Characterisation of a highly pathogenic influenza A virus of subtype H5N2 isolated from ostriches in South Africa in 2004

Abstract

Objectives: The HPAI H5N2 strain that caused an outbreak in ostriches of the Eastern Cape Province, South Africa in 2004 was characterized.

Design: Haemagglutination inhibition (HI) and agar gel immunodiffusion (AGID) were performed on sera from ostrich farms in the outbreak region, and intravenous pathogenicity (IVPI) tests, reverse-transcriptase-polymerase-chain reaction (RT-PCR), nucleic acid sequencing and phylogenetic comparisons were performed on the HPAI H5N2 virus isolated during the outbreak.

Results: The deduced amino acid sequence at the HA0 cleavage site determined by RT-PCR and nucleotide sequencing was PQREKRRKKRGLF and thus the virus fell within the definition of a highly pathogenic virus, but in an IVPI test in chickens on the virus isolated from the index case and a value of 0.63 was recorded, which is below the criterion for highly pathogenic viruses in this in vivo test. After a further passage in embryonated eggs a second IVPI was carried out and an elevated value of 1.19 was obtained. Cloacal swabs were taken from the initial IVPI birds, inoculated into embryonated chickens eggs and a third IVPI was then performed on the resulting haemagglutinating, infective allantoic fluid. An index of 2.73 was recorded.

Conclusions: HI tests appeared to be the more sensitive test compared to AGID when testing for antibodies to avian influenza in sera. An ostrich-derived virus with a virulent HA0 cleavage site was not initially virulent in chickens but after passage in the latter the virulence increased. Phylogenetic analyses demonstrated the link between AI viruses carried by wild ducks and those infecting ostriches.

Figure 1

Maximum likelihood phylogenetic tree of partial H5 sequences, generated using Phylogeny Inference Package (phylip) software and including sequences generated from isolates submitted to VLA Weybridge as part of its EU/OIE/FAO Reference Laboratory status or obtained from the Influenza Sequence Database (ISD).