Hemagglutinin Activity Research Articles

Purification of avian influenza virus (H9N2) from allantoic fluidby size-exclusion chromatography

Avian influenza viruses represent a rising threat of pandemic influenza. Allantoic fluid including influenza virus H9N2 was run on downstream processing to purify the virus. To do this, allantoic fluid was clarified and concentrated. It was applied to size exclusion chromatography. Different parameters such as ovalbumin, protein content, and hemagglutinin activity were determined to confirm the purity. The results may suggest that this method is quite efficient for achieving a purified H9N2 influenza virus and further reveal that size-exclusion chromatography is a practical method for purifying avian influenza viruses. In conclusion, this procedure can be used in vaccine production and antiserum preparation as an alternative to traditional methods and also can be considered in the purifying of other viruses when there is no facility of ultracentrifuge or zonal centrifuge.

Factors affecting the immunogenicity of the live attenuated influenza vaccine produced in continuous cell line

ABSTRACT The biological basis for the restricted immunogenicity of some live attenuated influenza vaccine strains generated on the backbone of the cold adapted (ca) A/Singapore/1/1957/ca (H2N2) influenza A virus master strain and produced in the Vero cells was investigated. According to our previous results the vaccine candidate made from A/Hong Kong/1035/1998 (H1N1) Vero-derived virus did not provoke a measurable antibody titers following the intranasal immunization of humans. We report here that the hemagglutinin (HA) of A/Hong Kong/1035/1998 virus contained the mutation 10Ile→Val in the HA2 subunit, that increased the pH threshold of HA conformational change (pH of activation) by 0.3 pH units and therefore might be responsible for the lack of immune response in humans. Similar effect was shown for the reassortant made from the Vero-derived A/Switzerland/5389/1995 (H1N1) (5389wt) virus which had the HA2 mutation 3Phe→Leu leading to the lack of immune response in mice. Another factor compromising the immunogenicity of a vaccine candidate is the incompatibility of epidemic virus HA with the M gene of the master strain. In mice the 6/2 A/Switzerland/5389/1995 reassortant induced antibodies that were directed predominantly to the HA2 subunit and were detectable by ELISA but not by a hemagglutination inhibition (HAI) test. In contrast, the 5/3 reassortant, bearing the HA, neuraminidase (NA), and M genes from the epidemic virus induced an equivalent amount of antibodies against the HA1 and HA2 subunits detected by HAI and ELISA. By comparing the sensitivity of the viruses to amantadine, we showed that the M2 ion channel of the master strain had lower activity than that of the A/Switzerland/5389/1995. These data suggest that M2 of the master strain was not sufficiently active to keep the pH of the trans- Golgi network high enough to prevent the conformational change of the acid sensitive HA to the low pH form. Overall, the adaptation mutations in the HA of the vaccine candidate that increase the pH of HA activation as well as the incompatibility of HA and M genes must be taken into consideration when constructing the reassortant strains for the live attenuated vaccine.

Changes in the phenotypic properties of highly pathogenic influenza A virus of H5N1 subtype induced by N186I and N186T point mutations in hemagglutinin

The change in the phenotypic properties resulting from amino acid substitutions in the hemagglutinin (HA) molecule is an important link in the evolutionary process of influenza viruses. It is believed to be one of the mechanisms of the emergence of highly pathogenic strains of influenza A viruses, including subtype H5N1. Using the site-directed mutagenesis, we introduced mutations in the HA gene of the H5N1 subtype of influenza A virus. The obtained virus variants were analyzed and compared using the following parameters: optimal pH of conformational transition (according to the results of the hemolysis test), specificity of receptor binding (using a set of synthetic analogues of cell surface sialooligosaccharides), thermoresistance (heat-dependent reduction of hemagglutinin activity), virulence in mice, and the kinetics of replication in chicken embryos, and reproductive activity at different temperatures (RCT-based). N186I and N186T mutations in the HA protein increased the virulence of the original virus in mice. These mutations accelerated virus replication in the early stages of infection in chicken embryos and increased the level of replication at late stages. In addition, compared to the original virus, the mutant variants replicated more efficiently at lower temperatures. The obtained data clearly prove the effect of amino acid substitutions at the 186 position of HA on phenotypic properties of the H5N1 subtype of influenza A.

