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PubMedDate not supplied

[Antigenic specificity of neuraminidase and structure of neuraminidase gene of influenza A(H1N1) serotype Hsw1N1 viruses isolated from man and animals].

The enzyme immunoassay and neuraminidase activity inhibition test using polyclonal and monospecific antineuraminidase sera were employed to establish the similarities and differences in the antigenic structure of neuraminidase of influenza A viruses (H1N1), serovariant Hsw1N1, isolated from man in Alma-Ata (USSR), 1984, New Jersey (USA), 1976, and Pazardjik (BPR), 1982, as well as from swine and birds. Oligonucleotide mapping revealed significant structural differences in the genes coding for neuraminidase of Hsw1N1 viruses. The experimental results indicate a high degree of the enzyme variability in this group of viruses.

Animals↗
PubMed1979

Adaptation of the influenza neuraminidase and neuraminidase-inhibition assays to the microtitrator system.

A micro-method based on Warren's colour reaction has been developed for influenza neuraminidase and neuraminidase-inhibition assays. Extraction with butanol is omitted and the assay is performed in wells of special plexiglass trays, where the reaction mixon and compared to controls in the same tray. The micro-assay, being inexpensive, time-saving and easy to perform even in poorly equipped laboratories, is suitable for large-scale serological screening and identification of the neuraminidase antigen of isolates.

Antibodies, Viral↗
PubMed2006

Amino acid change 335 E to K affects the sialic-acid-binding and neuraminidase activities of Urabe AM9 mumps virus hemagglutinin-neuraminidase glycoprotein.

A mutation coding for the amino acid change E335 to K is frequently found in the hemagglutinin-neuraminidase (HN) gene of Urabe AM9 mumps viruses isolated during post-vaccination meningitis cases. To identify if this mutation modifies the biological activities of the HN glycoprotein, two variants of Urabe AM9 vaccine differing at amino acid 335 (HN-E335 and HN-K335) were isolated and their receptor-binding specificity was determined by means of competence assays. Pre-incubation of the viruses with sialic acids inhibited both syncytia formation in Vero cells and replication in SH-SY5Y cells. Thus, HN-K335 showed higher affinity towards sialylalpha2,6lactose, whereas HN-G335 preferred sialylalpha2,3lactose. These results are relevant because a high expression of sialylalpha2,6lactose in nerve cells was confirmed by means of Sambucus nigra lectin-cytochemistry. In addition, kinetics assays showed that HN-K335 and HN-E335 also differ in their hydrolysis rate (Vmax values of 37.5 vs. 3.5 nmol min-1mg-1, respectively). Therefore, HN-K335 variant presented a neuraminidase activity level 11-fold higher than that of HN-E335 variant. In conclusion, the mutation affects the receptor-binding and neuraminidase activities of Urabe AM9 mumps virus variants.

Amino Acid Substitution↗
PubMed1995

A strategy for theoretical binding constant, Ki, calculations for neuraminidase aromatic inhibitors designed on the basis of the active site structure of influenza virus neuraminidase.

Neuraminidase (NA) is one of the two major surface antigens of influenza virus. It plays an indispensable role in the release and spread of progeny virus particles during infection. NA inhibitors reduce virus infection in animals. To improve the clinical efficacy of NA inhibitors, we have begun the design of non-carbohydrate inhibitors based on the active site structure of NA. The approach is an iterative process of ligand modeling and electrostatic calculations followed by chemical synthesis of compounds, biological testing, and NA-inhibitor complex structure determination by X-ray crystallography. A strategy has been developed to calculate Ki for newly designed inhibitors. The calculations using the DelPhi program were performed for carbohydrate inhibitors and three preliminary benzoic acid inhibitors of neuraminidase (BANA) that have been synthesized and shown to bind to the active site of NA in the crystal structure. The calculated Kis of these inhibitors have an enlightening agreement with their in vitro biological activities. This demonstrates that the calculations produce informative results on the affinity of modeled inhibitors. GRID maps were also calculated and several pockets were identified for accepting possible new ligands. The calculated Kis for newly designed ligands suggest that these potential compounds will have high inhibitory activities.

Aminobenzoates↗
PubMed2006

Importance of neuraminidase active-site residues to the neuraminidase inhibitor resistance of influenza viruses.

