Estimated incubation period distributions of mpox using cases from two international European festivals and outbreaks in a club in Berlin, May to June 2022.

BackgroundSince May 2022, an mpox outbreak affecting primarily men who have sex with men (MSM) has occurred in numerous non-endemic countries worldwide. As MSM frequently reported multiple sexual encounters in this outbreak, reliably determining the time of infection is difficult; consequently, estimation of the incubation period is challenging.AimWe aimed to provide valid and precise estimates of the incubation period distribution of mpox by using cases associated with early outbreak settings where infection likely occurred.MethodsColleagues in European countries were invited to provide information on exposure intervals and date of symptom onset for mpox cases who attended a fetish festival in Antwerp, Belgium, a gay pride festival in Gran Canaria, Spain or a particular club in Berlin, Germany, where early mpox outbreaks occurred. Cases of these outbreaks were pooled; doubly censored models using the log-normal, Weibull and Gamma distributions were fitted to estimate the incubation period distribution.ResultsWe included data on 122 laboratory-confirmed cases from 10 European countries. Depending on the distribution used, the median incubation period ranged between 8 and 9 days, with 5th and 95th percentiles ranging from 2 to 3 and from 20 to 23 days, respectively. The shortest interval that included 50% of incubation periods spanned 8 days (4-11 days).ConclusionCurrent public health management of close contacts should consider that in approximately 5% of cases, the incubation period exceeds the commonly used monitoring period of 21 days.

SARS-CoV-2 Prevalence on and Incidence after Arrival in Travelers on Direct Flights from Cape Town, South Africa to Munich, Germany Shortly after Occurrence of the Omicron Variant in November/December 2021: Results from the OMTRAIR Study.

The highly transmissible SARS-CoV-2-variant B.1.1.529 (Omicron) first appeared in South Africa in November 2021. In order to study Omicron entry to Germany, its occurrence related to incoming airline travel, symptomatology and compliance with entry regulations and recommendations, we conducted a cross-sectional study, followed by a retrospective cohort study among passengers and crew on 19 direct flights from Cape Town, South Africa, to Munich, Germany, between 26 November and 23 December 2021. Travelers were mandatorily PCR-tested on arrival and invited to complete an online questionnaire. SARS-CoV-2-prevalence on arrival was 3.3% (n = 90/2728), and 93% were Omicron. Of the passengers, 528 (19%) completed the questionnaire. Among participants who tested negative on arrival, self-reported SARS-CoV-2-incidence was 4.3% within 14 days, of whom 74% reported a negative PCR-test ≤ 48 h before boarding, 77% were fully vaccinated, and 90% reported wearing an FFP2/medical mask during flight. We found multiple associations between risk factors and infection on and after arrival, among which having a positive-tested travel partner was the most noteworthy. In conclusion, PCR testing before departure was insufficient to control the introduction of the Omicron variant. Additional measures (e.g., frequent testing, quarantine after arrival or travel ban) should be considered to delay virus introduction in such settings.

Epidemiological and Serological Analysis of a SARS-CoV-2 Outbreak in a Nursing Home: Impact of SARS-CoV-2 Vaccination and Enhanced Neutralizing Immunity Following Breakthrough Infection.

Background: Despite a vaccination rate of 82.0% (n = 123/150), a SARS-CoV-2 (Alpha) outbreak with 64.7% (n = 97/150) confirmed infections occurred in a nursing home in Bavaria, Germany. Objective: the aim of this retrospective cohort study was to examine the effects of the Corminaty vaccine in a real-life outbreak situation and to obtain insights into the antibody response to both vaccination and breakthrough infection. Methods: the antibody status of 106 fully vaccinated individuals (54/106 breakthrough infections) and epidemiological data on all 150 residents and facility staff were evaluated. Results: SARS-CoV-2 infections (positive RT-qPCR) were detected in 56.9% (n = 70/123) of fully vaccinated, compared to 100% (n = 27/27) of incompletely or non-vaccinated individuals. The proportion of hospitalized and deceased was 4.1% (n = 5/123) among fully vaccinated and therewith lower compared to 18.5% (n = 5/27) hospitalized and 11.1% (n = 3/27) deceased among incompletely or non-vaccinated. Ct values were significantly lower in incompletely or non-vaccinated (p = 0.02). Neutralizing antibodies were detected in 99.1% (n = 105/106) of serum samples with significantly higher values (p < 0.001) being measured post-breakthrough infection. α-N-antibodies were detected in 37.7% of PCR positive but not in PCR negative individuals. Conclusion: Altogether, our data indicate that SARS-CoV-2 vaccination does provide protection against infection, severe disease progression and death with regards to the Alpha variant. Nonetheless, it also shows that infection and transmission are possible despite full vaccination. It further indicates that breakthrough infections can significantly enhance α-S- and neutralizing antibody responses, indicating a possible benefit from booster vaccinations.

The role of helix 8 and of the cytosolic C-termini in the internalization and signal transduction of B(1) and B(2) bradykinin receptors.

