Lysosomal sialidase NEU1, its intracellular properties, deficiency, and use as a therapeutic agent.

Neuraminidase 1 (NEU1) is a lysosomal sialidase that cleaves terminal α-linked sialic acid residues from sialylglycans. NEU1 is biosynthesized in the rough endoplasmic reticulum (RER) lumen as an N-glycosylated protein to associate with its protective protein/cathepsin A (CTSA) and then form a lysosomal multienzyme complex (LMC) also containing ß-galactosidase 1 (GLB1). Unlike other mammalian sialidases, including NEU2 to NEU4, NEU1 transport to lysosomes requires association of NEU1 with CTSA, binding of the CTSA carrying terminal mannose 6-phosphate (M6P)-type N-glycan with M6P receptor (M6PR), and intralysosomal NEU1 activation at acidic pH. In contrast, overexpression of the single NEU1 gene in mammalian cells causes intracellular NEU1 protein crystallization in the RER due to self-aggregation when intracellular CTSA is reduced to a relatively low level. Sialidosis (SiD) and galactosialidosis (GS) are autosomal recessive lysosomal storage diseases caused by the gene mutations of NEU1 and CTSA, respectively. These incurable diseases associate with the NEU1 deficiency, excessive accumulation of sialylglycans in neurovisceral organs, and systemic manifestations. We established a novel GS model mouse carrying homozygotic Ctsa IVS6 + 1 g/a mutation causing partial exon 6 skipping with simultaneous deficiency of Ctsa and Neu1. Symptoms developed in the GS mice like those in juvenile/adult GS patients, such as myoclonic seizures, suppressed behavior, gargoyle-like face, edema, proctoptosis due to Neu1 deficiency, and sialylglycan accumulation associated with neurovisceral inflammation. We developed a modified NEU1 (modNEU1), which does not form protein crystals but is transported to lysosomes by co-expressed CTSA. In vivo gene therapy for GS and SiD utilizing a single adeno-associated virus (AAV) carrying modNEU1 and CTSA genes under dual promoter control will be created.

Structure of the immunoregulatory sialidase NEU1.

Sialic acids linked to glycoproteins and glycolipids are important mediators of cell and protein recognition events. These sugar residues are removed by neuraminidases (sialidases). Neuraminidase-1 (sialidase-1 or NEU1) is a ubiquitously expressed mammalian sialidase located in lysosomes and on the cell membrane. Because of its modulation of multiple signaling processes, it is a potential therapeutic target for cancers and immune disorders. Genetic defects in NEU1 or in its protective protein cathepsin A (PPCA, CTSA) cause the lysosomal storage diseases sialidosis and galactosialidosis. To further our understanding of this enzyme's function at the molecular level, we determined the three-dimensional structure of murine NEU1. The enzyme oligomerizes through two self-association interfaces and displays a wide substrate-binding cavity. A catalytic loop adopts an inactive conformation. We propose a mechanism of activation involving a conformational change in this loop upon binding to its protective protein. These findings may facilitate the development of selective inhibitor and agonist therapies.

Sialidase NEU3 and its pathological significance.

Sialidases (EC 3.2.1.18, also called neuraminidases) catalyze the removal of α-glycosidically linked sialic acid residues from glycoproteins and glycolipids; this is the initial step in the degradation of these glycoconjugates. Sialidases of mammalian origin have been implicated in not only lysosomal catabolism but also the modulation of functional molecules involved in many biological processes. To date, four types of mammalian sialidases have been cloned and designated as Neu1, Neu2, Neu3 and Neu4. These sialidases differ in their subcellular localization and enzymatic properties, as well as their chromosomal localization, and they are expressed in a tissue-specific manner. Among the sialidases, the plasma membrane-associated sialidase Neu3 appears to play particular roles in controlling transmembrane signaling through the modulation of gangliosides, and its aberrant expression is closely related to various pathogeneses, including that of cancer. Interestingly, the human orthologue NEU3 acts in two ways, catalytic hydrolysis of gangliosides and protein interactions with other signaling molecules. Aberrant NEU3 expression can induce various pathological conditions. This review briefly summarizes recent studies, focusing on the involvement of NEU3 in various pathological phenomena.

