Two highly pathogenic avian influenza (HPAI) virus clones that met the criteria for high-pathogenicity avian influenza viruses, by possessing a multibasic hemagglutinin (HA) cleavage site, were isolated from an H5N1 outbreak in Norfolk, England, in 1991–92. These two isolates, A/turkey/England/50-92/91 (50-92) and A/turkey/England/87-92/91 (87-92), displayed differences in virulence as determined by intravenous pathogenicity index-3 and -0, respectively. DNA sequencing of these two isolates identified 10 amino acid differences throughout the genome: three in HA and polymerase B2 (PB2) and two in polymerase B1 (PB1) and single mutations in nucleoprotein (NP) and polymerase A (PA). Serial intracerebral passages were performed in 1- or 2-day-old specific pathogen free (SPF) chicks with 87-92. Viruses reisolated from each bird passage displayed increases in intracerebral pathogenicity index values (from 0 to 1.9) and therefore virulence. Reverse transcriptase polymerase chain reaction and DNA sequencing on viruses isolated at each passage displayed nine out of the 10 mutations associated with the higher pathogenic genotype of 50-92, except for the mutation found in NP, which retained the amino acid residue associated with 87-92. Serial passage through 9-day-old SPF embryonated chicken eggs and serial intravenous passage in 6-wk-old birds could not reproduce these results. These results further highlight that nucleotide changes in the genome other than at the HA cleavage site can attenuate the virulence of HPAI viruses.

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