Neuraminidase inhibitors (NAIs) are antivirals designed to target conserved residues at the neuraminidase (NA) enzyme active site in influenza A and B viruses. The conserved residues that interact with NAIs are under selective pressure, but only a few have been linked to resistance. In the A/Wuhan/359/95 (H3N2) recombinant virus background, we characterized seven charged, conserved NA residues (R118, R371, E227, R152, R224, E276, and D151) that directly interact with the NAIs but have not been reported to confer resistance to NAIs. These NA residues were replaced with amino acids that possess side chains having similar properties to maintain their original charge. The NA mutations we introduced significantly decreased NA activity compared to that of the A/Wuhan/359/95 recombinant wild-type and R292K (an NA mutation frequently reported to confer resistance) viruses, which were analyzed for comparison. However, the recombinant viruses differed in replication efficiency when we serially passaged them in vitro; the growth of the R118K and E227D viruses was most impaired. The R224K, E276D, and R371K mutations conferred resistance to both zanamivir and oseltamivir, while the D151E mutation reduced susceptibility to oseltamivir only (approximately 10-fold) and the R152K mutation did not alter susceptibility to either drug. Because the R224K mutation was genetically unstable and the emergence of the R371K mutation in the N2 subtype is statistically unlikely, our results suggest that only the E276D mutation is likely to emerge under selective pressure. The results of our study may help to optimize the design of NAIs.

Animals↗
PubMed1987

Immunologic response to influenza virus neuraminidase is influenced by prior experience with the associated viral hemagglutinin. III. Reduced generation of neuraminidase-specific helper T cells in hemagglutinin-primed mice.

In BALB/c mice primed by influenza virus infection to H3 hemagglutinin and N2 neuraminidase, presentation of N2 in association with a heterosubtypic (H7) hemagglutinin results in production of a greater amount of N2 antibody than is found with homologous (H3N2) reimmunization. Titration of primed helper T cell (Th) activity by adoptive transfer of purified T cells to athymic mice given H6N2 vaccine demonstrates a lesser number of N2-specific Th cells in mice subjected to homologous reimmunization. We conclude that Th cells participate in the mediation of intermolecular (intravirionic) antigenic competition between influenza virus hemagglutinin and neuraminidase.

Animals↗
PubMed1973

Influenzavirus neuraminidase and neuraminidase-inhibition test procedures.

The adequate characterization of type A influenzaviruses requires the identification not only of the haemagglutinin but also of the neuraminidase. The neuraminidase-inhibition test, as performed in the two WHO international reference centres for influenza, is described and its use in other laboratories-particularly those collaborating in the WHO programme-is recommended.

Antigens, Viral↗
PubMedDate not supplied

Micro neuraminidase-inhibition assay for classification of influenza A virus neuraminidases.

A neuraminidase-inhibition (NI) assay performed in microtiter plates is described. This micro-NI assay is a modification of the NI assay recommended by the World Health Organization. It reduces the quantity of reagents required and permits antigenic classification of many isolates simultaneously. To determine the accuracy and sensitivity of this micro-NI assay, 110 influenza A viruses, representing all subtypes, based upon the nine known neuraminidases (NAs), were classified by both the micro-NI and macro-NI assays in two separate laboratories. The NAs were identified accurately by the micro-NI assay. Virus mixtures were detected by both assays, although the macro-NI was clearly more sensitive. The micro-NI assay was also suitable for testing sera for the presence of antibodies to the NAs. Although the micro-NI assay did not provide the quantitation of the macro-NI assay, it did prove to be a rapid method for virus classification and antibody studies on influenza A viruses.

Animals↗
PubMed2000

Efficacy and safety of the oral neuraminidase inhibitor oseltamivir in treating acute influenza: a randomized controlled trial. US Oral Neuraminidase Study Group.