Dual synergistic response for the electrochemical detection of H1N1 virus and viral proteins using high affinity peptide receptors.

Identifying alternatives to antibodies as bioreceptors to test samples feasibly is crucial for developing next-generation in vitro diagnostic methods. Here, we aimed to devise an analytical method for detecting H1N1 viral proteins (hemagglutinin [HA] and neuraminidase [NA]) as well as the complete H1N1 virus with high sensitivity and selectivity. By applying biopanning of M13 peptide libraries, high affinity peptides specific for HA or NA were successfully identified. After selection, three different synthetic peptides that incorporated gold-binding motifs were designed and chemically synthesized on the basis of the original sequence identified phage display technique with or without two repeat. Their binding interactions were characterized by enzyme-linked immunosorbent assay (ELISA), square wave voltammetry (SWV), Time of flight-secondary ion mass spectroscopy (ToF-SIMS) and X-ray photoelectron spectroscopy (XPS). The binding constants (Kd) of HA BP1, HA BP2 and NA BP1 peptides were found to be 169.72 nM, 70.02 nM and 224.49 nM for HA or NA proteins by electrochemical measurements (SWV). The single use of HA BP2 peptide enabled the detection of either H1N1 viral proteins or the actual H1N1 virus, while NA BP1 peptide exhibited lower binding for real H1N1 virus particles. Moreover, the use of both HA BP1 and BP2 as a divalent capturing reagent improved sensor performance as well as the strength of the electrochemical signal, thereby exhibiting a dual synergistic effect for the electrochemical detection of H1N1 antigens with satisfactory specificity and sensitivity (limit of detection of 1.52 PFU/mL).

Design of a Cytotoxic Neuroblastoma-Targeting Agent Using an Enzyme Acting on Polysialic Acid Fused to a Toxin.

Polysialic acid, an abundant cell surface component of the developing nervous system, which declines rapidly postnatally to virtual absence in the majority of adult tissues, is highly expressed in some malignant tumors including neuroblastoma. We found that the binding of a noncatalytic endosialidase to polysialic acid causes internalization of the complex from the surface of neuroblastoma kSK-N-SH cells, a subline of SK-N-SH, and leads to a complete relocalization of polysialic acid to the intracellular compartment. The binding and uptake of the endosialidase is polysialic acid-dependent as it is inhibited by free excess ligand or removal of polysialic acid by active endosialidase, and does not happen if catalytic endosialidase is used in place of inactive endosialidase. A fusion protein composed of the noncatalytic endosialidase and the cytotoxic portion of diphtheria toxin was prepared to investigate whether the cellular uptake observed could be used for the specific elimination of polysialic acid-containing cells. The conjugate toxin was found to be toxic to polysialic acid-positive kSK-N-SH with an IC50 of 1.0 nmol/L. Replacing the noncatalytic endosialidase with active endosialidase decreased the activity to the level of nonconjugated toxin. Normal nonmalignant cells were selectively resistant to the toxin conjugate. The results demonstrate that noncatalytic endosialidase induces a quantitative removal and cellular uptake of polysialic acid from the cell surface which, by conjugation with diphtheria toxin fragment, can be exploited for the selective elimination of polysialic acid-containing tumor cells.

Dual oxidase 1 promotes antiviral innate immunity.

Dual oxidase 1 (DUOX1) is an NADPH oxidase that is highly expre-ssed in respiratory epithelial cells and produces H2O2 in the airway lumen. While a line of prior in vitro observations suggested that DUOX1 works in partnership with an airway peroxidase, lactoperoxidase (LPO), to produce antimicrobial hypothiocyanite (OSCN-) in the airways, the in vivo role of DUOX1 in mammalian organisms has remained unproven to date. Here, we show that Duox1 promotes antiviral innate immunity in vivo. Upon influenza airway challenge, Duox1-/- mice have enhanced mortality, morbidity, and impaired lung viral clearance. Duox1 increases the airway levels of several cytokines (IL-1ß, IL-2, CCL1, CCL3, CCL11, CCL19, CCL20, CCL27, CXCL5, and CXCL11), contributes to innate immune cell recruitment, and affects epithelial apoptosis in the airways. In primary human tracheobronchial epithelial cells, OSCN- is generated by LPO using DUOX1-derived H2O2 and inactivates several influenza strains in vitro. We also show that OSCN- diminishes influenza replication and viral RNA synthesis in infected host cells that is inhibited by the H2O2 scavenger catalase. Binding of the influenza virus to host cells and viral entry are both reduced by OSCN- in an H2O2-dependent manner in vitro. OSCN- does not affect the neuraminidase activity or morphology of the influenza virus. Overall, this antiviral function of Duox1 identifies an in vivo role of this gene, defines the steps in the infection cycle targeted by OSCN-, and proposes that boosting this mechanism in vivo can have therapeutic potential in treating viral infections.

