Optimisation of a micro-neutralisation assay and its application in antigenic characterisation of influenza viruses.

OBJECTIVES: The identification of antigenic variants and the selection of influenza viruses for vaccine production are based largely on antigenic characterisation of the haemagglutinin (HA) of circulating viruses using the haemagglutination inhibition (HI) assay. However, in addition to evolution related to escape from host immunity, variants emerging as a result of propagation in different cell substrates can complicate the interpretation of HI results. The objective was to develop further a micro-neutralisation (MN) assay to complement the HI assay in antigenic characterisation of influenza viruses to assess the emergence of new antigenic variants and reinforce the selection of vaccine viruses. DESIGN AND SETTING: A 96-well-plate plaque reduction MN assay based on the measurement of infected cell population using a simple imaging technique. SAMPLE: Representative influenza A (H1N1) pdm09, A(H3N2) and B viruses isolated between 2004 and 2013 MAIN OUTCOME MEASURES AND RESULTS: Improvements to the plaque reduction MN assay included selection of the most suitable cell line according to virus type or subtype, and optimisation of experimental design and data quantitation. Comparisons of the results of MN and HI assays showed the importance of complementary data in determining the true antigenic relationships among recent human influenza A(H1N1)pdm09, A(H3N2) and type B viruses. CONCLUSIONS: Our study demonstrates that the improved MN assay has certain advantages over the HI assay: it is not significantly influenced by the cell-selected amino acid substitutions in the neuraminidase (NA) of A(H3N2) viruses, and it is particularly useful for antigenic characterisation of viruses which either grow to low HA titre and/or undergo an abortive infection resulting in an inability to form plaques in cultured cells.

A novel I221L substitution in neuraminidase confers high-level resistance to oseltamivir in influenza B viruses.

UNLABELLED: Influenza B viruses with a novel I221L substitution in neuraminidase (NA) conferring high-level resistance to oseltamivir were isolated from an immunocompromised patient after prolonged oseltamivir treatment. METHODS: Enzymatic characterization of the NAs (Km, Ki) and the in vitro fitness of viruses carrying wild-type or mutated (I221L) NA genes were evaluated. Proportions of wild-type and mutated NA genes were directly quantified in the patient samples. Structural characterizations by X-ray crystallography of a wild-type and I221L variant NA were performed. RESULTS: The Km and Ki revealed that the I221L variant NA had approximately 84 and 51 times lower affinity for oseltamivir carboxylate and zanamivir, respectively, compared with wild-type NA. Viruses with a wild-type or I221L variant NA had similar growth kinetics in Madin-Darby canine kidney (MDCK) cells, and 5 passages in MDCK cells revealed no reversion of the I221L substitution. The crystal structure of the I221L NA and oseltamivir complex showed that the leucine side chain protrudes into the hydrophobic pocket of the active site that accommodates the pentyloxy substituent of oseltamivir. CONCLUSIONS: Enzyme kinetic and NA structural analyses provide an explanation for the high level of resistance to oseltamivir while retaining good fitness of viruses carrying I221L variant NA.

Neuraminidase receptor binding variants of human influenza A(H3N2) viruses resulting from substitution of aspartic acid 151 in the catalytic site: a role in virus attachment?

Changes in the receptor binding characteristics of human H3N2 viruses have been evident from changes in the agglutination of different red blood cells (RBCs) and the reduced growth capacity of recently isolated viruses, particularly in embryonated eggs. An additional peculiarity of viruses circulating in 2005 to 2009 has been the poor inhibition of hemagglutination by postinfection ferret antisera for many viruses isolated in MDCK cells, including homologous reference viruses. This was shown not to be due to an antigenic change in hemagglutinin (HA) but was shown to be the result of a mutation in aspartic acid 151 of neuraminidase (NA) to glycine, asparagine, or alanine, which caused an oseltamivir-sensitive agglutination of RBCs. The D151G substitution was shown to cause a change in the specificity of NA such that it acquired the capacity to bind receptors, which were refractory to enzymatic cleavage, without altering its ability to remove receptors for HA. Thus, the inhibition of NA-dependent agglutination by the inclusion of oseltamivir carboxylate in the assay was effective in restoring the anti-HA specificity of the hemagglutination inhibition (HI) assay for monitoring antigenic changes in HA. Since the NA-dependent binding activity did not affect virus neutralization, and virus populations in clinical specimens possessed, at most, low levels of the "151 mutant," the biological significance of this feature of NA in, for example, immune evasion is unclear. It is apparent, however, that an important role of aspartic acid 151 in the activity of NA may be to restrict the specificity of the NA interaction and its receptor-destroying activity to complement that of HA receptor binding.