Development of a Rapid Live SARS-CoV-2 Neutralization Assay Based on a qPCR Readout.

Measuring SARS-CoV-2 neutralizing antibodies after vaccination or natural infection remains a priority in the ongoing COVID-19 pandemic to determine immunity, especially against newly emerging variants. The gold standard for assessing antibody-mediated immunity against SARS-CoV-2 are cell-based live virus neutralization assays. These assays usually take several days, thereby limiting test capacities and the availability of rapid results. In this study, therefore, we developed a faster live virus assay, which detects neutralizing antibodies through the early measurement of antibody-mediated intracellular virus reduction by SARS-CoV-2 qRT-PCR. In our assay, Vero E6 cells are infected with virus isolates preincubated with patient sera and controls. After 24 h, the intracellular viral load is determined by qRT-PCR using a standard curve to calculate percent neutralization. Utilizing COVID-19 convalescent-phase sera, we show that our novel assay generates results with high sensitivity and specificity as we detected antiviral activity for all tested convalescent-phase sera, but no antiviral activity in prepandemic sera. The assay showed a strong correlation with a conventional virus neutralization assay (rS = 0.8910), a receptor-binding domain ELISA (rS = 0.8485), and a surrogate neutralization assay (rS = 0.8373), proving that quantifying intracellular viral RNA can be used to measure seroneutralization. Our assay can be adapted easily to new variants, as demonstrated by our cross-neutralization experiments. This characteristic is key for rapidly determining immunity against newly emerging variants. Taken together, the novel assay presented here reduces turnaround time significantly while making use of a highly standardized and sensitive SARS-CoV-2 qRT-PCR method as a readout.

Development and Validation of a Burkholderia pseudomallei Core Genome Multilocus Sequence Typing Scheme To Facilitate Molecular Surveillance.

Burkholderia pseudomallei causes the severe disease melioidosis. Whole-genome sequencing (WGS)-based typing methods currently offer the highest resolution for molecular investigations of this genetically diverse pathogen. Still, its routine application in diagnostic laboratories is limited by the need for high computing power, bioinformatic skills, and variable bioinformatic approaches, with the latter affecting the results. We therefore aimed to establish and validate a WGS-based core genome multilocus sequence typing (cgMLST) scheme, applicable in routine diagnostic settings. A soft defined core genome was obtained by challenging the B. pseudomallei reference genome K96243 with 469 environmental and clinical genomes, resulting in 4,221 core and 1,351 accessory targets. The scheme was validated with 320 WGS data sets. We compared our novel typing scheme with single nucleotide polymorphism-based approaches investigating closely and distantly related strains. Finally, we applied our scheme for tracking the environmental source of a recent infection. The validation of the scheme detected >95% good cgMLST target genes in 98.4% of the genomes. Comparison with existing typing methods revealed very good concordance. Our scheme proved to be applicable to investigating not only closely related strains but also the global B. pseudomallei population structure. We successfully utilized our scheme to identify a sugarcane field as the presumable source of a recent melioidosis case. In summary, we developed a robust cgMLST scheme that integrates high resolution, maximized standardization, and fast analysis for the nonbioinformatician. Our typing scheme has the potential to serve as a routinely applicable classification system in B. pseudomallei molecular epidemiology.

Burkholderia pseudomallei triggers canonical inflammasome activation in a human primary macrophage-based infection model.

