A Quantitative Human Red Blood Cell Agglutination Assay for Characterisation of Galectin Inhibitors.

Galectins are a family of beta-galactoside-binding proteins that are characterised by their carbohydrate recognition domain (CRD) and include galectin-1 and galectin-3. These galectins have been implicated in numerous diseases due to their pleiotropic nature, including cancer and fibrosis, with therapeutic inhibitors being clinically developed to block the CRD. One of the early methods developed to characterise these galectins was the hemagglutination of red blood cells. Although it is insightful, this approach has been hampered by a lack of sensitivity and accurate quantification of the agglutination observed. In this study, we aimed to validate a more precise and quantitative method to enable the further investigation of differences between galectins in respect to agglutination induction in different blood groups, as well as the characterisation of small molecule inhibitors. Quantification of hemagglutination was shown to be optimal using U-bottom plates imaged and analysed with FIJI ImageJ rather than flat-bottom plates read for absorbance on an optical density plate reader. Galectin-3-induced red blood cell agglutination efficacy increased significantly from blood group O to A to B. However, for both the galectin-1 monomer and concatemer, a more comparable effect was observed between blood group B and O, but with more potent effects than in blood group A. Inhibition assays for both galectin-3 and galectin-1 induced-hemagglutination were able to demonstrate clear concentration responses and expected selectivity profiles for a set of small-molecule glycomimetics, confirming the historical profiles obtained in biochemical binding and functional cellular assays.

Reduction of hemagglutination induced by a SARS-CoV-2 spike protein fragment using an amyloid-binding benzothiazole amphiphile.

COVID-19 infection is associated with a variety of vascular occlusive morbidities. However, a comprehensive understanding of how this virus can induce vascular complications remains lacking. Here, we show that a peptide fragment of SARS-CoV-2 spike protein, S192 (sequence 192-211), is capable of forming amyloid-like aggregates that can induce agglutination of red blood cells, which was not observed with low- and non-aggregated S192 peptide. We subsequently screened eight amyloid-binding molecules and identified BAM1-EG6, a benzothiazole amphiphile, as a promising candidate capable of binding to aggregated S192 and partially inhibiting its agglutination activity. These results provide new insight into a potential molecular mechanism for the capability of spike protein metabolites to contribute to COVID-19-related blood complications and suggest a new therapeutic approach for combating microvascular morbidities in COVID-19 patients.

Recent H9N2 avian influenza virus lost hemagglutination activity due to a K141N substitution in hemagglutinin.

The H9N2 avian influenza virus (AIV) represents a significant risk to both the poultry industry and public health. Our surveillance efforts in China have revealed a growing trend of recent H9N2 AIV strains exhibiting a loss of hemagglutination activity at 37°C, posing challenges to detection and monitoring protocols. This study identified a single K141N substitution in the hemagglutinin (HA) glycoprotein as the culprit behind this diminished hemagglutination activity. The study evaluated the evolutionary dynamics of residue HA141 and studied the impact of the N141K substitution on aspects such as virus growth, thermostability, receptor-binding properties, and antigenic properties. Our findings indicate a polymorphism at residue 141, with the N variant becoming increasingly prevalent in recent Chinese H9N2 isolates. Although both wild-type and N141K mutant strains exclusively target α,2-6 sialic acid receptors, the N141K mutation notably impedes the virus's ability to bind to these receptors. Despite the mutation exerting minimal influence on viral titers, antigenicity, and pathogenicity in chicken embryos, it significantly enhances viral thermostability and reduces plaque size on Madin-Darby canine kidney (MDCK) cells. Additionally, the N141K mutation leads to decreased expression levels of HA protein in both MDCK cells and eggs. These findings highlight the critical role of the K141N substitution in altering the hemagglutination characteristics of recent H9N2 AIV strains under elevated temperatures. This emphasizes the need for ongoing surveillance and genetic analysis of circulating H9N2 AIV strains to develop effective control and prevention measures.IMPORTANCEThe H9N2 subtype of avian influenza virus (AIV) is currently the most prevalent low-pathogenicity AIV circulating in domestic poultry globally. Recently, there has been an emerging trend of H9N2 AIV strains acquiring increased affinity for human-type receptors and even losing their ability to bind to avian-type receptors, which raises concerns about their pandemic potential. In China, there has been a growing number of H9N2 AIV strains that have lost their ability to agglutinate chicken red blood cells, leading to false-negative results during surveillance efforts. In this study, we identified a K141N mutation in the HA protein of H9N2 AIV to be responsible for the loss of hemagglutination activity. This finding provides insight into the development of effective surveillance, prevention, and control strategies to mitigate the threat posed by H9N2 AIV to both animal and human health.

