Abstract
ObjectivesThe 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 settingA 96-well-plate plaque reduction MN assay based on the measurement of infected cell population using a simple imaging technique.SampleRepresentative influenza A (H1N1) pdm09, A(H3N2) and B viruses isolated between 2004 and 2013Main outcome measures and resultsImprovements 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.ConclusionsOur 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.
Highlights
Influenza viruses evolve constantly to escape human immunity and cause annual epidemics and occasional pandemics
A(H3N2) viruses were propagated in Madin–Darby canine kidney (MDCK)-SIAT1 cells, and its parent cells were used for propagating A(H1N1)pdm09 and influenza B viruses
MDCK-Parent, MDCK-ECACC, MDCK-SIAT1 and Mini-pig kidney (MPK) cells were examined for their ability to support virus replication and plaque formation of panels of A(H1N1) pdm09 (2009 -2012), A(H3N2) (1999–2007) and type B (2007–2012) influenza viruses (Table 1)
Summary
Influenza viruses evolve constantly to escape human immunity and cause annual epidemics and occasional pandemics. For over 60 years, the identification of antigenic variants has been determined largely by the haemagglutination inhibition (HI) assay to measure the ability of antibodies, raised against vaccine and reference viruses, to prevent the attachment of virus to red blood cells (RBCs), a process analogous to the binding of virus to host cell receptors. During the past decade, interpretation of HI results has become complicated: either because of changes in receptor binding properties as a result of virus evolution, or due to selection of variants during the isolation and passage of viruses in cell lines or eggs. The loss of the ability of A(H3N2) viruses to agglutinate chicken, and subsequently turkey, RBCs was caused by amino acid substitutions E190D and D225N in the haemagglutinin (HA), occurring around 1990 and 2005, respectively..
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