Most of the current knowledge on Burkholderia pseudomallei-induced inflammasome activation and cell death in macrophages is derived from murine systems. Little is known about the involved bacterial structures and mechanisms in primary human macrophages. This is of particular relevance since murine and human macrophages as well as primary cells and cell lines differ in many aspects of inflammasome activation, including the proteins involved in the recognition of bacterial patterns. In this study, we therefore aimed (i) to establish an in vitro B. pseudomallei infection model with human monocyte-derived primary macrophages from single donors as these cells more closely resemble macrophages in the human host and (ii) to analyze B. pseudomallei-triggered cell death and bacterial elimination in those cells. Our results show that B. pseudomallei-infected primary human macrophages not only release the inflammasome-independent pro-inflammatory cytokines IL-8 and TNF-α, but are also engaged in canonical inflammasome activation as evidenced by caspase-1 and gasdermin D processing. Absence of the B. pseudomallei T3SS-3 needle protein BsaL, a potent activator of the canonical inflammasome, abolished lytic cell death, reduced IL-1ß release, and caspase-1 and gasdermin D processing. IFN-γ, known to promote non-canonical inflammasome activation, did not influence pyroptosis induction or IL-1ß release from infected primary human macrophages. Nevertheless, it reduced intracellular B. pseudomallei loads, an effect which was partially antagonist by the inhibition of NADPH oxidase. Overall, our data implicate T3SS-3 dependent inflammasome activation and IFN-γ induced immune mechanisms as critical defense mechanisms of human macrophages against B. pseudomallei. In addition, our infection model provides a versatile tool to study human host-pathogen interactions and has the potential to elucidate the role of human individual genetic variations in B. pseudomallei infections.

Behavioral study of selected microorganisms in an aqueous electrohydrodynamic liquid bridge.

An aqueous electrohydrodynamic (EHD) floating liquid bridge is a unique environment for studying the influence of protonic currents (mA cm-2) in strong DC electric fields (kV cm-1) on the behavior of microorganisms. It forms in between two beakers filled with water when high-voltage is applied to these beakers. We recently discovered that exposure to this bridge has a stimulating effect on Escherichia coli.. In this work we show that the survival is due to a natural Faraday cage effect of the cell wall of these microorganisms using a simple 2D model. We further confirm this hypothesis by measuring and simulating the behavior of Bacillus subtilis subtilis, Neochloris oleoabundans, Saccharomyces cerevisiae and THP-1 monocytes. Their behavior matches the predictions of the model: cells without a natural Faraday cage like algae and monocytes are mostly killed and weakened, whereas yeast and Bacillus subtilis subtilis survive. The effect of the natural Faraday cage is twofold: First, it diverts the current from passing through the cell (and thereby killing it); secondly, because it is protonic it maintains the osmotic pressure in the cell wall, thereby mitigating cytolysis which would normally occur due to the low osmotic pressure of the surrounding medium. The method presented provides the basis for selective disinfection of solutions containing different microorganisms.

Prestimulus amplitudes modulate P1 latencies and evoked traveling alpha waves.

Traveling waves have been well documented in the ongoing, and more recently also in the evoked EEG. In the present study we investigate what kind of physiological process might be responsible for inducing an evoked traveling wave. We used a semantic judgment task which already proved useful to study evoked traveling alpha waves that coincide with the appearance of the P1 component. We found that the P1 latency of the leading electrode is significantly correlated with prestimulus amplitude size and that this event is associated with a transient change in alpha frequency. We assume that cortical background excitability, as reflected by an increase in prestimulus amplitude, is responsible for the observed change in alpha frequency and the initiation of an evoked traveling trajectory.

Alpha phase, temporal attention, and the generation of early event related potentials.

In the present study, we have investigated the influence of ongoing alpha phase on the generation of the P1 component of the visual ERP, recorded in a target detection task. Our hypothesis is that in trials where pre- or peristimulus alpha phase is already aligned in a way that voltage positive alpha peaks develop seamlessly into the P1, detection performance will be enhanced as compared to trials where alpha is not aligned. The findings supported our hypothesis and showed that target detection times for the subset of seamless alpha trials was significantly shorter than for trials that are not seamless. Our findings contradict the evoked model for the generation of early ERP components, which rests on the assumption of fixed latency, fixed polarity components. We found that in the non-seamless trials the 'candidate' component of the single trial P1 was at the opposite polarity. Despite this fact, alpha phase locking was at the same high level as was observed for the seamless trials. Finally, we found that prestimulus alpha phase was aligned already in a time window preceding the P1 by 400ms.