Improved in vitro Hemagglutination Assays Utilizing P-Type and Type 1 Uropathogenic Escherichia coli to Evaluate Bacterial Anti-Adhesion Activity of Cranberry Products.

Cranberries have a long history of use in the prevention of urinary tract infections. Cranberry products vary in proanthocyanidin content, a compound implicated in preventing the adhesion of uropathogenic Escherichia coli (E. coli) to uroepithelial cells. Testing is routinely done by cranberry product formulators to evaluate in vitro bacterial anti-adhesion bioactivity, shelf-life, and potential efficacy of cranberry products for consumer use to maintain urinary tract health. Hemagglutination assays evaluate the anti-adhesion bioactivity of cranberry products by determining how effectively the products prevent agglutination of specific red blood cells with E. coli expressing P-type and Type 1 fimbriae. The current study sought to improve upon an established anti-adhesion assay method by expanding the number of E. coli strains used to broaden potential in vivo efficacy implications and presenting results using photomicrographic data to improve accuracy and build databases on products that are routinely tested. Different lots of cranberry powder ingredient and two formulated products were tested independently for anti-adhesion activity using the established method and the improved method. Positive harmonization of results on the same samples using rigorous controls was achieved and provides the substantiation needed for the cranberry industry to utilize the improved, rapid in vitro testing method to standardize cranberry products for sufficient anti-adhesion bioactivity and maintain consumer confidence.

Click chemistry inspired facile one-pot synthesis of mannosyl triazoles and their hemagglutination inhibitory properties: The effect of alkyl chain spacer length between the triazole and phthalimide moieties in the aglycone backbone.

An efficient one-pot synthesis of a new series of mannosyl triazoles has been achieved through CuAAC reaction where the alkyl chain spacer between the phthalimide moiety and the triazole ring in the aglycone backbone is varied from one methylene to six methylene units. The target compounds were evaluated in terms of their inhibitory potency against FimH using hemagglutination inhibition (HAI) assay. It was found that the length of four methylene units was the optimum for the fitting/binding of the compound to FimH as exemplified by compound 11 (HAI = 1.9 µM), which was approximately 200 times more potent than the reference ligand 1(HAI = 385 µM). The successful implementation of one-pot protocol with building blocks 1-7 and the architecture of ligand 11 will be the subject of our future work for developing more potent FimH inhibitors.

Comparative study of HA and HNB staining RT-LAMP assays for peste des petits ruminants virus detection in West African Dwarf goats.

Peste des petits ruminants (PPR) cause severe economic losses to many countries of the world where the disease is endemic. It has been targeted for global eradication by 2030 following the successful eradication of rinderpest in 2011. The proposed eradication program would benefit from efficient and relatively reliable diagnostic tools for early PPR virus (PPRV) detection. A total of 33 eight to 12 months old West African Dwarf (WAD) goats were used. Nineteen goats infected by commingling with two PPR virus-positive animals formed the infected group (PPRV-infected goats) while 14 non-infected goats formed the control group (CTG). The suitability of hydroxyl naphthol blue (HNB) staining of reverse transcription loop-mediated isothermal amplification (RT-LAMP) and haemagglutination (HA) assays was compared for their sensitivity to detect the PPRV in PPRV-infected goats and non-infected CTG. PPR disease severity in WAD goats at different days post infection (dpi) was evaluated by clinical scoring and haemagglutination titre (HAT). HNB staining RT-LAMP reaction and HA showed sensitivities of 100% and 73.68%, respectively, for PPRV detection. Expression of PPR clinical signs began from 3 dpi, attained peak at 5 dpi, thereafter showed irregular patterns till 24 dpi. Evaluation of HAT in PPRV-infected goats at 12 dpi ranged from 2 to 64 haemagglutination units (HAU), while CTG goats had 0 HAU. In conclusion, HA could be a good tool for rapid diagnosis of PPRV in a developing country setting. However, HNB staining RT-LAMP assay demonstrated high sensitivity for accurate diagnoses of PPRV and as an important diagnostic tool when precise phenotyping is desired.