A Unique Multibasic Proteolytic Cleavage Site and Three Mutations in the HA2 Domain Confer High Virulence of H7N1 Avian Influenza Virus in Chickens.

In 1999, after circulation for a few months in poultry in Italy, low-pathogenic (LP) avian influenza (AI) H7N1 virus mutated into a highly pathogenic (HP) form by acquisition of a unique multibasic cleavage site (mCS), PEIPKGSRVRR*GLF (asterisk indicates the cleavage site), in the hemagglutinin (HA) and additional alterations with hitherto unknown biological function. To elucidate these virulence-determining alterations, recombinant H7N1 viruses carrying specific mutations in the HA of LPAI A/chicken/Italy/473/1999 virus (Lp) and HPAI A/chicken/Italy/445/1999 virus (Hp) were generated. Hp with a monobasic CS or carrying the HA of Lp induced only mild or no disease in chickens, thus resembling Lp. Conversely, Lp with the HA of Hp was as virulent and transmissible as Hp. While Lp with a multibasic cleavage site (Lp_CS445) was less virulent than Hp, full virulence was exhibited when HA2 was replaced by that of Hp. In HA2, three amino acid differences consistently detected between LP and HP H7N1 viruses were successively introduced into Lp_CS445. Q450L in the HA2 stem domain increased virulence and transmission but was detrimental to replication in cell culture, probably due to low-pH activation of HA. A436T and/or K536R restored viral replication in vitro and in vivo. Viruses possessing A436T and K536R were observed early in the HPAI outbreak but were later superseded by viruses carrying all three mutations. Together, besides the mCS, stepwise mutations in HA2 increased the fitness of the Italian H7N1 virus in vivo. The shift toward higher virulence in the field was most likely gradual with rapid optimization. In 1999, after 9 months of circulation of low-pathogenic (LP) avian influenza virus (AIV), a devastating highly pathogenic (HP) H7N1 AIV emerged in poultry, marking the largest epidemic of AIV reported in a Western country. The HPAIV possessed a unique multibasic cleavage site (mCS) complying with the minimum motif for HPAIV. The main finding in this report is the identification of three mutations in the HA2 domain that are required for replication and stability, as well as for virulence, transmission, and tropism of H7N1 in chickens. In addition to the mCS, Q450L was required for full virulence and transmissibility of the virus. Nonetheless, it was detrimental to virus replication and required A436T and/or K536R to restore replication, systemic spread, and stability. These results are important for better understanding of the evolution of highly pathogenic avian influenza viruses from low-pathogenic precursors.

Biochemical and Immunological Characterization of Truncated Fragments of the Receptor-Binding Domains of C. difficile Toxin A.

Clostridium difficile is an emerging pathogen responsible for opportunistic infections in hospitals worldwide and is the main cause of antibiotic-associated pseudo-membranous colitis and diarrhea in humans. Clostridial toxins A and B (TcdA and TcdB) specifically bind to unknown glycoprotein(s) on the surface of epithelial cells in the host intestine, disrupting the intestinal barrier and ultimately leading to acute inflammation and diarrhea. The C-terminal receptor-binding domain (RBD) of TcdA, which is responsible for the initial binding of the toxin to host glycoproteins, has been predicted to contain 7 potential oligosaccharide-binding sites. To study the specific roles and functions of these 7 putative lectin-like binding regions, a consensus sequence of TcdA RBD derived from different C. difficile strains deposited in the NCBI protein database and three truncated fragments corresponding to the N-terminal (residues 1–411), middle (residues 296–701), and C-terminal portions (residues 524–911) of the RBD (F1, F2 and F3, respectively) were designed and expressed in Escherichia coli. In this study, the recombinant RBD (rRBD) and its truncated fragments were purified, characterized biologically and found to have the following similar properties: (a) are capable of binding to the cell surface of both Vero and Caco-2 cells; (b) possess Toll-like receptor agonist-like adjuvant activities that can activate dendritic cell maturation and increase the secretion of pro-inflammatory cytokines; and (c) function as potent adjuvants in the intramuscular immunization route to enhance immune responses against weak immunogens. Although F1, F2 and F3 have similar repetitive amino acid sequences and putative oligosaccharide-binding domains, they do not possess the same biological and immunological properties: (i) TcdA rRBD and its fragments bind to the cell surface, but only TcdA rRBD and F3 internalize into Vero cells within 15 min; (ii) the fragments exhibit various levels of hemagglutinin (HA) activity, with the exception of the F1 fragment, which demonstrates no HA activity; and (iii) in the presence of alum, all fragments elicit various levels of anti-toxin A-neutralizing antibody responses, but those neutralizing antibodies elicited by F2 did not protect mice against a TcdA challenge. Because TcdA rRBD, F1 and F3 formulated with alum can elicit immune protective responses against the cytotoxicity of TcdA, they represent potential components of future candidate vaccines against C. difficile-associated diseases.

