoform of the prion protein (PrP) designated PrPSc is the major, or possibly the only, component of infectious prions. Structural studies of PrPSc have been impeded by its lack of solubility under conditions in which infectivity is retained. Among the many detergents examined, only treatment with the ionic detergent sodium dodecyl sulfate (SDS) or Sarkosyl followed by sonication dispersed prion rods which are composed of PrP 27-30, an N-terminally truncated form of PrPSc. After ultracentrifugation at 100,000 x g for 1 h, approximately 30% of the PrP 27-30 and scrapie infectivity were found in the supernatant, which was fractionated by sedimentation through 5 to 20% sucrose gradients. Near the top of the gradient, spherical particles with an observed sedimentation coefficient of approximately 6S, approximately 10 mm in diameter and composed of four to six PrP 27-30 molecules, were found. The spheres could be digested with proteinase K and exhibited little, if any, scrapie infectivity. When the prion rods were disrupted in SDS and the entire sample was fractionated by sucrose gradient centrifugation, a lipid-rich fraction at the meniscus composed of fragments of rods and heterogeneous particles containing high levels of prion infectivity was found. Fractions adjacent to the meniscus also contained spherical particles. Circular dichroism of the spheres revealed 60% alpha-helical content; addition of 25% acetonitrile induced aggregates high in beta sheet but remaining devoid of infectivity. Although the highly purified spherical oligomers of PrP 27-30 lack infectivity, they may provide an excellent substrate for determining conditions of renaturation under which prion particles regain infectivity.

Amount, type, and size of nucleic acid molecules associated with purified prion preparations were analyzed. Return refocusing gel electrophoresis (RRGE) was developed to detect homogeneous and heterogeneous nucleic acids extracted from highly purified scrapie prion preparations. With this method all types of nucleic acids in the size range from 13 to several thousand nucleotides could be analyzed. The recovery of all nucleic acids, after deproteinization and two-phase extraction was higher than 90%. Despite extensive nuclease digestions some small polynucleotides remained. Although a scrapie-specific nucleic acid cannot be excluded, the results further define the possible characteristics for such a hypothetical molecule. If it was homogeneous in size, then it would be80 nt in length at a particle-to-infectivity ratio (P/I) near unity; if the other extreme, i.e. totally heterogeneous scrapie-specific nucleic acids were assumed, then scrapie-specific nucleic acids would have to include molecules smaller than 240 nt. In order to exclude the possibility that unspecific background nucleic acid is entrapped in prion-rods, infectious material has to be prepared without a proteolysis and rod formation, and the analysis of nucleic acids performed with those preparations.