Analysis of the Evolution of Pandemic Influenza A(H1N1) Virus Neuraminidase Reveals Entanglement of Different Phenotypic Characteristics.

The influenza A virus (IAV) neuraminidase (NA) is essential for virion release from cells and decoy receptors and an important target of antiviral drugs and antibodies. Adaptation to a new host sialome and escape from the host immune system are forces driving the selection of mutations in the NA gene. Phylogenetic analysis shows that until 2015, 16 amino acid substitutions in NA became fixed in the virus population after introduction in the human population of the pandemic IAV H1N1 (H1N1pdm09) in 2009. The accumulative effect of these substitutions, in the order in which they appeared, was analyzed using recombinant proteins and viruses in combination with different functional assays. The results indicate that NA activity did not evolve to a single optimum but rather fluctuated within a certain bandwidth. Furthermore, antigenic and enzymatic properties of NA were intertwined, with several residues affecting multiple properties. For example, the substitution K432E in the second sialic acid binding site, next to the catalytic site, was shown to affect catalytic activity, substrate specificity, and the pH optimum for maximum activity. This substitution also altered antigenicity of NA, which may explain its selection. We propose that the entanglement of NA phenotypes may be an important determining factor in the evolution of NA.IMPORTANCE Since its emergence in 2009, the pandemic H1N1 influenza A virus (IAV) has caused significant disease and mortality in humans. IAVs contain two envelope glycoproteins, the receptor-binding hemagglutinin (HA) and the receptor-destroying neuraminidase (NA). NA is essential for virion release from cells and decoy receptors, is an important target of antiviral drugs, and is increasingly being recognized as an important vaccine antigen. Not much is known, however, about the evolution of this protein upon the emergence of the novel pandemic H1N1 virus, with respect to its enzymatic activity and antigenicity. By reconstructing the evolutionary path of NA, we show that antigenic and enzymatic properties of NA are intertwined, with several residues affecting multiple properties. Understanding the entanglement of NA phenotypes will lead to better comprehension of IAV evolution and may help the development of NA-based vaccines.

Rapid identification of isoprenylated flavonoids constituents with inhibitory activity on bacterial neuraminidase from root barks of paper mulberry (Broussonetia papyrifera).

This study was to assess the possibility of using competitive and slow binding experiments with affinity-based ultrafiltration UPLC-QTof-MS analysis to identify potent bacterial neuraminidase (bNA) inhibitors from the Broussonetia papyrifera roots extract. To isolate unbound compounds from the enzyme-binding complex, the root bark extracts were either incubated in the absence of bNA, in the presence of bNA, or with the time-dependent bNA before the ultrafiltration was performed. Thirteen flavonoids were separated from the target extract, and their inhibitory activities were tested against bNA. The isolated flavonoids exhibited potent inhibition against NA (IC50 = 0.7-54.0 µM). Our kinetic analysis of representative active flavonoids (1, 2, and 6) showed slow and time-dependent reversible inhibition. Additionally, chalcones exhibited noncompetitive inhibition characteristics, whereas flavonols and flavans showed mixed-type behavior. The computational results supported the experimental behaviors of flavonoids 2, 6, 10, and 12, indicating that bounded to the active site, but flavonoids 6 and 10 binds near but not accurately at the active site. Although this is mixed-type inhibition, their binding can be considered competitive.

Thermally Triggered, Cell-Specific Enzymatic Glyco-Editing: In Situ Regulation of Lectin Recognition and Immune Response on Target Cells.

