Analysis of viral and cellular parameters which affect the fusion process of influenza viruses

In the present investigation we studied the fusogenic process developed by influenza A, B and C viruses on cell surfaces and different factors associated with virus and cell membrane structures. The biological activity of purified virus strains was evaluated in hemagglutination, sialidase and fusion assays. Hemolysis by influenza A, B and C viruses ranging from 77.4 to 97.2 percent, from 20.0 to 65.0 percent, from 0.2 to 93.7 percent and from 9.0 to 76.1 percent was observed when human, chicken, rabbit and monkey erythrocytes, respectively, were tested at pH 5.5. At this pH, low hemolysis indexes for influenza A, B and C viruses were observed if horse erythrocytes were used as target cells for the fusion process, which could be explained by an inefficient receptor binding activity of influenza on N-glycolyl sialic acids. Differences in hemaglutinin receptor binding activity due to its specificity to N-acetyl or N-glycolyl cell surface oligosaccharides, density of these cellular receptors and level of negative charges on the cell surface may possibly explain these results, showing influence on the sialidase activity and the fusogenic process. Comparative analysis showed a lack of dependence between the sialidase and fusion activities developed by influenza B viruses. Influenza A viruses at low sialidase titers (<2) also exhibited clearly low hemolysis at pH 5.5 (15.8 percent), while influenza B viruses with similarly low sialidase titers showed highly variable hemolysis indexes (0.2 to 78.0 percent). These results support the idea that different virus and cell-associated factors such as those presented above have a significant effect on the multifactorial fusion process.

Hemagglutinating and fusogenic activities of Newcastle disease virus: studies on receptor binding specificity and pH-induced conformational changes

Vaccinal and wild strains of Newcastle Disease virus (NDV) were analyzed for cell receptor binding and fusogenic biological properties associated with their HN (hemagglutinin-neuraminidase) and F (fusion protein) surface structures respectively. The evaluation of the biological activities of HN and F was carried out respectively by determination of hemagglutinating titers and hemolysis percentages, using erythrocytes from various animal origins at different pH values. Significant differences in hemagglutination titers for some strains of NDV were detected, when interacting with goose, sheep, guinea-pip and human "O" group erythrocytes at neutral pH. Diversity of hemolysis percentagens was observed between different NDV strains at acid pH. These analysis were developed to evaluate particular aspects of the actual influence of the receptor specifity and pH on the receptor binding and fusogenic processes of Newcastle Disease viruses.

The role of antigenically different virus neuraminidases as structures implicated in receptor-binding processes

Influenza A viruses exhibit segmented nucleic acid coding for eight different proteins, two of them as glycoproteins exposed on their lipoprotein envelopes, hemagglutinin (HA) and neuraminidase (NA). Hemagglutinin exhibits recptor-binding activity while neuraminidase develps sialidase cleavage activity which acts on cell receptors. Influenza A strains responsible for human, avian, equine and porcine respiratory infections all over the world present antigenically different hemagglutinin (H1 to H14) and neutraminidase (N1 to N9) structures on their surface. The objective of the present investigation was study the role of N2, N8 and N9, anti-genically diverse neuraminidase structures of human (N2) and animal (N8 and N9) influenza viruses, in the receptor-binding process. REceptor-binding activity of N2 and N8 was anlyzed by crossed tests using H3N2 and H3N8 antisera and the hemagglutination inhibition test as a model. Hemangglutinating activity of antigenically different N2 and N8 structures was demonstrable and was inhibited by homologous antisera (N2-H3N2, N8-H3N8) but not by heterologous antisera (N2-H3-N8,N8-H3-N2). This previously demonstrated N9 hemagglutinating activity was analysed for receptor-binding specificity using hemagglutination test and NeuAc alpha2,3Gal and NeuAc alpha2,6Gal derivatized erythrocytes. This highly purified N9 strain was obtained from a virus strain isolated from terns by Dr. Peter Colman (CSIRO Division of Biomolecular Engineering, Parkville, Victoria, Australia)...