Determinants for desensitization and sequestration of G protein-coupled receptors often contain serine or threonine residues located in their C-termini. The sequence context, however, in which these residues have to appear, and the receptor specificity of these motifs are largely unknown. Mutagenesis studies with the B(2) bradykinin receptor (B(2)wt), stably expressed in HEK 293 cells, identified a sequence distal to N338 (NSMGTLRTSI, including I347 but not the basally phosphorylated S348) and in particular the TSI sequence therein, as a major determinant for rapid agonist-inducible internalization and the prevention of receptor hypersensitivity. Chimeras of the noninternalizing B(1) bradykinin receptor (B(1)wt) containing these B(2)wt sequences sequestered poorly, however, suggesting that additional motifs more proximal to N338 are required. In fact, further substitution of the B(1)wt C-terminus with corresponding B(2)wt regions either at C330(7.71) following putative helix 8 (B(1)CB(2)) or at the preceding Y312(7.53) in the NPXXY sequence (B(1)YB(2)) resulted in chimeras displaying rapid internalization. Intriguingly, however, exchange performed at K322(7.63) within putative helix 8 generated a slowly internalizing chimera (B(1)KB(2)). Detailed mutagenesis analysis generating additional chimeras identified the change of V323 in B(1)wt to serine (as in B(2)wt) as being responsible for this effect. The slowly internalizing chimera as well as a B(1)wt point-mutant V323S displayed significantly reduced inositol phosphate accumulation as compared to B(1)wt or the other chimeras. The slow internalization of B(1)KB(2) was also accompanied by a lack of agonist-induced phosphorylation, that in contrast was observed for B(1)YB(2) and B(1)CB(2), suggesting that putative helix 8 is either directly or indirectly (e.g. via G protein activation) involved in the interaction between the receptor and receptor kinases.

Alanine screening of the intracellular loops of the human bradykinin B receptor--effects on receptor maintenance, G protein activation and internalization.

The bradykinin B(2) receptor is coupled to G protein G(q/11) and becomes sequestered into intracellular compartments after activation. To more closely define the receptor sequences involved in these processes and their functions, we systematically mutated all three intracellular loops (ICLs), either as point mutations or in groups of three to five amino acids to Ala, obtaining a total of 14 mutants. All constructs were stably expressed in HEK 293 cells and, with the exception of triple mutant DRY --> AAA, retained the ability to specifically bind [(3)H]bradykinin. The binding affinities at 4 or 37 degrees C of several mutants differed considerably from those determined for the wild-type receptor, indicating an allosteric connection between the conformation of the binding site and that of the ICLs. Mutations in ICL-1 strongly reduced surface expression without affecting G protein signaling or [(3)H]bradykinin internalization. Two cluster mutants in the middle of ICL-2 containing basic residues displayed considerably reduced potencies, whereas two mutations in ICL-3 resulted in receptor conformations that were considered to be semi-active, based on the observation that they responded with phosphoinositide hydrolysis to compounds normally considered to be antagonists. This, and the fact that a cluster mutant at the C-terminal end of ICL-3 was signaling incompetent, hint at the involvement of ICL-2 and ICL-3 in G(q/11) activation, albeit with different functions. None of the mutants displayed reduced ligand-induced receptor internalization, indicating that the loops are not essential for this process. No conclusion could be drawn, however, with regard to the role of the DRY sequence, as the corresponding triplet mutation lacked binding capability.

C-terminal fusion of eGFP to the bradykinin B2 receptor strongly affects down-regulation but not receptor internalization or signaling.

A functional comparison was made between the wild-type bradykinin B2 receptor (B2wt) and the chimera B2eGFP (enhanced green-fluorescent protein fused to the C-terminus of B2wt), both stably expressed in HEK 293 cells. There was almost no difference in terms of ligand-inducible receptor phosphorylation and internalization, signal transduction (accumulation of inositol phosphates) or expression and affinity. However, stimulation for up to 8 h with 10 microM bradykinin (BK) resulted in a strong decrease in surface receptors (by 60% within 5 h) in B2wt, but not in B2eGFP. When the expression levels of both constructs where comparably reduced using a weaker promoter, long-term stimulation resulted in a reduction in surface receptors for B2wt(low) to less than 20% within 1 h, whereas the chimera B2eGFP(low) still displayed 50% binding activity after 2 h. A 1-h incubation in the absence of BK resulted in a recovery of 60% of the binding in B2wt(low) after 1-h stimulation with BK, but of only 20% after 7-h stimulation. In contrast, B2eGFP(low) levels were restored to more than 70%, even after 7-h stimulation. These data indicate that although the fusion of eGFP to B2wt does not affect its ligand-induced internalization, it strongly reduces the down-regulation, most likely by promoting receptor recycling over degradation.

Inhibition of sequestration of human B2 bradykinin receptor by phenylarsine oxide or sucrose allows determination of a receptor affinity shift and ligand dissociation in intact cells.

Depending on their interaction with intracellular proteins, G protein-coupled receptors (GPCR) often display different affinities for agonists at 37 degrees C. Determining the affinity at that temperature is often difficult in intact cells as most GPCRs are internalized after activation. When sequestration of the B2 bradykinin receptor (B2R) was inhibited by either 0.5 M sucrose or phenylarsine oxide (PAO), a shift in the affinity was detected when the incubation temperature was raised from 4 degrees C to 37 degrees C or lowered from 37 degrees C to 4 degrees C. In contrast, binding of the antagonist [3H]NPC 17731 was temperature-independent. B2R mutants displayed different affinity shifts allowing conclusions on the role of the involved amino acids. By inhibiting receptor sequestration it was possible to determine also dissociation of [3H]BK and of [3H]NPC 17731 from intact cells at 37 degrees C. Surprisingly, both dissociation rates were markedly enhanced by the addition of unlabeled ligand, most likely via prevention of reassociation of dissociated [3H]ligand. This suggests that dissociated [3H]ligand cannot move freely away from the receptor. In summary, our data demonstrate that inhibition of receptor internalization either by PAO or sucrose provides an excellent method to study receptor function and the effects of mutations in intact cells.