Nanoluciferase-based cell fusion assay for rapid and high-throughput assessment of SARS-CoV-2-neutralizing antibodies in patient samples.

After more than two years, COVID-19 still represents a global health burden of unprecedented extent and assessing the degree of immunity of individuals against SARS-CoV-2 remains a challenge. Virus neutralization assays represent the gold standard for assessing antibody-mediated protection against SARS-CoV-2 in sera from recovered and/or vaccinated individuals. Neutralizing antibodies block the interaction of viral spike protein with human angiotensin-converting enzyme 2 (ACE2) receptor in vitro and prevent viral entry into host cells. Classical viral neutralization assays using full replication-competent viruses are restricted to specific biosafety level 3-certified laboratories, limiting their utility for routine and large-scale applications. We developed therefore a cell-fusion-based assay building on the interaction between viral spike and ACE2 receptor expressed on two different cell lines, substantially reducing biosafety risks associated with classical viral neutralization assays. This chapter describes this simple, sensitive, safe and cost-effective approach for rapid and high-throughput evaluation of SARS-CoV-2 neutralizing antibodies relying on high-affinity NanoLuc® luciferase complementation technology (HiBiT). When applied to a variety of standards and patient samples, this method yields highly reproducible results in 96-well, as well as in 384-well format. The use of novel NanoLuc® substrates with increased signal stability like Nano-Glo® Endurazine™ furthermore allows for high flexibility in assay set-up and full automatization of all reading processes. Lastly, the assay is suitable to evaluate the neutralizing capacity of sera against the existing spike variants, and potentially variants that will emerge in the future.

Body fat assessment in youth with overweight or obesity by an automated bioelectrical impedance analysis device, in comparison with the dual-energy x-ray absorptiometry: a cross sectional study.

BACKGROUND: Bioelectrical impedance analysis (BIA) is a widely used method to assess total body fat (TBF) depots characterising obesity. Automated BIA devices provide an inexpensive and easy assessment of TBF, making them widely available to the general public and healthcare providers without specific qualification to assess body composition. The equations included in the automated BIA devices have been developed in very few specific populations, which means that they are not suitable to assess TBF for everyone and need to be validated before use in other populations. The aim of the present work is to evaluate the accuracy of the automated BIA device Tanita® BC-532 in youth of White European ethnicity, compared with the dual-energy x-ray absorptiometry (DEXA), gold standard measurement of TBF. METHODS: Total body fat percentage (TBF%) was measured with the BIA device Tanita® BC-532 and DEXA (Hologic® QDR4500W) in 197 youth of White European ethnicity (N = 104 girls), 7-17 years old, and visiting the Diabetes & Endocrinology Care Paediatrics Clinic, Centre Hospitalier de Luxembourg, for overweight or obesity management. RESULTS: TBF% evaluated with BIA was significantly correlated with TBF% measured with DEXA in both boys (r Pearson = 0.617) and girls (r Pearson = 0.648) (p <  10- 4). However, the residual mean between the assessment of TBF% by BIA and by DEXA [TBF BIA (%)-TBF DEXA (%)] is extremely high (mean ± standard deviation = 10.52% ± 5.22% in boys, respectively 9.96% ± 4.40% in girls). The maximal absolute residual value is also very high, about 24% in both genders. CONCLUSIONS: The automated BIA device Tanita® BC-532 appears to be not accurate to assess total body fat in youth with overweight or obesity. There is a need to calibrate the BIA device before its use in the populations where it was not previously validated.

Detection of Human Enteric Viruses in French Polynesian Wastewaters, Environmental Waters and Giant Clams.

Lack of wastewater treatment efficiency causes receiving seawaters and bivalve molluscan shellfish to become contaminated, which can lead to public health issues. Six wastewater samples, five seawater samples and three batches of giant clams from Tahiti (French Polynesia) were investigated for the presence of enteric viruses, but also if present, for the diversity, infectivity and integrity of human adenoviruses (HAdV). Enteroviruses (EV), sapoviruses (SaV) and human polyomaviruses (HPyV) were detected in all wastewater samples. In decreasing frequency, noroviruses (NoV) GII and HAdV, rotaviruses (RoV), astroviruses (AsV), NoV GI and finally hepatitis E viruses (HEV) were also observed. Nine types of infectious HAdV were identified. HPyV and EV were found in 80% of seawater samples, NoV GII in 60%, HAdV and SaV in 40% and AsV and RoV in 20%. NoV GI and HEV were not detected in seawater. Intact and infectious HAdV-41 were detected in one of the two seawater samples that gave a positive qPCR result. Hepatitis A viruses were never detected in any water types. Analysis of transcriptomic data from giant clams revealed homologues of fucosyltransferases (FUT genes) involved in ligand biosynthesis that strongly bind to certain NoV strains, supporting the giant clams ability to bioaccumulate NoV. This was confirmed by the presence of NoV GII in one of the three batches of giant clams placed in a contaminated marine area. Overall, all sample types were positive for at least one type of virus, some of which were infectious and therefore likely to cause public health concerns.

