PhyloFlu, a DNA Microarray for Determining the Phylogenetic Origin of Influenza A Virus Gene Segments and the Genomic Fingerprint of Viral Strains

Recent evidence suggests that most influenza A virus gene segments can contribute to the pathogenicity of the virus. In this re- gard, the hemagglutinin (HA) subtype of the circulating strains has been closely surveyed, but the reassortment of internal gene segments is usually not monitored as a potential source of an increased pathogenicity. In this work, an oligonucleotide DNA mi- croarray (PhyloFlu) designed to determine the phylogenetic origins of the eight segments of the influenza virus genome was con- structed and validated. Clades were defined for each segment and also for the 16 HA and 9 neuraminidase (NA) subtypes. Viral genetic material was amplified by reverse transcription-PCR (RT-PCR) with primers specific to the conserved 5= and 3= ends of the influenza A virus genes, followed by PCR amplification with random primers and Cy3 labeling. The microarray unambigu- ously determined the clades for all eight influenza virus genes in 74% (28/38) of the samples. The microarray was validated with reference strains from different animal origins, as well as from human, swine, and avian viruses from field or clinical samples. In most cases, the phylogenetic clade of each segment defined its animal host of origin. The genomic fingerprint deduced by the combined information of the individual clades allowed for the determination of the time and place that strains with the same genomic pattern were previously reported. PhyloFlu is useful for characterizing and surveying the genetic diversity and variation of animal viruses circulating in different environmental niches and for obtaining a more detailed surveillance and follow up of reassortant events that can potentially modify virus pathogenicity.