Effect of Different Levels of Maternally Derived Genotype VII Newcastle Disease Virus-Specific Hemagglutination Inhibition Antibodies on Protection against Virulent Challenge in Chicks.

Newcastle disease (ND), caused by the virulent Newcastle disease virus (NDV), is an acute, highly contagious, and economically significant avian disease worldwide. Vaccination is the most effective measure for controlling ND. In recent years, vaccines matched with the prevalent strains of genotype VII have been developed and are now commercially available. These vaccines can provide full protection for chickens against clinical disease and mortality after challenges with genotype VII viruses and significantly decrease virus shedding compared to conventional vaccines belonging to genotypes I and II. Vaccinated hens can transfer antibodies to their offspring through the egg yolk. Maternally derived antibodies can provide passive protection against diseases but can also interfere with vaccination efficacy early in life. This study was conducted on chicks hatched from hens vaccinated with a commercial genotype VII NDV-matched vaccine to investigate the correlation between hemagglutination inhibition (HI) antibody levels in chicks and hens and the decaying pattern of maternally derived HI antibodies, and to evaluate the protective efficacy of different levels of maternally derived HI antibodies against challenge with a virulent NDV strain of genotype VII based on survivability and virus shedding. The HI antibody titers in chicks at hatching were about 1.3 log2 lower than those in hens, indicating an antibody transfer rate of approximately 41.52%. The estimated half-life of these antibodies was about 3.2 days. The protective efficacy of maternally derived HI antibodies was positively correlated with the titer. These antibodies could effectively protect chicks against mortality when the titer was 7 log2 or higher, but they were unable to prevent virus shedding or infection even at a high titer of 11 log2. The obtained results will greatly assist producers in determining the immune status of chicks and formulating appropriate vaccination schedules against ND.

New insights into the neuraminidase-mediated hemagglutination activity of influenza A(H3N2) viruses.

Influenza virus neuraminidase (NA) can act as a receptor-binding protein, a role commonly attributed to hemagglutinin (HA). In influenza A(H3N2) viruses, three NA amino acid residues have previously been associated with NA-mediated hemagglutination: T148, D151, and more recently, H150. These residues are part of the 150-loop of the NA monomer. Substitutions at 148 and 151 arise from virus propagation in laboratory cell cultures, whereas changes at 150 occurred during virus evolution in the human host. In this study, we examined the effect of natural amino acid polymorphism at position 150 on NA-mediated hemagglutination. Using the A/Puerto Rico/8/34 backbone, we generated a comprehensive panel of recombinant A(H3N2) viruses that have different NAs but shared an HA that displays poor binding to red blood cells (RBCs). None of the tested substitutions at 150 (C, H, L, R, and S) promoted NA-binding. However, we identified two new determinants of NA-binding, Q136K and T439R, that emerged during virus culturing. Similar to T148I, both Q136K and T439R reduced NA enzyme activity by 48-86% and inhibition (14- to 173-fold) by the NA inhibitor zanamivir. NA-binding was observed when a virus preparation contained approximately 10% of NA variants with either T148I or T439R, highlighting the benefit of using deep sequencing in virus characterization. Taken together, our findings provide new insights into the molecular mechanisms underlying the ability of NA to function as a binding protein. Information gained may aid in the design of new and improved NA-targeting antivirals.

[Latex Agglutination as an Alternative to the Hemagglutination Reaction of Influenza Viruses].

As an alternative to the classical method of erythrocyte hemagglutination, a latex agglutination assay based on the interaction of influenza viruses with the sialoglycoprotein fetuin immobilized on the surface of polystyrene microspheres has been developed. Twelve influenza A virus strains of different subtypes and two influenza B viruses of different lines were tested. Simultaneous titration of viruses using the classical hemagglutination test and the proposed latex agglutination assay showed similar sensitivity and a high degree of correlation (R = 0.94). The obtained microspheres can be used for titration of viruses that recognize and bind sialylated glycans as receptors. In particular, latex aggregation was also induced by the Newcastle disease virus.

