Dissection of the Influenza A Virus Endocytic Routes Reveals Macropinocytosis as an Alternative Entry Pathway

Influenza A virus (IAV) enters host cells upon binding of its hemagglutinin glycoprotein to sialylated host cell receptors. Whereas dynamin-dependent, clathrin-mediated endocytosis (CME) is generally considered as the IAV infection pathway, some observations suggest the occurrence of an as yet uncharacterized alternative entry route. By manipulating entry parameters we established experimental conditions that allow the separate analysis of dynamin-dependent and -independent entry of IAV. Whereas entry of IAV in phosphate-buffered saline could be completely inhibited by dynasore, a specific inhibitor of dynamin, a dynasore-insensitive entry pathway became functional in the presence of fetal calf serum. This finding was confirmed with the use of small interfering RNAs targeting dynamin-2. In the presence of serum, both IAV entry pathways were operational. Under these conditions entry could be fully blocked by combined treatment with dynasore and the amiloride derivative EIPA, the hallmark inhibitor of macropinocytosis, whereas either drug alone had no effect. The sensitivity of the dynamin-independent entry pathway to inhibitors or dominant-negative mutants affecting actomyosin dynamics as well as to a number of specific inhibitors of growth factor receptor tyrosine kinases and downstream effectors thereof all point to the involvement of macropinocytosis in IAV entry. Consistently, IAV particles and soluble FITC-dextran were shown to co-localize in cells in the same vesicles. Thus, in addition to the classical dynamin-dependent, clathrin-mediated endocytosis pathway, IAV enters host cells by a dynamin-independent route that has all the characteristics of macropinocytosis

Second sialic acid-binding site of influenza A virus neuraminidase: Binding receptors for efficient release

Influenza A viruses (IAVs) are a major cause of human respiratory tract infections and cause significant disease and mortality. Human IAVs originate from animal viruses that breached the host species barrier. IAV particles contain sialoglycan receptor-binding haemagglutinin (HA) and receptor-destroying neuraminidase (NA) in their envelope. When IAV crosses the species barrier, the functional balance between HA and NA needs to be adjusted to the sialoglycan repertoire of the novel host species. Relatively little is known about the role of NA in host adaptation in contrast to the extensively studied HA. NA prevents virion aggregation and facilitates release of (newly assembled) virions from cell surfaces as well as from decoy receptors abundantly present in mucus and cell glycocalyx. In addition to a highly conserved catalytic site, NA carries a second sialic acid-binding site (2SBS). The 2SBS preferentially binds α2,3-linked sialic acids, and enhances activity of the neighboring catalytic site by bringing/keeping multivalent substrates in close contact with this site. In this way, the 2SBS contributes to the HA-NA balance of virus particles and affects virus replication. The 2SBS is highly conserved in all NA subtypes of avian IAVs, with some notable exceptions associated with changes in the receptor-binding specificity of HA and host tropism. Conservation of the 2SBS is invariably lost in human (pandemic) viruses as well as in several other viruses adapted to mammalian host species. Preservation or loss of the 2SBS is likely to be an important factor of the viral host range

Disturbed oscillatory brain dynamics in subcortical ischemic vascular dementia

<p>Abstract</p> <p>Background</p> <p>White matter hyperintensities (WMH) can lead to dementia but the underlying physiological mechanisms are unclear. We compared relative oscillatory power from electroencephalographic studies (EEGs) of 17 patients with subcortical ischemic vascular dementia, based on extensive white matter hyperintensities (SIVD-WMH) with 17 controls to investigate physiological changes underlying this diagnosis.</p> <p>Results</p> <p>Differences between the groups were large, with a decrease of relative power of fast activity in patients (alpha power 0.25 ± 0.12 versus 0.38 ± 0.13, p = 0.01; beta power 0.08 ± 0.04 versus 0.19 ± 0.07; p<0.001) and an increase in relative powers of slow activity in patients (theta power 0.32 ± 0.11 versus 0.14 ± 0.09; p<0.001 and delta power 0.31 ± 0.14 versus 0.23 ± 0.09; p<0.05). Lower relative beta power was related to worse cognitive performance in a linear regression analysis (standardized beta = 0.67, p<0.01).</p> <p>Conclusions</p> <p>This pattern of disturbance in oscillatory brain activity indicate loss of connections between neurons, providing a first step in the understanding of cognitive dysfunction in SIVD-WMH.</p

Next generation single-domain antibodies against respiratory zoonotic RNA viruses

The global impact of zoonotic viral outbreaks underscores the pressing need for innovative antiviral strategies, particularly against respiratory zoonotic RNA viruses. These viruses possess a high potential to trigger future epidemics and pandemics due to their high mutation rate, broad host range and efficient spread through airborne transmission. Recent pandemics caused by coronaviruses and influenza A viruses underscore the importance of developing targeted antiviral strategies. Single-domain antibodies (sdAbs), originating from camelids, also known as nanobodies or VHHs (Variable Heavy domain of Heavy chain antibodies), have emerged as promising tools to combat current and impending zoonotic viral threats. Their unique structure, coupled with attributes like robustness, compact size, and cost-effectiveness, positions them as strong alternatives to traditional monoclonal antibodies. This review describes the pivotal role of sdAbs in combating respiratory zoonotic viruses, with a primary focus on enhancing sdAb antiviral potency through optimization techniques and diverse administration strategies. We discuss both the promises and challenges within this dynamically growing field

