Comparison between denaturing gradient gel electrophoresis and phylogenetic analysis for characterization of A/H3N2 influenza samples detected during the 1999-2004 epidemics in Brazil.

In a preliminary study, a denaturing gradient gel electrophoresis method (DGGE) was described for influenza virus variants screening [Motta, F.C., Rosado, A.S., Couceiro, J.N.S.S., 2002. Standardization of denaturing gradient gel electrophoresis for mutant screening of influenza A (H3N2) virus samples. J. Virol. Meth. 105, 105-115]. Such a protocol has confirmed its usefulness, discriminating closely related samples by the evaluation of the HA1 portion of haemagglutinin coding RNA segment. In this study, the HA1 sequence/phylogenetic analysis was compared with DGGE results to evaluate the degree of agreement between these methods. Forty-one influenza clinical samples characterized as the A/H3 subtype by a multiplex-PCR throughout 1999-2004 epidemics were chosen at random. The 569 bp DGGE amplicons were generated by nested-PCR using the first round multiplex-PCR product as template. The amplicons were analyzed on a 6% polyacrylamide gel with a urea-formamide gradient (25-35%) at 60 degrees C/150 V/5 h, being differentiated by their melting profiles. Even with the multiple melting domains characteristic of the region used in this study, the 41 samples could be grouped in 7 distinct clusters by DGGE. Five of the clusters reproduced exactly the phylogenetic tree topology, including the most external branches. Although the other two clusters demonstrated a poorer match, the internal genetic correlations were conserved, and just four samples were grouped incorrectly in comparison with the phylogenetic results. The results demonstrated the usefulness of this method for screening of variant samples throughout or in subsequent epidemics, thus improving the detection of influenza variants.