Regions Important for Hemagglutination Activity and Serotypes of Avibacterium paragallinarum HMTp210 Protein.

Avibacterium paragallinarum is an important respiratory pathogen of domestic chickens. Avibacterium paragallinarum has been subtyped into three serogroups and nine serovars according to the Page and revised Kume schemes. The major hemagglutinin antigen of A. paragallinarum is HMTp210, which is a large protein of about 2000 amino acids (aa), including a 70-aa signal peptide at its N-terminal end. However, the regions important for the hemagglutination (HA) activity and serotypes of HMTp210 remain unclear. In this study we constructed a series of A. paragallinarum strains expressing HMTp210 in-frame deletion mutants and determined their HA titers to identify the regions important for the HA activity and serotypes of HMTp210. Two distinct types of HA activities were found in HMTp210. The type 1 HA activity resided in the region spanning the full-length HA (aa 71-2084), whereas the type 2 resided in the region spanning aa 1003-2084. The putative ligand binding of the type 1 HA activity was located at aa 176-360, which had a structure similar to YadA of Yersinia enterocolitica. The putative ligand binding site of the type 2 HA activity was located at aa 1003-1125, which had a structure similar to UspA1 from Moraxella catarrhalis. The type 1 HA activity appeared to be Page serogroup specific, whereas type 2 appeared to be Kume serovar specific. Finally, sequence analyses of the regions spanning aa 1-400 and aa 1100-1600 of HMTp210 could be useful for the molecular serotyping (the Page and revised Kume schemes) of A. paragallinarum isolates.

Regiones importantes para la actividad de hemaglutinación y serotipos de la proteína HMTp210 de Avibacterium paragallinarum. La bacteria Avibacterium paragallinarum es un patógeno respiratorio importante de los pollos domésticos. Avibacterium paragallinarum se subtipificó en tres serogrupos y nueve serovares de acuerdo con los esquemas revisados de Page y Kume. El principal antígeno de la hemaglutinina de A. paragallinarum es la proteína HMTp210, que es una proteína grande de unos 2000 aminoácidos (aa), que incluye un péptido señal de 70 aminoácidos en su extremo N-terminal. Sin embargo, las regiones importantes para la actividad de hemaglutinación (HA) y de los serotipos de la proteína HMTp210 siguen sin estar determinados. En este estudio, se construyó una serie de cepas de A. paragallinarum que expresaban mutantes de deleción en marco de lectura de HMTp210 y se determinaron sus títulos de hemaglutinación para identificar las regiones importantes para la actividad de hemaglutinación y de los serotipos de HMTp210. Se encontraron dos tipos distintos de actividades hemaglutinación en la proteína HMTp210. La actividad de hemaglutinación de tipo 1 residía en la región que abarcaba la longitud completa (aminoácidos 71­2084), mientras que la de tipo 2 residía en la región que abarcaba entre los aminoácidos 1003­2084. El sitio supuesto de unión al ligando de la actividad de hemaglutinación tipo 1 se ubicó entre los aminoácidos 176­360, que tenía una estructura similar a la proteína YadA de Yersinia enterocolitica. El supuesto sitio de unión del ligando de la actividad de hemaglutinación tipo 2 se ubicó entre los aminoácidos 1003­1125, que tenía una estructura similar a la proteína UspA1 de Moraxella catarrhalis. La actividad de hemaglutinación tipo 1 parecía ser específica del serogrupo Page, mientras que la hemaglutinación tipo 2 parecía ser específica del serovar Kume. Finalmente, los análisis de secuencias de las regiones que abarcan los aminácidos 1­400 y aminoácidos 1100­1600 de HMTp210 podrían ser útiles para la serotipificación molecular (por el esquema revisado de Page y Kume revisado) de aislamientos de A. paragallinarum.

Hemagglutination Assay via Optical Density Characterization in 3D Microtrap Chips.