Influenza virus activating host proteases: Identification, localization and inhibitors as potential therapeutics

Cellular proteases are reponsible for activation of influenza virus hemagglutinin (HA) in epithelial tissues of the respiratory tract. The trans-Golgi network (TGN) is the main subcellular compartment where HA cleavage occurs during its biosynthesis. The proteolytic HA cleavage is an indispensable prerequisite for the fusion of viral with endosomal membrane and the delivery of the virus genome into the cell. Both, the structure and accessibility of the HA cleavage site determine the responsible host protease(s) for cutting. Most influenza virus strains contain a HA sequence with a single arginine at the cleavage site suitable for processing by the trypsin-like serine proteases human airway trypsin-like protease (HAT) and transmembrane protease serine 2 (TMPRSS2), albeit a minority of viruses possesses HA cleavage site motifs that are processed by other proteases. TMPRSS2-deficient mice demonstrated the relevance of TMPRSS2 for pneumotropism and pathogenicity of H1N1 and H7N9 virus infections. In contrast, H3N2 virus infections are promoted by an additional not yet identified protease. Highly pathogenic avian H5 and H7 viruses are characterized by an enlarged cleavage site loop containing a multibasic amino acid motif, where the eukaryotic subtilases furin or PC5/6 cleave. Their ubiquitous presence in the organism allows a systemic virus infection. Peptidomimetic inhibitors derived from the HA cleavage site inhibit the HA-activating proteases and thus virus propagation.

Critical assessment of influenza VLP production in Sf9 and HEK293 expression systems.

BackgroundEach year, influenza is responsible for hundreds of thousand cases of illness and deaths worldwide. Due to the virus’ fast mutation rate, the World Health Organization (WHO) is constantly on alert to rapidly respond to emerging pandemic strains. Although anti-viral therapies exist, the most proficient way to stop the spread of disease is through vaccination. The majority of influenza vaccines on the market are produced in embryonic hen’s eggs and are composed of purified viral antigens from inactivated whole virus. This manufacturing system, however, is limited in its production capacity. Cell culture produced vaccines have been proposed for their potential to overcome the problems associated with egg-based production. Virus-like particles (VLPs) of influenza virus are promising candidate vaccines under consideration by both academic and industry researchers.MethodsIn this study, VLPs were produced in HEK293 suspension cells using the Bacmam transduction system and Sf9 cells using the baculovirus infection system. The proposed systems were assessed for their ability to produce influenza VLPs composed of Hemagglutinin (HA), Neuraminidase (NA) and Matrix Protein (M1) and compared through the lens of bioprocessing by highlighting baseline production yields and bioactivity. VLPs from both systems were characterized using available influenza quantification techniques, such as single radial immunodiffusion assay (SRID), HA assay, western blot and negative staining transmission electron microscopy (NSTEM) to quantify total particles.ResultsFor the HEK293 production system, VLPs were found to be associated with the cell pellet in addition to those released in the supernatant. Sf9 cells produced 35 times more VLPs than HEK293 cells. Sf9-VLPs had higher total HA activity and were generally more homogeneous in morphology and size. However, Sf9 VLP samples contained 20 times more baculovirus than VLPs, whereas 293 VLPs were produced along with vesicles.ConclusionsThis study highlights key production hurdles that must be overcome in both expression platforms, namely the presence of contaminants and the ensuing quantification challenges, and brings up the question of what truly constitutes an influenza VLP candidate vaccine.Electronic supplementary materialThe online version of this article (doi:10.1186/s12896-015-0152-x) contains supplementary material, which is available to authorized users.

