The H5 hemagglutinin (HA) of a highly virulent avian influenza virus, A/Turkey Ontario/7732/66 (H5N9), was previously shown to have five neutralizing epitopes, and escape mutants within one epitope (group 1) were markedly attenuated (M. Philpott, B. C. Easterday, and V. S. Hinshaw, J. Virol. 63:3453-3458, 1989). To define the genetic changes related to these antigenic and biologic properties, the HA genes of mutants within each of the epitope groups were sequenced by using the polymerase chain reaction. The mutations in the attenuated group 1 mutants were located near the distal tip of the HA molecule in close proximity to the receptor-binding site, on the basis of alignment with the three-dimensional structure of the H3 HA. All group 1 mutations involved charged amino acids. The group 1 mutants, similar to the wild-type virus, spread systemically and were recovered from the spleens of infected chickens but, unlike the wild-type virus, failed to produce severe necrosis in the spleens. Viral replication in the spleens was investigated by in situ hybridization of spleen sections from chickens infected with the wild-type or attenuated mutants. Wild-type virus replication was demonstrated in large, mononuclear, macrophagelike cells; however, group 1 mutant virus was detected attached only to erythrocytes within the red pulp. These results suggest that the attenuated mutants differ in their cell tropism within the spleen
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