CONTEXT: Previous studies have shown oseltamivir, a neuraminidase inhibitor, to be effective in preventing influenza and treating experimental influenza. OBJECTIVE: To evaluate the efficacy and safety of oseltamivir in the treatment of naturally acquired influenza infection. DESIGN: Randomized, placebo-controlled, double-blind study conducted January through March 1998. SETTING: Sixty primary care and university health centers throughout the United States. PARTICIPANTS: A total of 629 healthy nonimmunized adults aged 18 to 65 years with febrile respiratory illness of no more than 36 hours' duration with temperature of 38 degrees C or more plus at least 1 respiratory symptom and 1 constitutional symptom. INTERVENTIONS: Individuals were randomized to 1 of 3 treatment groups with identical appearing pills: oral oseltamivir phosphate, 75 mg twice daily (n = 211) or 150 mg (n = 209) twice daily, or placebo (n = 209). MAIN OUTCOME MEASURES: Duration and severity of illness in individuals infected with influenza. RESULTS: Two individuals withdrew before receiving medication and were excluded from further analyses. A total of 374 individuals (59.6%) were infected with influenza. Their duration of illness was reduced by more than 30% with both oseltamivir, 75 mg twice daily (median, 71.5 hours; P < .001), and oseltamivir, 150 mg twice daily (median, 69.9 hours; P = .006), compared with placebo (median, 103.3 hours). Severity of illness was reduced by 38% (median score, 597 score-hours; P < .001) with oseltamivir, 75 mg twice daily, and by 35% (median score, 626 score-hours; P < .001) with oseltamivir, 150 mg twice daily, vs placebo (median score, 963 score-hours). Oseltamivir treatment reduced the duration of fever and oseltamivir recipients returned to usual activities 2 to 3 days earlier than placebo recipients (P < or = .05). Secondary complications such as bronchitis and sinusitis occurred in 15% of placebo recipients compared with 7% of combined oseltamivir recipients (P = .03). Among all 629 subjects, oseltamivir reduced illness duration (76.3 hours and 74.3 hours for 75 mg and 150 mg, respectively, vs 97.0 hours for placebo; P = .004 for both comparisons) and illness severity (686 score-hours and 629 score-hours for 75 mg and 150 mg, respectively, vs 887 score-hours for placebo; P < .001 for both comparisons). Nausea and vomiting occurred more frequently in both oseltamivir groups (combined, 18.0% and 14.1%, respectively; P = .002) than in the placebo group (7.4% and 3.4%; P < .001). CONCLUSIONS: Our data suggest that oral oseltamivir treatment reduces the duration and severity of acute influenza in healthy adults and may decrease the incidence of secondary complications.

Acetamides↗
PubMed1999

Influenza type B neuraminidase can replace the function of type A neuraminidase.

Influenza A and B viruses do not form reassortants with each other, presumably due to selection at either the RNA or protein level. Although differences in the promoter sequences of type A and B viruses have been studied, selection at the protein level has not been addressed. In this paper we describe experiments to determine whether differences in structure and/or function of the neuraminidase (NA) protein preclude formation of A/B NA reassortants. Influenza type A (N9) NA or B/Lee/40 NA expressed from plasmids can support multicycle growth of a NA-deficient type A virus (NWS-Mvi), indicating that their function in tissue culture is similar. To determine whether the type A or B NA supplied in trans can be incorporated into the virion of NWS-Mvi, the virus grown in NA-expressing cells was purified by sucrose gradient centrifugation. In each case there was a peak of NA activity coincident with the virus peak, indicating that some NA protein is packaged into the virion. The experiments suggest that, in spite of large sequence differences, the functions of the head, stalk, signal-anchor, and cytoplasmic domains of type A and B NAs are similar in tissue culture. Thus, lack of formation of A/B NA reassortant viruses is not due to restriction at the protein level.

Animals↗
PubMed1985

Action of influenza virus neuraminidase on gangliosides. Haemagglutinin inhibits viral neuraminidase.

The action of partly purified neuraminidase (NA) of influenza A virus, a mixture of detergent solubilized NA and haemagglutinin (HA) and of intact virions on gangliosides GT1b, GD1a, GD1b, GM1 was studied. The viral NA transformed GT1b mainly into GD1b with formation of only minor amounts of GM1. HA was found to inhibit the hydrolysis activity of viral NA. At the same time viral NA transformed GD1a quantitatively into GM1 which was not hydrolyzed by the enzyme. These results suggest that the function of NA is to transfer the 'primary' receptor (such as GT1b) into the proper carbohydrate sequence (GD1b-like) which is proposed to serve as the minimal structure required for influenza virus reception.