In situ glyco-editing on the cell surface can endow cellular glycoforms with new structures and properties; however, the lack of cell specificity and dependence on cells' endogenous functions plague the revelation of cellular glycan recognition properties and hamper the application of glyco-editing in complicated authentic biosystems. Herein, we develop a thermally triggered, cell-specific glyco-editing method for regulation of lectin recognition on target live cells in both single- and cocultured settings. The method relies on the aptamer-mediated anchoring of microgel-encapsulated neuraminidase on target cells and subsequent thermally triggered enzyme release for localized sialic acid (Sia) trimming. This temperature-based enzyme accessibility modulation strategy exempts genetic or metabolic engineering operations and, thus for the first time, enables tumor-specific desialylation on complicated tissue slices. The proposed method also provides an unprecedented opportunity to potentiate the innate immune response of natural killer cells toward target tumor cells through thermally triggered cell-specific desialylation, which paves the way for in vivo glycoimmune-checkpoint-targeted cancer therapeutic intervention.

Masking terminal neo-epitopes of linear peptides through glycosylation favours immune responses towards core epitopes producing parental protein bound antibodies.

Glycosylation of hydrophobic peptides at one terminus effectively increases their water-solubility, and conjugation through the opposing end to a carrier protein, renders them more immunogenic. Moreover, the glycosylation minimizes antibody responses to potentially deleterious, non-productive terminal neo-epitope regions of the peptides, and consequently shifts peptide immunogenicity towards the core amino acid residues. As proof of concept, glycopeptide-protein conjugates related to influenza hemagglutinin (HA), neuraminidase (NA), and the dimerization loop region of human epidermal growth factor receptor 2 (Her2), demonstrated a favorable production of core peptide specific antibodies as determined by ELISA studies. Furthermore, glycosylated Her2 peptide conjugate antisera were also shown to recognize full length Her2 protein by ELISA and at the cell surface through flow cytometry analysis. In contrast, unmasked peptide conjugates generated significant antibody populations that were specific to the terminal neo-epitope of the peptide immunogen that are notably absent in parental proteins. Antibodies generated in this manner to peptides in the dimerization loop of Her2 are also functional as demonstrated by the growth inhibition of Her2 expressing SKBR3 carcinoma cells. This method provides a technique to tailor-make epitope-specific antibodies that may facilitate vaccine, therapeutic and diagnostic antibody development.

Efficient chemoenzymatic synthesis of fluorinated sialyl Thomsen-Friedenreich antigens and investigation of their characteristics.

A set of fluorinated sialyl-T derivatives were efficiently synthesized using one-pot multi-enzyme (OPME) chemoenzymatic approach. The P. multocida α2-3-sialyltransferase (PmST1) involved in the synthesis showed extremely flexible donor and acceptor substrate specificities. These sialosides have been successfully investigated with stability towards Clostridium perfringens sialidase substrate specificity assay using 1H NMR spectroscopy. Hydrolysis studies monitored by 1H NMR clearly demonstrated that the fluorine substitution obviously reduced hydrolysis rate of Clostridium perfringens sialidase. To further investigate the fluorine influence, structure-dependent variation of sialoside-lectin binding was observed for MAL and different sialoside-immobilized surfaces. Subtle changes on the ligand of carbohydrate-binding protein were distinguished by SPR. These fluorinated sialyl-T derivatives obtained are valuable probes for further biological studies or antitumor drug design.

Identification of novel fish sialidase genes responsible for KDN-cleaving activity.

2-keto-3-deoxy-D-glycero-D-galacto-nononic acid (KDN) is a minor component of sialic acids detected in vertebrates, such as human cancer cells, rat liver, and fish tissues. Although the enzyme activity of KDN-cleaving sialidase (KDN-sialidase) has been detected in rainbow trout, the gene responsible for its expression has not been identified in vertebrates. We evaluated sialidases in human and various fish for their KDN-cleaving activity using an artificial substrate, methylumbelliferyl-KDN (MU-KDN). Four of the human sialidases tested (NEU1, NEU2, NEU3, and NEU4) did not hydrolyze MU-KDN. Although most fish Neu1s showed negligible KDN-sialidase activity, two Neu1b sialidases from Oreochromis niloticus and Astyanax mexicanus, a paralog of Neu1, exhibited a potent KDN-sialidase activity. Further, O. niloticus and Oryzias latipes Neu3a exhibited a drastically high KDN-sialidase activity, while Danio rerio Neu3.1 showed moderate activities and other Neu3 proteins exhibited little activity. All the Neu4 sialidases tested in fish cleaved KDN and Neu5Ac from MU-KDN and MU-Neu5Ac, respectively, with equivalent potential. To our knowledge, this is the first report to identify KDN-sialidase genes in vertebrates and we believe that KDN-sialidase activity could be conserved among fish Neu4s.