Purification and Characterization of a Novel Hemagglutinin with Inhibitory Activity toward Osteocarcinoma Cells from Northeast China Black Beans.

In the present study, we isolated a novel hemagglutinin from an edible legume and explored its growth-inhibitory effect on osteocarcinoma and liver cancer cells. The protein was purified by liquid chromatography techniques which entailed affinity chromatography on Affi-gel blue gel, ion-exchange chromatography on Mono Q, and gel filtration on Superdex 75 with an FPLC system. The hemagglutinating activity of this hemagglutinin was demonstrated to be ion dependent and stable over a wide range of temperature and pH values. Antiproliferative activity was observed in the tumor cell lines MG-63 and HepG2 but not in the normal cell line WRL 68. Osteocarcinoma cells treated with the hemagglutinin underwent obvious cell shrinkage, chromatin condensation, mitochondrial membrane depolarization, and apoptosis. The mRNA expression level of interleukin-2 (IL-2), interleukin-6 (IL-6), interleukin-1 beta (IL-1β), interferon-gamma (IFN-γ), and tumor necrosis factor alpha (TNF-α) were found to be up-regulated to different extents after treatment of this hemagglutinin.

Pandemic Swine H1N1 Influenza Viruses with Almost Undetectable Neuraminidase Activity Are Not Transmitted via Aerosols in Ferrets and Are Inhibited by Human Mucus but Not Swine Mucus.

A balance between the functions of the influenza virus surface proteins hemagglutinin (HA) and neuraminidase (NA) is thought to be important for the transmission of viruses between humans. Here we describe two pandemic H1N1 viruses, A/swine/Virginia/1814-1/2012 and A/swine/Virginia/1814-2/2012 (pH1N1low-1 and -2, respectively), that were isolated from swine symptomatic for influenza. The enzymatic activity of the NA of these viruses was almost undetectable, while the HA binding affinity for α2,6 sialic acids was greater than that of the highly homologous pH1N1 viruses A/swine/Pennsylvania/2436/2012 and A/swine/Minnesota/2499/2012 (pH1N1-1 and -2), which exhibited better-balanced HA and NA activities. The in vitro growth kinetics of pH1N1low and pH1N1 viruses were similar, but aerosol transmission of pH1N1low-1 was abrogated and transmission via direct contact in ferrets was significantly impaired compared to pH1N1-1, which transmitted by direct and aerosol contact. In normal human bronchial epithelial cells, pH1N1low-1 was significantly inhibited by mucus but pH1N1-1 was not. In Madin-Darby canine kidney cell cultures overlaid with human or swine mucus, human mucus inhibited pH1N1low-1 but swine mucus did not. These data show that the interaction between viruses and mucus may be an important factor in viral transmissibility and could be a barrier for interspecies transmission between humans and swine for influenza viruses. A balance between the functions of the influenza virus surface proteins hemagglutinin (HA) and neuraminidase (NA) is thought to be important for transmission of viruses from swine to humans. Here we show that a swine virus with extremely functionally mismatched HA and NAs (pH1N1low-1) cannot transmit via aerosol in ferrets, while another highly homologous virus with HA and NAs that are better matched functionally (pH1N1-1) can transmit via aerosol. These viruses show similar growth kinetics in Madin-Darby canine kidney (MDCK) cells, but pH1N1low-1 is significantly inhibited by mucus in normal human bronchial epithelial cells whereas pH1N1-1 is not. Further, human mucus could inhibit these viruses, but swine mucus could not. These data show that the interaction between viruses and mucus may be an important factor in viral transmissibility and could be a species barrier between humans and swine for influenza viruses.

Inhibitors targeting the influenza virus hemagglutinin.