G(M1) Ganglioside↗
PubMed1991

Transfer of the hemagglutinin activity of influenza virus neuraminidase subtype N9 into an N2 neuraminidase background.

It has previously been shown that influenza virus neuraminidase (NA) of the N9 subtype is unusual in that it possesses hemagglutinin activity as well as NA activity. Loss of red cell binding in certain escape mutants suggested that the hemagglutinating site is separate from the NA active site and involves at least two of the polypeptide loops found on the surface of the molecule (Webster et al., 1987. J. Virol. 61, 2910-2916). We have used site-directed mutagenesis to transfer the amino acids in these loops at positions 368-370 and 399-403 of N9 NA (A/tern/Australia/G70c/75), separately and together, into subtype N2 NA (A/Tokyo/3/67). The three mutant proteins were expressed from an SV40 transient expression system (Fuerst et al., 1986. Proc. Natl. Acad. Sci. USA. 83, 8122-8126). The mutant which contained both loops of N9 NA had acquired the hemagglutinin activity of N9. The agglutinated red cells are released by the enzyme activity of N9 NA, indicating that the agglutination involves binding to sialic acid in the same configuration as does the parental N9 NA, and an inhibitor of NA did not affect hemagglutination, indicating that this site is separate from the NA site as in parental N9.

Agglutination Tests↗
PubMed2001

A novel liposomal influenza vaccine (INFLUSOME-VAC) containing hemagglutinin-neuraminidase and IL-2 or GM-CSF induces protective anti-neuraminidase antibodies cross-reacting with a wide spectrum of influenza A viral strains.

A liposomal influenza vaccine (INFLUSOME-VAC) was developed with the objective of overcoming the major drawbacks of the currently used influenza vaccines: their relatively low efficacy in certain high-risk groups (the elderly, infants, the immunosuppressed) and the need for annual immunization. INFLUSOME-VAC consists of liposomes containing the viral surface proteins hemagglutinin (HA) and neuraminidase (NA) derived from various influenza strains and IL-2 or GM-CSF, as an adjuvant. Vaccination of mice showed that, whereas conventional vaccines induced a low- and short-term response against HA and very low or no anti-NA response, INFLUSOME-VAC produced high titers of both anti-HA and anti-NA antibodies (Abs) in young and old mice that persisted for at least 6 months. Moreover, the anti-NA Abs efficiently cross-reacted with several N2 viral subtypes spanning 20 years, and such vaccines afforded partial protection against heterosubtypic viral infection.

Animals↗
PubMed1991

Evolutionary pathways of N2 neuraminidases of swine and human influenza A viruses: origin of the neuraminidase genes of two reassortants (H1N2) isolated from pigs.

The complete nucleotide sequences of the neuraminidase (NA) genes of two reassortant (H1N2) and two H3N2 influenza A viruses isolated from pigs were determined and phylogenetic relationships between these and previously reported N2 NA genes were investigated. On the basis of pairwise nucleotide sequence identity, the NA genes of two reassortants, A/sw/Kanagawa/2/78 and A/sw/Ehime/1/80, were most closely related to those of human influenza A virus strains isolated in 1972 and the earliest available swine H3N2 influenza A viruses, respectively. Phylogenetic trees showed that the NA genes can be segregated into three groups, including lineages for (i) swine strains, (ii) the earliest human strain and (iii) recent human strains. The evolutionary tree for the 11 nucleotide and amino acid sequences suggested that the NAs of A/sw/HK/4/76 and A/sw/Kanagawa/2/78 belong to the lineage for recent human viruses. In contrast, the NA genes of the A/sw/HK/3/76 and H1N2 reassortant A/sw/Ehime/1/80 viruses were found to be of a swine lineage. The swine virus NA genes were further characterized by the cocirculation of two distinct lineages. Although the rates of synonymous (silent) substitutions for the swine and human viruses were nearly identical (0.00946 to 0.00884 per site per year), the rate of non-synonymous (amino acid changing) substitutions for swine virus NA genes was about 60% of that for the human virus.

Amino Acid Sequence↗
PubMed1998

The position 4 nucleotide at the 3' end of the influenza virus neuraminidase vRNA is involved in temporal regulation of transcription and replication of neuraminidase RNAs and affects the repertoire of influenza virus surface antigens.