Targeted glycan degradation potentiates the anticancer immune response in vivo.

Currently approved immune checkpoint inhibitor therapies targeting the PD-1 and CTLA-4 receptor pathways are powerful treatment options for certain cancers; however, most patients across cancer types still fail to respond. Consequently, there is interest in discovering and blocking alternative pathways that mediate immune suppression. One such mechanism is an upregulation of sialoglycans in malignancy, which has been recently shown to inhibit immune cell activation through multiple mechanisms and therefore represents a targetable glycoimmune checkpoint. Since these glycans are not canonically druggable, we designed an αHER2 antibody-sialidase conjugate that potently and selectively strips diverse sialoglycans from breast cancer cells. In syngeneic breast cancer models, desialylation enhanced immune cell infiltration and activation and prolonged the survival of mice, an effect that was dependent on expression of the Siglec-E checkpoint receptor found on tumor-infiltrating myeloid cells. Thus, antibody-sialidase conjugates represent a promising modality for glycoimmune checkpoint therapy.

Characterization of a novel O-acetyl sialic acid specific lectin from the hemolymph of the marine crab, Atergatis integerrimus (Lamarck, 1818).

An O-acetyl sialic acid specific lectin was purified from the hemolymph of the marine crab Atergatis integerrimus by affinity chromatography using BSM (Bovine Submaxillary Mucin) coupled to cyanogen bromide activated Sepharose 4B and biospecific adsorption using formalinized buffalo erythrocytes. The purified AiL (Atergatis integerrimus lectin) showed an 1218 fold increase in specific activity when compared to the crude hemolymph agglutinin. The lectin, on non - denaturing PAGE showed a single band of 216 kDa and when subjected to SDS - PAGE, the lectin resolved into three subunits of molecular weight 70, 72 and 74 kDa. Physico chemical characterization revealed the lectin as pH and temperature sensitive, calcium dependent and sensitive to calcium chelators. Based on the calcium dependency of the lectin, AiL could be classified as a C-type lectin. The purified lectin agglutinated buffalo erythrocytes with greater avidity and was inhibited by the glycoproteins BSM, thyroglobulin, fetuin, PSM, and sugars raffinose, trehalose, l - fucose, α - Lactose, melibiose and GluNAc suggesting the affinity of the lectin to sialic acid. Reduction in HA with asialo buffalo erythrocytes and HAI titer with desialylated BSM, confirms the sialic acid specificity of the lectin. The reduction in HAI following de - O - acetylation confirms the specificity of the lectin for O - acetyl sialic acid. FTIR analysis confirms the purified lectin as a glycoprotein with spectral bands corresponding to amide bands and saccharides. Thus this study paves way to assess the therapeutic application of this lectin that could be targeted to modified sialic acid moieties that are expressed on the malignant cells and pathogenic microbes and also deduce the crystal structure of the lectin.

Production and characterization of a monoclonal antibody against the sialidase of Gardnerella vaginalis using a synthetic peptide in a MAP8 format.

Bacterial vaginosis is one of the most frequent vaginal infections. Its main etiological agent is Gardnerella vaginalis, which produces several virulence factors involved in vaginal infection and colonization, in particular, sialidase (SLD), a potential clinical biomarker that participates in immune response modulation and mucus degradation. The main objective of this work was the production and evaluation of a monoclonal antibody against G. vaginalis sialidase and its validation in immunoassays. For immunization of mice, a synthetic multiantigenic peptide was used, and hybridomas were generated. After fusion, hybridomas were evaluated for antibody production and cloned by limited dilution. One clone producing IgG1 was selected and characterized by indirect ELISA, dot blot, and Western blot, and we also tested clinical isolates and HeLa cells infected with G. vaginalis. The results showed that the anti-SLD antibody recognized a single protein of ~90 kDa that correlated with the estimated molecular weight of SLD. In addition, anti-SLD antibody recognized SLD from complete bacteria and from culture supernatants of infected Hela cells. In conclusion, our results showed that the anti-SLD antibody recognized SLD from different sources and could be considered a new tool for the diagnosis of bacterial vaginosis. KEY POINTS: • Anti-sialidase mAb was generated using a synthetic peptide • The mAb recognizes synthetic peptide and intact protein from multiple sources • The antibody was characterized by several immunological methods.