The annual flu season causes thousands of deaths and millions of hospitalizations, which pose a great burden to global health and economy. Moreover, a flu pandemic arising from reassortment viruses, such as H5N1 and H1N1, raises even greater concern due to the lack of effective vaccines at the initial stage of flu outbreak. The influenza virus is the causative agent of flu infection. Currently there are four drugs in use to combat influenza infection. Amantadine and rimantadine are M2 proton channel blockers that inhibit virus uncoating; oseltamivir and zanamivir are neuraminidase (NA) inhibitors that inhibit virus release. However, recent years have witnessed a drastic increase in instances of drug resistance, and flu strains that are resistant to both classes of drugs have been reported. Thus, there is a pressing need to develop the next generation of anti-influenza drugs. Among a handful of anti-influenza drug targets, the viral fusion protein hemagglutinin (HA) is one of the most advanced. This review discusses the biological roles of HA during viral replication and highlights peptide- and small molecule-based HA inhibitors, including recent computationally designed HA binders. The text is organized into four sections based on the maturation stages of HA: inhibitors targeting the glycosylation of HA, the proteolytic activation of HA, the attachment of HA to host cell receptors, and peptide- and small molecule-based inhibitors targeting HA-mediated membrane fusion. Of particular interest are advances in the areas of developing dual inhibitors targeting both HA and NA and broad-spectrum HA inhibitors targeting both groups of HAs.

Dynamic Changes during Acid-Induced Activation of Influenza Hemagglutinin

Influenza hemagglutinin (HA) mediates virus attachment to host cells and fusion of the viral and endosomal membranes during entry. While high-resolution structures are available for the pre-fusion HA ectodomain and the post-fusion HA2 subunit, the sequence of conformational changes during HA activation has eluded structural characterization. Here, we apply hydrogen-deuterium exchange with mass spectrometry to examine changes in structural dynamics of the HA ectodomain at various stages of activation, and compare the soluble ectodomain with intact HA onvirions. At pH conditions approaching activation (pH 6.0-5.5) HA exhibits increased dynamics at the fusion peptide and neighboring regions, while the interface between receptor binding subunits (HA1) becomes stabilized. In contrast to many activation models, these data suggest that HA responds to endosomal acidification by releasing the fusion peptide prior to HA1 uncaging and the spring-loaded refolding of HA2. This staged process may facilitate efficient HA-mediated fusion.

Biophysical Measurement of the Balance of Influenza A Hemagglutinin and Neuraminidase Activities

The interaction of influenza A viruses with the cell surface is controlled by the surface glycoproteins hemagglutinin (HA) and neuraminidase (NA). These two glycoproteins have opposing activities: HA is responsible for binding the host receptor (sialic acid) to allow infection, and NA is responsible for cleaving the receptor to facilitate virus release. Several studies have demonstrated that compatible levels of HA and NA activity are required for a virus to replicate efficiently. This is consequently of great interest for determining virus transmissibility. The concurrent role of these two proteins in receptor binding has never been directly measured. We demonstrate a novel biophysical approach based on bio-layer interferometry to measure the balance of the activities of these two proteins in real time. This technique measures virus binding to and release from a surface coated with either the human-like receptor analog α2,6-linked sialic acid or the avian-like receptor analog α2,3-linked sialic acid in both the presence and absence of NA inhibitors. Bio-layer interferometry measurements were also carried out to determine the effect of altering HA receptor affinity and NA stalk length on receptor binding.

A mutant H3N2 influenza virus uses an alternative activation mechanism in TMPRSS2 knockout mice by loss of an oligosaccharide in the hemagglutinin stalk region.

The host protease TMPRSS2 plays an essential role in proteolytic activation of the influenza A virus (IAV) hemagglutinin (HA) protein possessing a monobasic cleavage site. However, after passages in TMPRSS2 knockout mice, an H3N2 subtype IAV began to undergo cleavage activation of HA, showing high virulence in the mice due to the loss of an oligosaccharide at position 8 in the HA stalk region. Thus, the H3N2 IAV acquired cleavability by an alternative HA activation mechanism/protease(s).

