Passage Of Influenza Research Articles

Modeling influenza virus infection in mature Wistar rats

Passage of influenza A virus (IAV) in embryonated chicken eggs, intranasal inoculation of rats with virus-containing material s, production of pulmonary homogenate, determination of IAV titer in embryonated chicken eggs, detection of histological changes in lung and pulmonary vessels. The article presents the results of the adaptation of influenza virus A/St. Petersburg/48/16 H1N1(pdm09) to mature Wistar rats. The infectious titer of the virus in the homogenates of infected rats lungs at the last stage of adaptation was 7.0 lg EID50/ml. Histological studies revealed pronounced changes in the respiratory tract (spasm of bronchioles, submucosal edema, desquamation of ciliated epithelium of bronchioles) and pulmonary vessels (spasm, desquamation and swelling of endotheliocytes, dissociation and swelling of the elastic membrane and media). In order to identify IAV in blood vessels and lung tissues, an immunohistochemical study was performed using monoclonal antibodies to NP antigen of IAV. The data obtained allow us to conclude that the strain of influenza virus A/St. Petersburg/48/16 H1N1(pdm09) was adapted to mature Wistar rats maintaining virulent properties. The infectious titer of the virus at the last stage of adaptation was 7.0 lg EID50/ml. IAV identification is confirmed by immunohistochemical examination.

Passage of influenza A/H3N2 viruses in human airway cells removes artefactual variants associated with neuraminidase-mediated binding.

Serological assays with modern influenza A/H3N2 viruses have become problematic due to the progressive reduction in the ability of viruses of this subtype to bind and agglutinate red blood cells (RBCs). This is due to reduced ability of the viral haemagglutinin (HA) glycoprotein to bind to the sialic acid-containing receptors presented by these cells. Additionally, as a result of reduced HA-mediated binding in cell culture, modern A/H3N2 viruses often acquire compensatory mutations during propagation that enable binding of cellular receptors through their neuraminidase (NA) surface protein. Viruses that have acquired this NA-mediated binding agglutinate RBCs through their NA, confusing the results of serological assays designed to assess HA antigenicity. Here we confirm with a large dataset that the acquisition of mutations that confer NA binding of RBCs is a culture artefact, and demonstrate that modern A/H3N2 isolates with acquired NA-binding mutations revert to a clinical-like NA sequence after a single passage in human airway epithelial (HAE) cells.

Passage in egg culture is a major cause of apparent positive selection in influenza B hemagglutinin

Several studies have identified residues apparently under positive selection in influenza B virus hemagglutinin. Host immune evasion is the main mechanism proposed to exert this selection pressure. However, these reports have not considered the culture history of the strains used to calculate positive selection. This article shows that passage of influenza B virus through egg culture is a strong contributory factor to the strength and statistical significance of positive selection on hemagglutinin. Non-synonymous mutations resulting in the loss of the N-glycosylation site at positions 197-199 of hemagglutinin have been positively selected to a far greater degree in egg-cultured strains than in other strains. Once egg-cultured strains are removed from the analysis, positive selection is found to be far weaker, less statistically significant, and more diffusely localized along the protein. Caution should therefore be exercised both in claims for the existence of positive selection in influenza B hemagglutinin, and in attribution of host immune evasion as its cause. The major cause of molecular adaptation in influenza B hemagglutinin proteins may well be laboratory eggs rather than natural hosts.

Selection of antigenically distinct variants of influenza C viruses by the host cell

Five strains of influenza C virus were isolated and passaged in the amniotic sacs of embryonated hens' eggs, or in the HMV-II line of human malignant melanoma cells, and were tested for reactivity with a panel of monoclonal antibodies to the hemagglutinin-esterase (HE) glycoprotein. It was observed with two strains (C/Yamagata/4/88, C/Yamagata/7/88) that the HE of virus passaged in HMV-II cells was antigenically distinguishable from that of virus cultivated in eggs. Virus clones obtained after repeated passages of these two strains in HMV-II cells all showed a significant increase in the ability to replicate in the cell culture compared to clones derived from viruses grown in eggs. No difference was seen, by contrast, in the ability to grow in eggs between HMV-II- and egg-derived virus clones. It was also found that HMV-II-grown viruses but not egg-grown viruses could agglutinate glutaraldehyde-fixed chicken erythrocytes at 23°. These observations, taken together, suggest that isolation and passage of influenza C virus in HMV-II cells sometimes result in selection of antigenically distinct variants which have an advantage in binding to the cell surface receptors. Sequence analyses of the HE genes revealed that compared to egg-grown viruses, HMV-II-adapted variant of the Yamagata/4/88 strain had a single amino acid substitution in the HE molecule at position 283 (Asp → Asn) and that of the Yamagata/7/88 strain had two substitutions at positions 212 (Glu → Lys) and 519 (Asn → Asp).

Aminoadamantane-resistant strains of influenza A2 virus.

After one passage of influenza A2/Singapore/1/57 virus in mice treated with 150 mg./kg./day of aminoadamantane, a partially drug-resistant strain of virus was detected in 1 of 12 mice. The isolation rate of aminoadamantane-resistant viruses increased to 8 after three passages in drug-treated mice. Some virus strains showed a 500-fold increase in resistance to aminoadamantane and to the structurally related compounds alpha-methyl-1-adamantane methylamine and 2-adamantanamine sulphate. No aminoadamantane-resistant viruses were detected after passage of influenza four times in mice treated with lower (15 or 1.5 mg./kg./day) concentrations of aminoadamantane. Aminoadamantane had no detectable effect on the development of lung lesions in mice infected with the drug-resistant influenza strain, whereas lung lesions were reduced in aminoadamantane treated mice infected with a control strain of influenza A2/Singapore virus. No differences were detected in the buoyant density in caesium chloride, morphology or serology between control and aminoadamantane-resistant strains of virus. These drug-resistant influenza viruses may be useful for detailed studies of the mode of action of aminoadamantane.