Effect of the Shellfish Proteinase K Digestion Method on Norovirus Capsid Integrity.

Norovirus outbreaks are associated with the consumption of contaminated shellfish, and so efficient methods to recover and detect infectious norovirus in shellfish are important. The Proteinase K digestion method used to recover norovirus from shellfish, as described in the ISO 15216, would be a good candidate but its impact on the virus capsid integrity and thus infectivity was never examined. The aim of this study was to assess the impact of the Proteinase K digestion method, and of the heat treatment component of the method alone, on norovirus (genogroups I and II) and MS2 bacteriophage capsid integrity. A slightly modified version of the ISO method was used. RT-qPCR was used for virus detection following digestion of accessible viral RNA using RNases. MS2 phage infectivity was measured using a plaque assay. The effect of shellfish digestive glands (DG) on recovery was evaluated. In the presence of shellfish DG, a reduction in MS2 phage infectivity of about 1 log10 was observed after the Proteinase K digestion method and after heat treatment component alone. For norovirus GII and MS2 phage, there was no significant loss of genome following the Proteinase K digestion method but there was a significant 0.24 log10 loss of norovirus GI. In the absence of shellfish DG, the reduction in MS2 phage infectivity was about 2 log10, with the addition of RNases resulting in a significant loss of genome for all tested viruses following complete Proteinase K digestion method and the heat treatment alone. While some protective effect from the shellfish DG on viruses was observed, the impact on capsid integrity and infectivity suggests that this method, while suitable for norovirus genome detection, may not completely preserve virus infectivity.

A 1-Year Study on the Detection of Human Enteric Viruses in New Caledonia.

Human enteric viruses occur in high concentrations in wastewater and can contaminate receiving environmental waters. Due to the lack of data on the prevalence of enteric viruses in New Caledonia, the presence and the concentrations of enteric viruses in wastewater and seawater were determined. Untreated wastewater and seawater samples were collected monthly for 1 year from a wastewater treatment plant (WWTP) and from the WWTP's outlet, located directly on a popular recreational beach. Samples were tested for norovirus genogroups I and II (NoV GI and GII), astroviruses (AsV), sapoviruses (SaV), enteroviruses (EV), hepatitis A viruses (HAV), rotaviruses (RoV), human adenoviruses (HAdV) and human polyomaviruses (HPyV). To support these data, faecal samples from cases of gastroenteritis were tested for the first time for NoV and detected in the population. NoV GI, NoV GII, EV, SaV, HAdV and HPyV were detected in all wastewaters, RoV in 75% and AsV in 67%. HAV were not detected in wastewater. Overall, 92% of seawater samples were positive for at least one virus. HPyV were detected most frequently in 92% of samples and at concentrations up to 7.7 × 10(3) genome copies/L. NoV GI, NoV GII, EV, SaV, RoV and HAdV were found in 33, 66, 41, 33, 16 and 66% of seawater samples, respectively. AsV were not detected in seawater. This study reports for the first time the presence of NoV and other enteric viruses in New Caledonia and highlights the year-round presence of enteric viruses in the seawater of a popular beach.

Binding-Based RT-qPCR Assay to Assess Binding Patterns of Noroviruses to Shellfish.

Outbreaks of norovirus (NoV) gastroenteritis are often associated with consumption of shellfish contaminated with human NoVs. Strong non-specific binding and specific binding between NoVs and histo-blood group antigens (HBGAs) present in shellfish tissues may explain why depuration is ineffective. Recent studies on NoV-binding patterns in shellfish have examined the attachment of NoV virus-like particles (VLPs) to HBGAs present in shellfish using enzyme-linked immunosorbent assays (ELISAs). As NoVs are genetically diverse, it is not practical to produce a range of VLPs and specific antibodies for binding studies. Tank-based bioaccumulation experiments for binding studies also require laboratory space and time. The aim of this study was to develop an alternative method to determine binding patterns for a range of shellfish species and NoV genotypes without using VLPs, antibodies, or tanks. Pacific oysters, green-lipped mussels, two GI and four GII NoV genotypes were selected for assay development. Shellfish gut homogenates were coated onto microwell plates, then purified NoV suspensions were added to each well. Blocking and wash steps using similar reagents as used in ELISAs were carried out. RNA was extracted directly in each well, then RNA copies were quantified by RT-qPCR. Diluent buffer-coated wells spiked with NoVs were used as controls. Different binding patterns were observed. NoV binding was always higher with oysters than with mussels. The highest NoV binding was found with GI.3 and oysters, with 97 % NoV GI.3 bound to oyster homogenate compared with 5 % bound to mussel homogenate. GI.4 did not bind to mussels.