Resting state alpha frequency is associated with menstrual cycle phase, estradiol and use of oral contraceptives.

Ongoing intrinsic brain activity in resting, but awake humans is dominated by alpha oscillations. In human, individual alpha frequency (IAF) is associated with cognitive performance. Noticeable, performance in cognitive and emotional tasks in women is associated with menstrual cycle phase and sex hormone levels, respectively. In the present study, we correlated frequency of alpha oscillation in resting women with menstrual cycle phase, sex hormone level, or use of oral contraceptives. Electroencephalogram (EEG) was recorded from 57 women (aged 24.07 ± 3.67 years) having a natural menstrual cycle as well as from 57 women (aged 22.37 ± 2.20 years) using oral contraceptives while they sat in an armchair with eyes closed. Alpha frequency was related to the menstrual cycle phase. Luteal women showed highest and late follicular women showed lowest IAF or center frequency. Furthermore, IAF as well as center frequency correlated negatively with endogenous estradiol level, but did not reveal an association with endogenous progesterone. Women using oral contraceptives showed an alpha frequency similar to women in the early follicular phase. We suggest that endogenous estradiol modulate resting alpha frequency.

Lexical access and evoked traveling alpha waves.

Retrieval from semantic memory is usually considered within a time window around 300-600ms. Here we suggest that lexical access already occurs at around 100ms. This interpretation is based on the finding that semantically rich and frequent words exhibit a significantly shorter topographical latency difference between the site with the shortest P1 latency (leading site) and that with the longest P1 latency (trailing site). This latency difference can be described in terms of an evoked traveling alpha wave as was already shown in earlier studies.

Alpha entrainment is responsible for the attentional blink phenomenon.

The attentional blink phenomenon is the reduced ability to report a second target (T2) after identifying a first target (T1) in a rapid serial visual presentation (RSVP) of stimuli (e.g., letters), which are presented at approximately 10 items per second. Several explanations have been proposed, which focus primarily on cognitive aspects, such as attentional filter-, capacity limitation- and retrieval failure-processes. Here, we focus on the hypothesis that an entrainment of alpha oscillations (with a frequency of about 10Hz) is a critical factor for the attentional blink phenomenon. Our hypothesis is based on the fact that item presentation rate in the RSVP typically lies in the alpha frequency range and is motivated by theories assuming an inhibitory function for alpha. We predict that entrainment--during the time window of T2 presentation--is larger for attentional blink (AB) items (when T2 cannot be reported) than for NoAB trials (when T2 cannot be reported). The results support our hypothesis and show that alpha entrainment as measured by the amplitude of the alpha evoked response and the extent of alpha phase concentration is larger for AB than for NoAB trials. Together with the lack of differences in alpha power these findings demonstrate that the differences between AB and NoAB trials--during presentation onset of T2--are due to an entrainment of alpha phase and not due to an amplitude modulation. Thus, we conclude that alpha entrainment may be considered the critical factor underlying the attentional blink phenomenon.

Evoked traveling alpha waves predict visual-semantic categorization-speed.

In the present study we have tested the hypothesis that evoked traveling alpha waves are behaviorally significant. The results of a visual-semantic categorization task show that three early ERP components including the P1-N1 complex had a dominant frequency characteristic in the alpha range and behaved like traveling waves do. They exhibited a traveling direction from midline occipital to right lateral parietal sites. Phase analyses revealed that this traveling behavior of ERP components could be explained by phase-delays in the alpha but not theta and beta frequency range. Most importantly, we found that the speed of the traveling alpha wave was significantly and negatively correlated with reaction time indicating that slow traveling speed was associated with fast picture-categorization. We conclude that evoked alpha oscillations are functionally associated with early access to visual-semantic information and generate--or at least modulate--the early waveforms of the visual ERP.