The evolution of the major surface hemagglutinin (HA) antigen of type A H5N1 influenza viruses is explored at the amino acid level using a new proteotyping approach. Alignments of translated hemagglutinin gene sequences of all characterised type A H5N1 strains, or subsets thereof, has enabled the presence of signature peptides of conserved sequence and unique mass to be investigated from the perspective of the host, period and region from which strains were isolated. Consistent with the rapid, cross species transmission of H5N1 strains among migratory birds, poultry and humans throughout south-east Asia, no signatures unique to the host or region were found. Nevertheless, several period-specific signature peptides were identified that enable strains associated with the 1997 H5N1 pandemic to be rapidly differentiated from those in circulation across the subsequent decade.

The application of a rapid and direct proteotyping approach with which to identify the gene origin of viral antigens in a reassortant influenza strain is demonstrated. The reassortant strain, constructed for a vaccine against type A 2009 H1N1 pandemic influenza, contains genes derived from a wild-type pandemic strain (A/California/7/2009) and an egg adapted high-growth strain (denoted NYMC X-157) derived from an earlier A/Puerto Rico/8/34 strain. The proteotyping approach employs modern proteomics methods and high resolution mass spectrometry to correctly establish that the genes of the surface antigens, hemagglutinin and neuraminidase, are derived from the A/California/7/2009 strain while those for nucleoprotein and matrix protein M1 antigens are derived from the NYMC X-157 strain. This is achieved for both gel-separated antigens and those from a whole vaccine digest. Furthermore, signature peptides detected in the mass spectra of the digested antigens enable the engineered reassortant strain to be identified as a type A virus of the H1N1 subtype in accord with earlier studies. The results demonstrate that proteotyping approach provides a more direct and rapid approach over RT-PCR with which to characterize reassortant strains of the influenza virus at the molecular protein level. Given that these strains pose the greatest risk to human and animal health and have been responsible for all human pandemics of the 20th and 21st centuries, there is a vital need for the origins and evolutionary history of these strains to be rapidly established.

This study describes the first recorded outbreak of HPAI in the city of Seoul, in captive birds held in an exhibition for public viewing at a local district office. The index cases were two pheasants, which had been introduced into the exhibit on 24 April, 4 days prior to death, from a store in a local market in Gyeonggi-do. Ducks and chickens from an HPAI outbreak farm, subsequently confirmed on 4 May, had also been held in this store. This outbreak highlights the potential role of local markets in AIV transmission. This outbreak led to considerable public health concern in Korea, however, no human cases were reported. The non-commercial poultry sector needs to be considered in national plans for preparedness and response.