Kinetic analysis of paramyxovirus-sialoglycan receptor interactions reveals virion motility

Many viruses initiate infection by binding to sialoglycan receptors at the cell surface. Binding to such receptors comes at a cost, however, as the sheer abundance of sialoglycans e.g. in mucus, may immobilize virions to non-functional decoy receptors. As a solution, sialoglycan-binding as well as sialoglycan-cleavage activities are often present in these viruses, which for paramyxoviruses are combined in the hemagglutinin-neuraminidase (HN) protein. The dynamic interactions of sialoglycan-binding paramyxoviruses with their receptors are thought to be key determinants of species tropism, replication and pathogenesis. Here we used biolayer interferometry to perform kinetic analyses of receptor interactions of animal and human paramyxoviruses (Newcastle disease virus, Sendai virus, and human parainfluenza virus 3). We show that these viruses display strikingly different receptor interaction dynamics, which correlated with their receptor-binding and -cleavage activities and the presence of a second sialic acid binding site. Virion binding was followed by sialidase-driven release, during which virions cleaved sialoglycans until a virus-specific density was reached, which was largely independent of virion concentration. Sialidase-driven virion release was furthermore shown to be a cooperative process and to be affected by pH. We propose that paramyxoviruses display sialidase-driven virion motility on a receptor-coated surface, until a threshold receptor density is reached at which virions start to dissociate. Similar motility has previously been observed for influenza viruses and is likely to also apply to sialoglycan-interacting embecoviruses. Analysis of the balance between receptor-binding and -cleavage increases our understanding of host species tropism determinants and zoonotic potential of viruses

H3N2 influenza A virus gradually adapts to human-type receptor binding and entry specificity after the start of the 1968 pandemic

To become established upon zoonotic transfer, influenza A viruses (IAV) need to switch binding from "avian-type" α2-3-linked sialic acid receptors (2-3Sia) to "human-type" Siaα2-6-linked sialic acid receptors (2-6Sia). For the 1968 H3N2 pandemic virus, this was accomplished by two canonical amino acid substitutions in its hemagglutinin (HA) although a full specificity shift had not occurred. The receptor repertoire on epithelial cells is highly diverse and simultaneous interaction of a virus particle with a range of low- to very low-affinity receptors results in tight heteromultivalent binding. How this range of affinities determines binding selectivity and virus motility remains largely unknown as the analysis of low-affinity monovalent HA-receptor interactions is technically challenging. Here, a biolayer interferometry assay enabled a comprehensive analysis of receptor-binding kinetics evolution upon host-switching. Virus-binding kinetics of H3N2 virus isolates slowly evolved from 1968 to 1979 from mixed 2-3/2-6Sia specificity to high 2-6Sia specificity, surprisingly followed by a decline in selectivity after 1992. By using genetically tuned HEK293 cells, presenting either a simplified 2-3Sia- or 2-6Sia-specific receptor repertoire, receptor-specific binding was shown to correlate strongly with receptor-specific entry. In conclusion, the slow and continuous evolution of entry and receptor-binding specificity of seasonal H3N2 viruses contrasts with the paradigm that human IAVs need to rapidly acquire and maintain a high specificity for 2-6Sia. Analysis of the kinetic parameters of receptor binding provides a basis for understanding virus-binding specificity, motility, and HA/neuraminidase balance at the molecular level

Serological screening of influenza A virus antibodies in cats and dogs indicates frequent infection with different subtypes

Influenza A viruses (IAVs) infect humans and a variety of other animal species. Infections with some subtypes of IAV were also reported in domestic cats and dogs. Besides animal health implications, close contact between companion animals and humans also poses a potential risk of zoonotic IAV infections. In this study, serum samples from different cat and dog cohorts were analyzed for IAV antibodies against 7 IAV subtypes, using three distinctive IAV-specific assays differing in IAV subtype-specific discriminatory power and sensitivity. Enzyme-linked immunosorbent assays against the complete hemagglutinin (HA) ectodomain or the HA1 domain were used, as well as a novel nanoparticle-based, virus-free hemagglutination inhibition (HI) assay. Using these three assays, we found cat and dog sera from different cohorts to be positive for antibodies against one or more IAV subtypes/strains. Cat and dog serum samples collected after the 2009 pandemic H1N1 outbreak exhibit much higher seropositivity against H1 compared with samples from before 2009. Cat sera furthermore displayed higher reactivity for avian IAVs than dog sera. Our findings show the added value of using complementary serological assays, which are based on reactivity with different numbers of HA epitopes, to study IAV antibody responses and for improved serosurveillance of IAV infections. We conclude that infection of cats and dogs with both human and avian IAVs of different subtypes is prevalent. These observations highlight the role of cats and dogs in IAV ecology and indicate the potential of these companion animals to give rise to novel (reassorted) viruses with increased zoonotic potential