Within the sequence motif conserved at the extreme ends of the influenza virus vRNAs, a unique natural variation, U or C, is observed at position 4 of the 3' end. To test the role of this nucleotide, two isogenic A/WSN/33 viruses, carrying either C4 or U4 nucleotide at the 3' end of the neuraminidase (NA) gene, were generated. Compared with the C4 virus, the U4 virus exhibited delayed synthesis of vRNA and stimulation of mRNA synthesis with prolonged accumulation in influenza virus-infected cells. The mRNA/ vRNA ratio was increased up to 20-fold by the C4 --> U4 substitution suggesting that the U4 nucleotide greatly stimulated transcription of the vRNA template. In isolated virion, the U4 virus had higher NA activity than the C4 virus. In MDBK cells, the U4 virus grew to lower haemagglutination (HA) titres but with higher infectivity than the C4 virus, with a corresponding increase in the ratio of p.f.u./HA units of about 10- to 40-fold. Western blot analysis of isolated virion showed that the ratio of two surface proteins, HA/NA, was greatly decreased in the U4 virus. This suggests that the position 4 nucleotide is a genetic determinant for the repertoire of surface antigens and their ratio could be changed without detrimental effects on virus growth. Results could be used to design genetically engineered influenza virus for vaccination. The observed down-regulation of transcription by C4 nucleotide is consistent with its potential role in segment-specific regulation of influenza virus gene expression, especially PB1, PB2 and PA proteins, during virus infection.

Animals↗
PubMed1967

Intracellular synthesis of myxovirus neuraminidase in chick embryo cell monolayer culture. I. Neuraminidase activity, hemagglutinin synthesis, and content of cellbound sialic acid in Newcastle disease virus-infected cells.

Neuraminidase (Nase) activity of chick embryo monolayer cell homogenates was determined by its rate of splitting of neuraminlactose, free neuraminic acid (NA) being determined by the thiobarbituric acid assay. Noninfected cells were found to have no detectable amount of Nase activity. Newcastle disease virus (NDV)-infected cells (multiplicity of infection, 20 to 75 plaque-forming units per cell) displayed a high level of Nase synthesis, the rate of synthesis being parallel to that of hemagglutinin (HA) synthesis (with a 1.5 hr delay in the latter). An "eclipse" of the Nase and HA activities associated with the virus that was adsorbed onto cells was observed. The data provide evidence that the Nase is not incorporated into the viral envelope from a pre-existing cell supply but that its synthesis is coded by the viral genome. The content of cell-bound sialic acid, determined simultaneously in infected-cell homogenates, showed characteristic features allowing certain conclusions concerning the renewal of NA-terminating cell receptors during the course of infection, and the intracellular action of the Nase of the virus introduced into cells by the inoculum and that of the newly synthesized Nase at different stages of infection.

Animals↗
PubMed1994

Antigenicity of the N8 influenza A virus neuraminidase: existence of an epitope at the subunit interface of the neuraminidase.

To locate antigenic epitopes on the N8 neuraminidase (NA), we generated a panel of 97 monoclonal antibodies (MAbs), 66 of which inhibited NA activity (NI antibodies). Three groups of NI MAbs were identified from their different reactivities with escape mutants. Group 1 antibodies recognized the peptide loop containing residues 344 to 346, which appears to be an immunodominant region on the rim of the enzyme center of the N8 NA. Group 2 antibodies recognized a novel epitope containing residues 150, 199, 367, 399, and 400 (N2 numbering). From the location of these residues on the three-dimensional structure of the N8 NA, the epitope appears to be located at the interface of two adjacent monomers in the tetrameric NA, one contributing residues 150 and 199 and the other contributing residues 367 and 399 to 400. The available evidence indicates that the MAbs of this group react with the NA only after it is fully assembled. The third group of antibodies recognized the peptide loops containing residues 367 and 399 to 400. All of the amino acid substitutions in N8 escape mutants which affect the NI activity of antibodies were located in the peptide loops known to form epitopes in the N2 and N9 subtypes, indicating that antigenic regions in the NA head inducing NI antibodies appear to be similar among different subtypes of influenza A viruses. The MAbs used in this study will be valuable in studying the role of each N8 NA epitope in host immune defense systems and in the kinetics analysis of the biosynthesis of the enzyme.

Amidohydrolases↗