Improvement of Bioactive Potential of Canavalia Beans of Coastal Sand Dunes by Solid-Substrate Fermentation Using Rhizopus oligosporus

Cooked beans of two wild legumes (Canavalia cathartica and C. maritima) were subjected to solid-substrate fermentation by Rhizopus oligosporus and bioactive potential has been compared with raw and cooked beans. Flavonoids and canavanine in raw beans were significantly decreased on cooking, while they were significantly enhanced in fermented beans. Phytic acid content was signficantlty decreased from raw vs. cooked vs. fermented beans. Raw, cooked and fermented beans were devoid of trypsin inhibitors. Hemagglutinin activity of raw beans was completely eliminated on cooking, while SSF increased its activity about 50% of raw beans against A+ve and or B+ve blood groups. Solid-substrate fermentation of cooked beans with R. oligosporus resulted in better nutraceutical potential compared to raw and mearly cooked beans qualifying as important nutraceutical source to develop indigenous value-added products.

English

Cereals are the staple diet of most of the world’s population. The millets are very important staple food in the rural parts of India. Millets can secure India’s food and farming in future because it is amazing in their nutrition contents. Echinochloa frumentacea (Sanwa) millet is good source of energy and provide protein, fatty acid, minerals, vitamins, dietary fibre and polypheonals. Proteins present in various foods differ in their nutritive value on account of the difference in the amino acid contents. The amino acid content, fatty acid content (TSFA and TUFA) and anti nutritional factor ranged from 0.0008 to 0.522%, 24.2 to 26.0%, 73.5 to 75.4% and 0.301 to 0.302, 0.0202 to 0.0204 g/100 g and 31.95 mg/100 g respectively. No cyanide content and haemagglutinin activity were found. Nutritionally the seeds of E. frumentacea variety DFM-1 and HR-374 are rich in aspartic acid (essential amino acid) content and total unsaturated fatty acid content. Key words: Amino acid, fatty acid content, anti nutritional factor, minor millets variety of Echinochloa frumentacea.

Proteinquakes in the evolution of influenza virus hemagglutinin (A/H1N1) under opposing migration and vaccination pressures.

Influenza virus contains two highly variable envelope glycoproteins, hemagglutinin (HA) and neuraminidase (NA). Here we show that, while HA evolution is much more complex than NA evolution, it still shows abrupt punctuation changes linked to punctuation changes of NA. HA exhibits proteinquakes, which resemble earthquakes and are related to hydropathic shifting of sialic acid binding regions. HA proteinquakes based on shifting sialic acid interactions are required for optimal balance between the receptor-binding and receptor-destroying activities of HA and NA for efficient virus replication. Our comprehensive results present a historical (1945–2011) panorama of HA evolution over thousands of strains and are consistent with many studies of HA and NA interactions based on a few mutations of a few strains.

Biochemical and Immunological Characterizations of the Receptor Binding Domain of C. difficile Toxin B

Clostridium difficile (Cd) is an emerging nosocomial pathogen responsible for antibiotic-associated pseudo-membranous colitis and diarrhea in hospital acquired infections. Clostridial toxins A (TcdA) and B (TcdB) which specifically bind to unknown glycoprotein(s) on the surface of epithelial cells disrupt the intestinal barrier and ultimately lead to acute inflammation and diarrhea . There is still debate as to whether the receptor binding domains (RBD) of toxins can individually elicit protection in the hamster challenge model. In this study, a TcdB RBD which was derived from C.difficile strain VPI10463 with >95% amino acid sequence identity to hyper-virulent strain BI/NAP1/027 was designed and expressed in Escherichia coli. Recombinant RBD (rRBD) was purified, characterized biologically and immunologically and found to have the following properties: (a) capable of binding to the cell surface of both Vero and Caco-2 cells and entering into the cytosol; (b) devoid of hemagglutinin activity (HA); (c) the ability to up-regulate cell surface markers expressions and cytokines secretions from dendritic cells; (d) eliciting anti-TcdB neutralizing antibody responses that could weakly cross-neutralize TcdA in the absence of adjuvant; (e) and inducing weak protection against a lethal dose of Cd spores in the hamster challenge model. Therefore, rRBD shows potential as an immunogen to be included in the development of vaccines against Clostridium difficile-associated diseases.