Hemagglutination assay has been used for blood typing and detecting viruses, thus applicable for the diagnosis of infectious diseases, including COVID-19. Therefore, the development of microfluidic devices for fast detection of hemagglutination is on-demand for point-of-care diagnosis. Here, we present a way to detect hemagglutination in 3D microfluidic devices via optical absorbance (optical density, OD) characterization. 3D printing is a powerful way to build microfluidic structures for diagnostic devices. However, mixing liquid in microfluidic chips is difficult due to laminar flow, which hampers practical applications such as antigen-antibody mixing. To overcome the issue, we fabricated 3D microfluidic chips with embedded microchannel and microwell structures to induce hemagglutination between red blood cells (RBCs) and antibodies. We named it a 3D microtrap chip. We also established an automated measurement system which is an integral part of diagnostic devices. To do this, we developed a novel way to identify RBC agglutination and non-agglutination via the OD difference. By adapting a 3D-printed aperture to the microtrap chip, we obtained a pure absorbance signal from the microchannels by eliminating the background brightness of the microtrap chip. By investigating the underlying optical physics, we provide a 3D device platform for detecting hemagglutination.

Linker remodels human Galectin-8 structure and regulates its hemagglutination and pro-apoptotic activity.

Numerous articles have reported the involvement of linker in regulating bioactivity of tandem-repeat galectins. We hypothesize that linker interacts with N/C-CRDs to regulate the bioactivity of tandem-repeat galectins. To further investigate structural molecular mechanism of linker in regulating bioactivity of Gal-8, Gal-8LC was crystallized. Gal-8LC structure revealed formation of ß-strand S1 by Asn174 to Pro176 from linker. S1-strand interacts with C-terminal of C-CRD via hydrogen bond interactions, mutually influencing their spatial structures. Our Gal-8 NL structure have demonstrated that linker region from Ser154 to Gln158 interacts with the N-terminal of Gal-8. Ser154 to Gln158 and Asn174 to Pro176 are likely involved in regulation of Gal-8's biological activity. Our preliminary experiment results revealed different hemagglutination and pro-apoptotic activities between full-length and truncated forms of Gal-8, indicating involvement of linker in regulating these activities. We generated several mutant and truncated forms of Gal-8 (Gal-8 M3, Gal-8 M5, Gal-8TL1, Gal-8TL2, Gal-8LC-M3 and Gal-8_177-317). Ser154 to Gln158 and Asn174 to Pro176 were found to be involved in regulating hemagglutination and pro-apoptotic activities of Gal-8. Ser154 to Gln158 and Asn174 to Pro176 are critical functional regulatory regions within linker. Our study holds significant importance in providing a profound understanding of how linker regulates biological activity of Gal-8.

Haemagglutination inhibition and virus microneutralisation serology assays: use of harmonised protocols and biological standards in seasonal influenza serology testing and their impact on inter-laboratory variation and assay correlation: A FLUCOP collaborative study.

Introduction: The haemagglutination inhibition assay (HAI) and the virus microneutralisation assay (MN) are long-established methods for quantifying antibodies against influenza viruses. Despite their widespread use, both assays require standardisation to improve inter-laboratory agreement in testing. The FLUCOP consortium aims to develop a toolbox of standardised serology assays for seasonal influenza. Building upon previous collaborative studies to harmonise the HAI, in this study the FLUCOP consortium carried out a head-to-head comparison of harmonised HAI and MN protocols to better understand the relationship between HAI and MN titres, and the impact of assay harmonisation and standardisation on inter-laboratory variability and agreement between these methods. Methods: In this paper, we present two large international collaborative studies testing harmonised HAI and MN protocols across 10 participating laboratories. In the first, we expanded on previously published work, carrying out HAI testing using egg and cell isolated and propagated wild-type (WT) viruses in addition to high-growth reassortants typically used influenza vaccines strains using HAI. In the second we tested two MN protocols: an overnight ELISA-based format and a 3-5 day format, using reassortant viruses and a WT H3N2 cell isolated virus. As serum panels tested in both studies included many overlapping samples, we were able to look at the correlation of HAI and MN titres across different methods and for different influenza subtypes. Results: We showed that the overnight ELISA and 3-5 day MN formats are not comparable, with titre ratios varying across the dynamic range of the assay. However, the ELISA MN and HAI are comparable, and a conversion factor could possibly be calculated. In both studies, the impact of normalising using a study standard was investigated, and we showed that for almost every strain and assay format tested, normalisation significantly reduced inter-laboratory variation, supporting the continued development of antibody standards for seasonal influenza viruses. Normalisation had no impact on the correlation between overnight ELISA and 3-5 day MN formats.

H3N2 influenza hemagglutination inhibition method qualification with data driven statistical methods for human clinical trials.