Characterization of the hemagglutinin receptor specificity and neuraminidase substrate specificity of clinical isolates of human influenza A viruses

Six clinical isolates of influenza A viruses were examined for hemagglutinin receptor specificity and neuraminidase substrate specificity. All of the viral isolates minimally passaged in mammalian cells demonstrated preferential agglutination of human erythrocytes enzymatically modified to contain NeuAc alpha 2,6Gal sequences, with no agglutination of cells bearing NeuAc alpha 2,3Gal sequences. This finding is consistent with the hemagglutination receptor specificity previously demonstrated for laboratory strains of influenza A viruses. The neuraminidase substrate specificities of the clinical isolates examined were also identical to that described for the N2 neuraminidase of recent laboratory strains of human influenza viruses. The H3N2 viruses all displayed the ability to release sialic acid from both alpha 2, 3 and alpha 2, 6 linkages. In addition, two clinical isolates of H1N1 viruses also demonstrated this dual neuraminidase substrate specificity, a characteristic which has not been previously described for the N1 neuraminidase. These results demonstrate that complementary hemagglutinin and neuraminidase specificities are found in recent isolates of both H1N1 and H3N2 influenza viruses.

Comparative evaluation of a simple and sensitive assay for detection of orthomyxo and paramyxoviruses

Studies were done to evaluate comparatively the traditional HA assay and a more recently introduced lectin-neuraminidase (LN) methodologyin search of a simple and sensitive assay for virus detection during laboratorial diagnosis. The results proved the value of LN assay as a sensitive methodologyfor detection of virus particles, presenting results at least equal to those obtained by HA (hemagglutination) assay, with significant values of accumulated frequencies for LN/HA factors (ratios between LN and HA titers) higher than two. The accumulated values of frequencies for LN/HA factors as high as four were very significant, 72.7 (per cent) for influenzavirus and 60.7 (per cent) for Newcastle disease virus (NDV), moreover accumulated frequencies for LN/HA factors even as high as 32 were due to influenzavirus (45.4 per cent) and NDV (7.2 per cent) samples. After the storage period, most of those concentraded samples that even did not present HA titers could be detected through LN assay, demonstrating a lower threshold for virus detection

Hemagglutinating and sialidase activities of subpopulations of influenza A viruses

The present study was conducted to investigate the characteristics of two samples of influenza A/England/42/72 (H3N2) virus, one of them selected by an adsorption-elution technique, to determine the possible existence of virus variants or subpopulations. Based on specificity of virulence-related cell receptor-binding and sialidase activities, this selection technique using human O group erythrocytes revealed the presence of variants within a standard virus sample with diversity for their hemagglutinating and sialidase activities. The standard-like (E1) sample exhibited titers of 4 and 32 HAU (hemagglutinating units in 25 microliters) with human O group and chicken erythrocytes, respectively, while the sample obtained by the adsorption-elution process (E2) exhibited titers of 32 and 4 HAU, respectively, with these same types of erythrocytes. The E2 sample showed higher sialidase activity at pH values between 5.4 and 6.6 with human erythrocytes (128-256 HAU), but the E1 sample did not exhibit significant sialidase activity with either human or chicken erythrocytes. The different pH optima for hemolysis (5.2) and sialidase (5.4-6.6) activities and the higher hemolysis indexes present in samples with sialidase activity inhibited by heating (at 56 degrees C for 30 min) or by treatment with EDTA (dilution in buffer containing 2 mM EDTA, a chelating agent on calcium-dependent sialidase activity) demonstrate the independence of these activities in the selected sample: native E2 (absorbance = 0.18), EDTA-treated native E2 (absorbance = 0.28), heated E2 (absorbance = 0.26), EDTA-treated heated E2 (absorbance = 0.41).(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of low pH on hemagglutinating and hemolytic activities of west equine encephalomyelitis virus

1. The hemagglutinating (HA) and hemolytic (HL) activities mediated by egg-propagated west equine encephalomyelitis (WEE) virus preparations were investigated. 2. The purified virus preparation exhibited the best HA and HL activity at pH 6.0 and 6.0-6.2, respectively, as observed in the HA and HL tests. 3. In the virus preparations, both HA and HL activities were completely lost upon pretreatment at low pH (6.0). 4. The present results suggest that the alphavirus-mediated HA and HL activities against chicken erythrocytes can be considered to be a fusion from without

A combined staining technique developed for virus particle observation in the electron microscope

Descrevemos uma tecnica de coloracao dupla para a visualizacao direta de particulas virais ao microscopio eletronico. Os virus foram contrastados com acido fosfotundstico (PIA), juntamente, com acetato de uranila (UA). Para a concentracao das particulas virais, antes da contrastacao, fizemos uso de uma tecnica descrita por Yunis et al. (1978). Esta tecnica, juntamente com o metodo de colocacao dupla, melhorou consideravelmente a visualizacao e o contraste das particulas virais encontradas nos liquidos de cultura de tecidos infectados e alantoico de ovos embrionados inoculados, tornando o exame mais confiavel, rapido e de facil execucao