Expression of Factor X in BHK-21 Cells Promotes Low Pathogenic Influenza Viruses Replication.

A cDNA clone for factor 10 (FX) isolated from chicken embryo inserted into the mammalian cell expression vector pCDNA3.1 was transfected into the baby hamster kidney (BHK-21) cell line. The generated BHK-21 cells with inducible expression of FX were used to investigate the efficacy of the serine transmembrane protease to proteolytic activation of influenza virus hemagglutinin (HA) with monobasic cleavage site. Data showed that the BHK-21/FX stably expressed FX after ten serial passages. The cells could proteolytically cleave the HA of low pathogenic avian influenza virus at multiplicity of infection 0.01. Growth kinetics of the virus on BHK-21/FX, BHK-21, and MDCK cells were evaluated by titrations of virus particles in each culture supernatant. Efficient multicycle viral replication was markedly detected in the cell at subsequent passages. Virus titration demonstrated that BHK-21/FX cell supported high-titer growth of the virus in which the viral titer is comparable to the virus grown in BHK-21 or MDCK cells with TPCK-trypsin. The results indicate potential application for the BHK-21/FX in influenza virus replication procedure and related studies.

The haemagglutinin of Avibacterium paragallinarum is a trimeric autotransporter adhesin that confers haemagglutination, cell adherence and biofilm formation activities

The haemagglutinin (HA) protein plays a key role in the immunogenicity and pathogenicity of Avibacterium paragallinarum. A 210-kDa protein (HMTp210) was previously reported to be the HA of Av. paragallinarum, but the biological function of HMTp210 is not well defined. In this study, mutant strains that lacked HMTp210 were constructed using the TargeTron® gene knockout system. Haemagglutination and haemagglutination-inhibition (HI) assays showed that the HMTp210-deficient mutants exhibited no HA activity and failed to elicit HI antibodies in immunized chickens. Additionally, HMTp210-deficient mutants exhibited reduced ability to adhere to HeLa cells and to form biofilms on abiotic surfaces. Virulence assays showed that HMTp210-deficient mutants are less virulent than their isogenic wild-type strains. HMTp210 bears significant similarity to proteins of the trimeric autotransporter adhesin (TAA) family, and recombinant HMTp210 expressed in E. coli formed a trimeric structure. Taken together, these results indicated that HMTp210 is a trimeric autotransporter adhesin that confers haemagglutination, cell adherence and biofilm formation activities. These results should prove valuable to further elucidate the biological function of HA and the mechanism of pathogenicity of Av. paragallinarum.

Functional dissection of the Clostridium botulinum type B hemagglutinin complex: identification of the carbohydrate and E-cadherin binding sites.

Botulinum neurotoxin (BoNT) inhibits neurotransmitter release in motor nerve endings, causing botulism, a condition often resulting from ingestion of the toxin or toxin-producing bacteria. BoNTs are always produced as large protein complexes by associating with a non-toxic protein, non-toxic non-hemagglutinin (NTNH), and some toxin complexes contain another non-toxic protein, hemagglutinin (HA), in addition to NTNH. These accessory proteins are known to increase the oral toxicity of the toxin dramatically. NTNH has a protective role against the harsh conditions in the digestive tract, while HA is considered to facilitate intestinal absorption of the toxin by intestinal binding and disruption of the epithelial barrier. Two specific activities of HA, carbohydrate and E-cadherin binding, appear to be involved in these processes; however, the exact roles of these activities in the pathogenesis of botulism remain unclear. The toxin is conventionally divided into seven serotypes, designated A through G. In this study, we identified the amino acid residues critical for carbohydrate and E-cadherin binding in serotype B HA. We constructed mutants defective in each of these two activities and examined the relationship of these activities using an in vitro intestinal cell culture model. Our results show that the carbohydrate and E-cadherin binding activities are functionally and structurally independent. Carbohydrate binding potentiates the epithelial barrier-disrupting activity by enhancing cell surface binding, while E-cadherin binding is essential for the barrier disruption.