AN ARG-LYS INSERTION AT THE HEMAGGLUTININ CLEAVAGE SITE OF AN H5N2 AVIAN INFLUENZA ISOLATE

Recent isolations of H5N2 subtype avian influenza (AI) viruses in Nort h America have raised questions concerning their origin, transmission to commercial poultry, and potential for virulence. One ratite-origin isolate of low pathogenicity, A/emu/TX/39924/93 (H5N2), was subjected to a procedure that rapidly selects and/or amplifies highly pathogenic (HP) strains. The resulting highly virulent derivative had an altered hemagglutinin (HA) gene containing an additional six nucleotides at p osition 970-975 in the HA(1) coding region. This resulted in an arg-ly s insertion near the proteolytic cleavage site of the HA protein. The remainder of the HA sequence differed by an additional seven amino aci ds from the parent. The HA precursor of the derivative, but not the pa rent, was readily cleaved during replication in cell culture without a ddition of trypsin. In experimentally infected chickens, the derivativ e produced lesions typical of highly pathogenic avian influenza. A rev erse transcriptase-polymerase chain reaction (RT-PCR) primer set was d esigned to amplify exclusively from molecules with the inserted six nu cleotides. The set yielded product only from the selected derivative s amples and not the parent. Thus, the levels of the HP variants in the parent stock were undetectable, or the insertion occurred rapidly duri ng the selection process.