Introduction: Hemagglutination inhibition (HAI) antibody titers to seasonal influenza strains are important surrogates for vaccine-elicited protection. However, HAI assays can be variable across labs, with low sensitivity across diverse viruses due to lack of standardization. Performing qualification of these assays on a strain specific level enables the precise and accurate quantification of HAI titers. Influenza A (H3N2) continues to be a predominant circulating subtype in most countries in Europe and North America since 1968 and is thus a focus of influenza vaccine research. Methods: As a part of the National Institutes of Health (NIH)-funded Collaborative Influenza Vaccine Innovation Centers (CIVICs) program, we report on the identification of a robust assay design, rigorous statistical analysis, and complete qualification of an HAI assay using A/Texas/71/2017 as a representative H3N2 strain and guinea pig red blood cells and neuraminidase (NA) inhibitor oseltamivir to prevent NA-mediated agglutination. Results: This qualified HAI assay is precise (calculated by the geometric coefficient of variation (GCV)) for intermediate precision and intra-operator variability, accurate calculated by relative error, perfectly linear (slope of -1, R-Square 1), robust (<25% GCV) and depicts high specificity and sensitivity. This HAI method was successfully qualified for another H3N2 influenza strain A/Singapore/INFIMH-16-0019/2016, meeting all pre-specified acceptance criteria. Discussion: These results demonstrate that HAI qualification and data generation for new influenza strains can be achieved efficiently with minimal extra testing and development. We report on a qualified and adaptable influenza serology method and analysis strategy to measure quantifiable HAI titers to define correlates of vaccine mediated protection in human clinical trials.

Novel blood typing method by discrimination of hemagglutination and rouleaux using an erythrocyte aggregometer.

BACKGROUND: In pretransfusion blood typing, pretreatments such as centrifugation and suspension of red blood cells (RBCs) and mixing them with sufficient amounts of reagents are required, but these steps are time-consuming and costly. OBJECTIVE: Aiming to develop a new blood typing method that requires no dilution and only a small amount of reagent, we attempted to determine blood type using syllectometry, an easy-to-use and rapid optical method for measuring the RBC aggregation that occurs when blood flow is abruptly stopped in a flow channel. METHODS: Samples of whole blood from 20 healthy participants were mixed with antibody reagents for blood typing at mixing ratios of 2.5% to 10% and measured with a syllectometry device. RESULTS: Amplitude (AMP), one of the aggregation parameters, showed significant differences between agglutination and non-agglutination samples at mixing ratios from 2.5% to 10%. Although there were significant individual differences in aggregation parameters, calculation of AMP relative to that of blood before reagent mixing reduced the individual differences and enabled determination of blood type in all participants. CONCLUSIONS: This new method enables blood typing with a small amount of reagent, without the time-consuming and labor-intensive pretreatments such as centrifugation and suspension of RBCs.

Efficacy of Newcastle disease LaSota vaccine-induced hemagglutination inhibition antibodies against challenges with heterologous virulent strains of genotypes VII and IX.

Newcastle disease (ND), caused by virulent Newcastle disease virus (NDV), still remains one of the most important avian diseases affecting the poultry industry worldwide, despite intensive vaccination programs have been implemented in many countries. All NDV isolates characterized to date are of one serotype and classified into classes I and II, with class II being further divided into twenty-one genotypes. Antigenic and genetic diversity is observed among the different genotypes. Current commercially available vaccines belonging to genotypes I and II are genetically divergent from strains that caused ND outbreaks worldwide in the last two decades. Reports of vaccination failures on their insufficient ability to inhibit infection or virus shedding have created renewed interest in developing vaccines homologous to virulent NDV circulating in the field. In this study, after vaccination with the most widely used LaSota vaccine (genotype II), chickens with different hemagglutination inhibition (HI) antibody levels were challenged with heterologous virulent NDV strains of genotypes VII and IX to evaluate how antibody levels relate to clinical protection and infection or virus shedding. Under the experimental condition, LaSota vaccine could fully protect birds from morbidity and mortality, but higher antibody levels were required to inhibit virus shedding. The number of birds shedding virus generally tended to decrease as the HI antibody titers increase in vaccinated birds. When the HI antibody titers reached ≥ 13 log2 and ≥ 10 log2, the virus shedding from JSC0804 strain (genotype VII) and F48E8 strain (genotype IX) could be completely inhibited, respectively, but it may be difficult to ensure that all individuals reach and maintain those levels in chicken flocks vaccinated according to routine procedure. Furthermore, the virus shedding in vaccinated birds was correlated with the amino acid similarity between the vaccine and challenge strains; more similarity, less virus shedding. The results obtained highlight that stringent biosecurity measures combined with vaccination are crucial for chicken farms to maintain a virulent NDV-free status.