Glyco-Modification of Mucin Hydrogels to Investigate Their Immune Activity.

Mucins are multifunctional glycosylated proteins that are increasingly investigated as building blocks of novel biomaterials. An attractive feature is their ability to modulate the immune response, in part by engaging with sialic acid binding receptors on immune cells. Once assembled into hydrogels, bovine submaxillary mucins (Muc gels) were shown to modulate the recruitment and activation of immune cells and avoid fibrous encapsulation in vivo. However, nothing is known about the early immune response to Muc gels. This study characterizes the response of macrophages, important orchestrators of the material-mediated immune response, over the first 7 days in contact with Muc gels. The role of mucin-bound sialic acid sugar residues was investigated by first enzymatically cleaving the sugar and then assembling the mucin variants into covalently cross-linked hydrogels with rheological and surface nanomechanical properties similar to nonmodified Muc gels. Results with THP-1 and human primary peripheral blood monocytes derived macrophages showed that Muc gels transiently activate the expression of both pro-inflammatory and anti-inflammatory cytokines and cell surface markers, for most makers with a maximum on the first day and loss of the effect after 7 days. The activation was sialic acid-dependent for a majority of the markers followed. The pattern of gene expression, protein expression, and functional measurements did not strictly correspond to M1 or M2 macrophage phenotypes. This study highlights the complex early events in macrophage activation in contact with mucin materials and the importance of sialic acid residues in such a response. The enzymatic glyco-modulation of Muc gels appears as a useful tool to help understand the biological functions of specific glycans on mucins which can further inform on their use in various biomedical applications.

Improvement in predicting drug sensitivity changes associated with protein mutations using a molecular dynamics based alchemical mutation method.

While molecular-targeted drugs have demonstrated strong therapeutic efficacy against diverse diseases such as cancer and infection, the appearance of drug resistance associated with genetic variations in individual patients or pathogens has severely limited their clinical efficacy. Therefore, precision medicine approaches based on the personal genomic background provide promising strategies to enhance the effectiveness of molecular-targeted therapies. However, identifying drug resistance mutations in individuals by combining DNA sequencing and in vitro analyses is generally time consuming and costly. In contrast, in silico computation of protein-drug binding free energies allows for the rapid prediction of drug sensitivity changes associated with specific genetic mutations. Although conventional alchemical free energy computation methods have been used to quantify mutation-induced drug sensitivity changes in some protein targets, these methods are often adversely affected by free energy convergence. In this paper, we demonstrate significant improvements in prediction performance and free energy convergence by employing an alchemical mutation protocol, MutationFEP, which directly estimates binding free energy differences associated with protein mutations in three types of a protein and drug system. The superior performance of MutationFEP appears to be attributable to its more-moderate perturbation scheme. Therefore, this study provides a deeper level of insight into computer-assisted precision medicine.

Diversity of sialidases found in the human body - A review.

Sialidases are enzymes essential for numerous organisms including humans. Hydrolytic sialidases (EC 3.2.1.18), trans-sialidases and anhydrosialidases (intramolecular trans-sialidases, EC 4.2.2.15) are glycoside hydrolase enzymes that cleave the glycosidic linkage and release sialic acid residues from sialyl substrates. The paper summarizes diverse sialidases present in the human body and their potential impact on development of antiviral compounds - inhibitors of viral neuraminidases. It includes a brief overview of catalytic mechanisms of action of sialidases and describes the origin of sialidases in the human body. This is followed by description of the structure and function of sialidase families with a special focus on the GH33 and GH34 families. Various effects of sialidases on human body are also briefly described. Modulation of sialidase activity may be considered a useful tool for effective treatment of various diseases. In some cases, it is desired to completely suppress the activity of sialidases by suitable inhibitors. Specific sialidase inhibitors are useful for the treatment of influenza, epilepsy, Alzheimer's disease, diabetes, different types of cancer, or heart defects. Challenges and future directions are shortly depicted in the final part of the paper.

