Journal of Medical Virology 74:141–146 (2004) Antigenic Characterisation of Influenza B Virus With A New Microneutralisation Assay: Comparison to Haemagglutination and Sequence Analysis Filippo Ansaldi,* Sabrina Bacilieri, Daniela Amicizia, Laura Valle, Federica Banfi, Paolo Durando, Laura Sticchi, Roberto Gasparini, Giancarlo Icardi, and Pietro Crovari C.I.R.I., Department of Health Sciences, University of Genoa, Genoa, Italy Although the haemagglutination inhibition assay is considered the ‘‘gold standard’’ for antigenic characterisation of influenza viruses, some lim- itations of this technique are well known. A new microneutralisation assay, as a tool for antigenic characterisation of influenza B viruses, has been standardised and its performance evaluated in comparison with the haemagglutination inhibi- tion test in the light of molecular characterisation of the haemagglutinin. Twelve B viruses belong- ing to the two lineages and the four sub-lineages discriminated by phylogenetic analysis of HA were tested. The microneutralisation assay clear- ly distinguishes viruses belonging to different lineages and, in addition, discriminates strains belonging to different sub-lineages that are poor- ly or not discriminated using the haemagglutina- tion inhibition test. This new microneutralisation assay could provide a useful tool for antigenic characterisation of circulating influenza viruses and contribute, together with the haemaggluti- nation inhibition test and sequence analysis of the haemagglutinin and neuraminidase, in the choice of the strain for use in vaccine composi- tion. J. Med. Virol. 74:141 – 146, 2004. ß 2004 Wiley-Liss, Inc. KEY WORDS: microneutralisation; lineages; haemagglutination inhibition assay INTRODUCTION The main option for reducing the impact of influenza is immunoprophylaxis with a vaccine, a preventive measure particularly recommended for groups at ‘‘high-risk’’ of complications. The effectiveness of vac- cine prophylaxis depends largely on the matching between the vaccine strains and the circulating viruses. Influenza viruses are characterised by a high antigenic variability and the need of a vaccine including the main circulating viruses requires adequate epidemiological and virological surveillance [de Jong et al., 2000; Kilbourne et al., 2002]. The current global surveillance system consists in a network of 110 National Influenza Centres in 83 countries, working in close collaboration with the four World Health Organization Collaborating Centres [Lavanchy, 1999]. Influenza virus strains were characterised antigenically by their reactivity in hae- magglutination inhibition (HI) tests with post-infection ferret antisera. Based on the results of HI assays and molecular analysis of the main surface glycoproteins of the virus, the haemagglutinin (HA) and neuraminadase (NA), vaccine formulation is adapted yearly to include those variants that will probably circulate in the coming season. The HI test reveals the greatest changes in antigenicity that are observed when a drift occurs, but isolates with few amino acid changes over antigenic sites are not discriminated. The effect of these changes on the antigenicity of the virus and on the effectiveness of the vaccine is poorly understood. In this report, a new microneutralisation assay is described for antigenic characterisation of influenza B viruses and its perfor- mance is compared with that of the HI test in the light of molecular characterisation of HA. Influenza B viruses have evolved into two antigenically distinct lineages, including B/Yamagata/16/88-like and B/Victoria/2/87- like viruses [Nerome et al., 1998]. Furthermore, phylo- genetic analysis allows discrimination of the virus belonging to both Victoria- and Yamagata-lineage into two clusters, i.e., B/Hong Kong/330/01 and B/Shang- dong/7/97 for the former lineage, B/Harbin/7/94 and B/ Sichuan/379/99 for the latter, that are either poorly or not discriminated using the HI test [Hay et al., 2002]. Filippo Ansaldi and Sabrina Bacilieri contributed equally to this study. *Correspondence to: Dr. Filippo Ansaldi, Department of Health Sciences, University of Genoa, Via Pastore 1, 16132 Genoa, Italy. E-mail: filippo.ansaldi@unige.it Accepted 28 April 2004 DOI 10.1002/jmv.20157 Published online in Wiley InterScience (www.interscience.wiley.com) ß 2004 WILEY-LISS, INC.