Direct Binding of Bovine IgG-Containing Immune Complexes to Human Monocytes and Their Putative Role in Innate Immune Training

Bovine milk IgG (bIgG) was shown to bind to and neutralize the human respiratory synovial virus (RSV). In animal models, adding bIgG prevented experimental RSV infection and increased the number of activated T cells. This enhanced activation of RSV-specific T cells may be explained by receptor-mediated uptake and antigen presentation after binding of bIgG-RSV immune complexes (ICs) with FcγRs (primarily CD32) on human immune cells. This indirect effect of bIgG ICs on activation of RSV-specific T cells was confirmed previously in human T cell cultures. However, the direct binding of ICs to antigen-presenting cells has not been addressed. As bovine IgG can induce innate immune training, we hypothesized that this effect could be caused more efficiently by ICs. Therefore, we characterized the expression of CD16, CD32, and CD64 on (peripheral blood mononuclear cells (PBMCs), determined the optimal conditions to form ICs of bIgG with the RSV preF protein, and demonstrated the direct binding of these ICs to human CD14 + monocytes. Similarly, bIgG complexed with a murine anti-bIgG mAb also bound efficiently to the monocytes. To evaluate whether the ICs could induce innate immune training more efficiently than bIgG itself, the resulted ICs, as well as bIgG, were used in an in vitro innate immune training model. Training with the ICs containing bIgG and RSV preF protein-but not the bIgG alone-induced significantly higher TNF-α production upon LPS and R848 stimulation. However, the preF protein itself nonsignificantly increased cytokine production as well. This may be explained by its tropism to the insulin-like growth factor receptor 1 (IGFR1), as IGF has been reported to induce innate immune training. Even so, these data suggest a role for IgG-containing ICs in inducing innate immune training after re-exposure to pathogens. However, as ICs of bIgG with a mouse anti-bIgG mAb did not induce this effect, further research is needed to confirm the putative role of bIgG ICs in enhancing innate immune responses in vivo

Passive inhalation of dry powder influenza vaccine formulations completely protects chickens against H5N1 lethal viral challenge

Bird to human transmission of high pathogenicity avian influenza virus (HPAIV) poses a significant risk of triggering a flu pandemic in the human population. Therefore, vaccination of susceptible poultry during an HPAIV outbreak might be the best remedy to prevent such transmissions. To this end, suitable formulations and an effective mass vaccination method that can be translated to field settings needs to be developed. Our previous study in chickens has shown that inhalation of a non-adjuvanted dry powder influenza vaccine formulation during normal breathing results in partial protection against lethal influenza challenge. The aim of the present study was to improve the effectiveness of pulmonary vaccination by increasing the vaccine dose deposited in the lungs and by the use of suitable adjuvants. Two adjuvants, namely, Bacterium-like Particles (BLP) and Advax, were spray freeze dried with influenza vaccine into dry powder formulations. Delivery of dry formulations directly at the syrinx revealed that BLP and Advax had the potential to boost either systemic or mucosal immune responses or both. Upon passive inhalation of dry influenza vaccine formulations in an optimized set-up, BLP and Advax/BLP adjuvanted formulations induced significantly higher systemic immune responses than the non-adjuvanted formulation. Remarkably, all vaccinated animals not only survived a lethal influenza challenge, but also did not show any shedding of challenge virus except for two out of six animals in the Advax group. Overall, our results indicate that passive inhalation is feasible, effective and suitable for mass vaccination of chickens if it can be adapted to field settings.</p

A bivalent spike-targeting nanobody with anti-sarbecovirus activity

The continued emergence and zoonotic threat posed by coronaviruses highlight the urgent need for effective antiviral strategies with broad reactivity to counter new emerging strains. Nanobodies (or single-domain antibodies) are promising alternatives to traditional monoclonal antibodies, due to their small size, cost-effectiveness and ease of bioengineering. Here, we describe 7F, a llama-derived nanobody, targeting the spike receptor binding domain of sarbecoviruses and SARS-like coronaviruses. 7F demonstrates potent neutralization against SARS-CoV-2 and cross-neutralizing activity against SARS-CoV and SARS-like CoV WIV16 pseudoviruses. Structural analysis reveals 7F's ability to induce the formation of spike trimer dimers by engaging with two SARS-CoV-2 spike RBDs, targeting the highly conserved class IV region, though concentration dependent. Bivalent 7F constructs substantially enhance neutralization potency and breadth, up to more recent SARS-CoV-2 variants of concern. Furthermore, we demonstrate the therapeutic potential of bivalent 7F against SARS-CoV-2 in the fully differentiated 3D tissue cultures mirroring the epithelium of the human airway ex vivo. The broad sarbecovirus activity and distinctive structural features of bivalent 7F underscore its potential as promising antiviral against emerging and evolving sarbecoviruses