Mutations in the Neuraminidase-Like Protein of Bat Influenza H18N11 Virus Enhance Virus Replication in Mammalian Cells, Mice, and Ferrets.

To characterize bat influenza H18N11 virus, we propagated a reverse genetics-generated H18N11 virus in Madin-Darby canine kidney subclone II cells and detected two mammal-adapting mutations in the neuraminidase (NA)-like protein (NA-F144C and NA-T342A, N2 numbering) that increased the virus titers in three mammalian cell lines (i.e., Madin-Darby canine kidney, Madin-Darby canine kidney subclone II, and human lung adenocarcinoma [Calu-3] cells). In mice, wild-type H18N11 virus replicated only in the lungs of the infected animals, whereas the NA-T342A and NA-F144C/T342A mutant viruses were detected in the nasal turbinates, in addition to the lungs. Bat influenza viruses have not been tested for their virulence or organ tropism in ferrets. We detected wild-type and single mutant viruses each possessing NA-F144C or NA-T342A in the nasal turbinates of one or several infected ferrets, respectively. A mutant virus possessing both the NA-F144C and NA-T342A mutations was isolated from both the lung and the trachea, suggesting that it has a broader organ tropism than the wild-type virus. However, none of the H18N11 viruses caused symptoms in mice or ferrets. The NA-F144C/T342A double mutation did not substantially affect virion morphology or the release of virions from cells. Collectively, our data demonstrate that the propagation of bat influenza H18N11 virus in mammalian cells can result in mammal-adapting mutations that may increase the replicative ability and/or organ tropism of the virus; overall, however, these viruses did not replicate to high titers throughout the respiratory tract of mice and ferrets.IMPORTANCE Bats are reservoirs for several severe zoonotic pathogens. The genomes of influenza A viruses of the H17N10 and H18N11 subtypes have been identified in bats, but no live virus has been isolated. The characterization of artificially generated bat influenza H18N11 virus in mammalian cell lines and animal models revealed that this virus can acquire mammal-adapting mutations that may increase its zoonotic potential; however, the wild-type and mutant viruses did not replicate to high titers in all infected animals.

Presence of N-acetylgalactosamine/galactose residues on bronchioloalveolar cells during rat postnatal development.

In mammals, the alveolarization process develops predominantly after birth. Airway cells display a complex assemblage of glycans on their surface. These glycans, particularly terminal glycan extensions, are important effective carriers of information that change during the differentiation process. Nevertheless, few systematic data are reported about the cell surface sugar residue content during post-natal lung development. In the present work, we aimed to identify and semi-quantify N-acetylgalactosamine (GalNAc)/galactose (Gal) residues on the bronchioloalveolar cell surface in rat lung sections from 1-, 4-, 8- day old and adult animals and link these data with the lung glycocalyx composition. Horseradish peroxidase-conjugated lectin from Glycine max (soybean agglutinin, SBA) was used, and light microscopy methodologies were performed. SBA labelling intensity was studied before and after sialidase pre-treatment, at one-, four- and eight-day-old animals and adult animals. For semi-quantitative evaluation of SBA binding intensity, two investigators performed the analysis independently, blinded to the type of experiment. Reactivity of the lectin was assessed in bronchiolar and respiratory portion/alveolar epithelial cell surfaces. We evidenced a stronger positive reaction when lung sections were pre-treated with neuraminidase before incubation with the lectin in one- and four-day-old animals and adult animals. These results were not so manifest in eight-day-old animals. This binding pattern, generally points towards the presence of terminal but mainly sub-terminal GalNAc/Gal residues probably capped by sialic acids on the rat bronchiolar/respiratory tract epithelial cells. As this glycan extension is common in O- and N-glycans, our results suggest that these glycan classes can be present in bronchioloalveolar cells immediately after birth and exist during the postnatal period. The results observed in eight-day-old rat lung sections may be due to the dramatic lung morphologic changes and the possible underlying biological mechanisms that occur during this age-moment.