Concentration and diversity of noroviruses detected in Luxembourg wastewaters in 2008-2009.

Noroviruses (NoV) in 78 wastewater samples from Luxembourg were quantified, cloned, and sequenced in 2008-2009. The concentrations of NoV genogroup II and the relative occurrences of certain genotypes changed significantly during the winter season. NoV genogroup I was frequently detected by real-time reverse transcription-PCR (RT-PCR), albeit at 30-fold lower concentrations than for genogroup II, hampering attempts to assess overall genetic diversity by the cloning/sequencing approach.

Aggregation and surface properties of F-specific RNA phages: implication for membrane filtration processes.

We report an experimental investigation of the electrokinetic properties and size variations of four F-specific bacteriophages of the types MS2, GA, Qbeta and SP (21-30 nm in diameter) over a broad range of pH values (1.5-7.5) and NaNO3 electrolyte concentrations (1-100 mM). The results obtained by dynamic light scattering show that the aggregation of SP and GA particles takes place over the whole range of pH and ionic strength conditions examined. For MS2 phages, the aggregation of MS2 particles is not observed for pH higher than the isoelectric point (pI) and large ionic strengths for which interparticular repulsive electrostatic interactions are however expected to be sufficiently screened. Aggregation of the MS2 phages, dispersed in 1 and 100 mM electrolyte concentration, occurs at pH 4, which basically corresponds to the pI as determined by electrophoresis measurements. The Qbeta particles suspended in solutions of low electrolyte concentrations aggregate at low pH values (pI approximately 3) and, unlike MS2, at large ionic strengths over the whole range of pH conditions considered in this study. These elements allow the determination of the hydrophobic sequence for the four phages SP approximately GA>Qbeta>MS2. Close inspection of the electrokinetic results reveals small to significant variations of the pI values-depending on the phage considered-with respect to the concentration of indifferent NaNO3 electrolyte. This indicates that features other than chemical and electrostatic in nature play a key role in determining the pI and more generally the electrophoretic mobility mu of viral particles. A qualitative interpretation is given and is based on the consideration of inner electro-osmotic flow within the isolated or aggregated particles. The impact of the flow properties within the particles is further in agreement with recent theoretical formalism developed for the electrokinetics of soft multiplayer particles, the phages analyzed here being some illustrative examples. The determination and qualitative interpretation of the surface properties of the viral particles as reported in the current study are commented within the context of water treatment especially concerning viral removal by membrane filtration processes.

Impact of chemical and structural anisotropy on the electrophoretic mobility of spherical soft multilayer particles: the case of bacteriophage MS2.

We report a theoretical investigation of the electrohydrodynamic properties of spherical soft particles composed of permeable concentric layers that differ in thickness, soft material density, chemical composition, and flow penetration degree. Starting from a recent numerical scheme developed for the computation of the direct-current electrophoretic mobility (mu) of diffuse soft bioparticles, the dependence of mu on the electrolyte concentration and solution pH is evaluated taking the known three-layered structure of bacteriophage MS2 as a supporting model system (bulk RNA, RNA-protein bound layer, and coat protein). The electrokinetic results are discussed for various layer thicknesses, hydrodynamic flow penetration degrees, and chemical compositions, and are discussed on the basis of the equilibrium electrostatic potential and hydrodynamic flow field profiles that develop within and around the structured particle. This study allows for identifying the cases where the electrophoretic mobility is a function of the inner structural and chemical specificity of the particle and not only of its outer surface properties. Along these lines, we demonstrate the general inapplicability of the notions of zeta potential (zeta) and surface charge for quantitatively interpreting electrokinetic data collected for such systems. We further shed some light on the physical meaning of the isoelectric point. In particular, numerical and analytical simulations performed on structured soft layers in indifferent electrolytic solution demonstrate that the isoelectric point is a complex ionic strength-dependent signature of the flow permeation properties and of the chemical and structural details of the particle. Finally, the electrophoretic mobilities of the MS2 virus measured at various ionic strength levels and pH values are interpreted on the basis of the theoretical formalism aforementioned. It is shown that the electrokinetic features of MS2 are to a large extent determined not only by the external proteic capsid but also by the chemical composition and hydrodynamic flow permeation of/within the inner RNA-protein bound layer and bulk RNA part of the bacteriophage. The impact of virus aggregation, as revealed by decreasing diffusion coefficients for decreasing pH values, is also discussed.