An external quality assessment feasibility study; cross laboratory comparison of haemagglutination inhibition assay and microneutralization assay performance for seasonal influenza serology testing: A FLUCOP study.

Introduction: External Quality Assessment (EQA) schemes are designed to provide a snapshot of laboratory proficiency, identifying issues and providing feedback to improve laboratory performance and inter-laboratory agreement in testing. Currently there are no international EQA schemes for seasonal influenza serology testing. Here we present a feasibility study for conducting an EQA scheme for influenza serology methods. Methods: We invited participant laboratories from industry, contract research organizations (CROs), academia and public health institutions who regularly conduct hemagglutination inhibition (HAI) and microneutralization (MN) assays and have an interest in serology standardization. In total 16 laboratories returned data including 19 data sets for HAI assays and 9 data sets for MN assays. Results: Within run analysis demonstrated good laboratory performance for HAI, with intrinsically higher levels of intra-assay variation for MN assays. Between run analysis showed laboratory and strain specific issues, particularly with B strains for HAI, whilst MN testing was consistently good across labs and strains. Inter-laboratory variability was higher for MN assays than HAI, however both assays showed a significant reduction in inter-laboratory variation when a human sera pool is used as a standard for normalization. Discussion: This study has received positive feedback from participants, highlighting the benefit such an EQA scheme would have on improving laboratory performance, reducing inter laboratory variation and raising awareness of both harmonized protocol use and the benefit of biological standards for seasonal influenza serology testing.

Direct evidence of fiber-protein-directed hemagglutination by canine adenoviruses.

Canine adenoviruses (CAdVs) are divided into two serotypes, CAdV1 and CAdV2, whose members mainly cause infectious hepatitis and laryngotracheitis, respectively, in canids. To gain insight into the molecular basis of viral hemagglutination, we constructed chimeric viruses whose fiber proteins or their knob domains, which play a role in viral attachment to cells, were swapped among CAdV1, CAdV2, and bat adenovirus via reverse genetics. The results revealed that, in each case, viral hemagglutination was specifically mediated by the fiber protein or knob domain, providing direct evidence for fiber-protein-directed receptor-binding characteristics of CAdVs.

SARS-CoV-2 Spike Protein Induces Hemagglutination: Implications for COVID-19 Morbidities and Therapeutics and for Vaccine Adverse Effects.

Experimental findings for SARS-CoV-2 related to the glycan biochemistry of coronaviruses indicate that attachments from spike protein to glycoconjugates on the surfaces of red blood cells (RBCs), other blood cells and endothelial cells are key to the infectivity and morbidity of COVID-19. To provide further insight into these glycan attachments and their potential clinical relevance, the classic hemagglutination (HA) assay was applied using spike protein from the Wuhan, Alpha, Delta and Omicron B.1.1.529 lineages of SARS-CoV-2 mixed with human RBCs. The electrostatic potential of the central region of spike protein from these four lineages was studied through molecular modeling simulations. Inhibition of spike protein-induced HA was tested using the macrocyclic lactone ivermectin (IVM), which is indicated to bind strongly to SARS-CoV-2 spike protein glycan sites. The results of these experiments were, first, that spike protein from these four lineages of SARS-CoV-2 induced HA. Omicron induced HA at a significantly lower threshold concentration of spike protein than the three prior lineages and was much more electropositive on its central spike protein region. IVM blocked HA when added to RBCs prior to spike protein and reversed HA when added afterward. These results validate and extend prior findings on the role of glycan bindings of viral spike protein in COVID-19. They furthermore suggest therapeutic options using competitive glycan-binding agents such as IVM and may help elucidate rare serious adverse effects (AEs) associated with COVID-19 mRNA vaccines, which use spike protein as the generated antigen.