Pathogenicity shifts in experimental avian influenza virus infections in chickens

The last two outbreaks of highly pathogenic (T-IP) avian influenza (AI) in North America involved H5 AI viruses (AIV). Both outbreaks began as mild respiratory disease in poultry and abruptly changed to severe, systemic diseases which coincided with specific nucleotide changes in the hemagglutinin gene. Fifteen low pathogenicity (LP) H5 AIV isolates obtained from poultry and ratites were manipulated in a 14-day-chicken-embryo-adult-laying-hen modeling system. Seven AIV isolates changed from producing a few deaths (LP) to killing greater than or equal to 75% of chickens (HP) in an intravenous pathogenicity test. The LP parent H5 viruses of these seven derivatives had hemagglutinin proteolytic cleavage site sequences of -P-Q-K-K-K-R down arrow G- (n=3), -P-Q-R-K-T-R down arrow G- (n=3) or -P-Q-R-K-R-K-T-R down arrow G- (n=1). Three of the seven HP derivatives had the loss of a oligosaccharide site at amino acid residue 11 (n=2) or an insertion of two basic amino acids at the proteolytic cleavage site (n=1). However, four derivatives lacked a consistent change in the hemagglutinin to explain the pathogenicity shift from parent to derivative. One H5 AIV gave rise to derivatives that had high plaquing efficiency in trypsin-free chicken embryo fibroblast cultures and was considered potential HP, but was not tested for in vivo pathogenicity. The seven parent H5 AIV isolates that did not change pathogenic phenotype had a cleavage site sequence of -P-Q-R-E-T-R down arrow G- (n=5), -P-Q-K-K-K-R down arrow G- (n=1), or -P-Q-R-K-T-R down arrow G- (n=1) and usually had a oligosaccharide site at amino acid residue 11. Seven H7 AIVs were tested in the model system and one isolate became HP, but this virus was essentially already a highly lethal virus, as show by repeated IV pathotyping at the NVSL of the parent virus. Examination of genes other than the hemagglutinin may be necessary to fully understand and predict pathogenicity shifts of H5 AIVs.