Molecular Analysis of H7 Avian Influenza Viruses from Australia and New Zealand: Genetic Diversity and Relationships from 1976 to 2007

Full-genome sequencing of 11 Australian and 1 New Zealand avian influenza A virus isolate (all subtype H7) has enabled comparison of the sequences of each of the genome segments to those of other subtype H7 avian influenza A viruses. The inference of phylogenetic relationships for each segment has been used to develop a model of the natural history of these viruses in Australia. Phylogenetic analysis of the hemagglutinin segment indicates that the Australian H7 isolates form a monophyletic clade. This pattern is consistent with the long-term, independent evolution that is, in this instance, associated with geographic regions. On the basis of the analysis of the other H7 hemagglutinin sequences, three other geographic regions for which similar monophyletic clades have been observed were confirmed. These regions are Eurasia plus Africa, North America, and South America. Analysis of the neuraminidase sequences from the H7N1, H7N3, and H7N7 genomes revealed the same region-based relationships. This pattern of independent evolution of Australian isolates is supported by the results of analysis of each of the six remaining genomic segments. These results, in conjunction with the occurrence of five different combinations of neuraminidase subtypes (H7N2, H7N3, H7N4, H7N6, H7N7) among the 11 Australian isolates, suggest that the maintenance host(s) is nearly exclusively associated with Australia. The single lineage of Australian H7 hemagglutinin sequences, despite the occurrence of multiple neuraminidase types, suggests the existence of a genetic pool from which a variety of reassortants arise rather than the presence of a small number of stable viral clones. This pattern of evolution is likely to occur in each of the regions mentioned above.The emergence of highly pathogenic avian influenza viruses of subtype H5N1 as a potential human pandemic disease threat has focused attention on the roles that wild birds play in the maintenance and distribution of avian influenza viruses (18, 22). Moreover, the H5 and H7 subtypes of avian influenza A virus are major causes of economic loss in poultry production through disease. In Australia, there have been five documented outbreaks of H7 subtype avian influenza A virus disease, with evidence of adaptation to the poultry host being provided by sequence data supporting the presence of high-pathogenicity avian influenza virus (HPAI) isolates in poultry. Waterfowl (Anseriformes order, particularly ducks, geese, and swans) and the waders and gulls (Charadriiformes order, particularly gulls, terns, and waders) have been found to be the major global natural reservoirs of influenza A viruses. Transmission of avian influenza viruses from wild birds to production poultry and geographic spread are dependent upon the migratory behavior of the wild bird reservoir hosts. Members of the Anseriformes and Charadriiformes orders undertake both irregular and regular transcontinental and intercontinental migrations. During these migrations, large numbers of birds congregate at aquatic feeding locations, providing ideal sites for cross-species transmission of avian influenza viruses. A variety of mechanisms have been observed whereby influenza A viruses adapt rapidly. These include genetic shifts facilitated through genome segment reassortment, as well as genetic drift through the insertion, deletion, and substitution of nucleotides. The error-prone RNA replication and a lack of error correction are the causes of drift. In vivo, this results in viral genetic diversity within any viral sample, or a quasispecies, thus providing a pool of closely related variant viruses from enabling events, such as viral adaptation to new hosts (25). Long-term sampling of water birds in North America and Europe has started to elucidate the ecology and biology of the avian influenza A virus types in the natural reservoirs in these regions (8, 18, 22). There is a suggestion that two superfamilies, the Eurasia (which in the context of this paper includes Europe, Asia, and Africa) and the Americas superfamilies, exist; however, the extent of overlap and the rate of transfer of influenza viruses between these two regions are not well-defined. Recent studies suggest that intercontinental virus exchange is slow and limited (17), while a detailed analysis of the differences between H7 hemagglutinin (HA) segments circulating in Europe and China showed that the H7 hemagglutinin segments shared a recent common ancestor and limited sequence divergence on a background of multiple reassortant virus genotypes between 1999 and 2005 (7).Avian influenza A viruses of the Oceania region (Australia, New Zealand, and southwest Pacific) have been far less well studied (3). Australia and New Zealand are at the southern extremity of a number of major bird migration pathways. Waders in the Charadriidae family migrate to south and southeastern Australia and New Zealand from their summer breeding grounds in Arctic regions of Siberia and Alaska, where they freely mix with the same or other species which migrate into the shared breeding grounds of Eurasia and the Americas (30). Pelagic seabirds of the Procellariformes order breed on and around Australian and New Zealand coasts during the southern hemisphere summer and migrate to maritime regions of the northern Pacific associated with Japan, Russia, and Alaska. Some move as far as the west coasts of North and South America (28). Unlike North and South America and Europe, where regular migrations of ducks, geese, swans, etc., are established, the members of the Anatidae family (ducks, etc.) in Australia and New Zealand are mainly endemic residents (30). However, within Australia, ducks undertake long-distance movements in response to water availability. Movements of waterfowl from northern Australia to nearby areas of Southeast Asia are believed to occur but are limited, as suggested by Wallace''s Line (19). Generally, these waterfowl movements have not been well studied (30). The risks to Australian poultry production systems by movement of H5N1 via migratory shorebirds and nomadic wildfowl have been assessed to be low using risk-based analysis techniques (9, 10).Regular and extensive surveillance sampling of migratory birds has been undertaken in North America and northern Europe (17, 18). The findings have shed significant insights into the ecology of the viruses and their hosts (8, 17). In contrast, surveillance sampling of wild birds in Asia and Oceania has been spasmodic and sparse, until the recent emergence of H5N1 highly pathogenic avian influenza virus as a poultry and human disease threat. Spasmodic and small-scale outbreaks of highly pathogenic avian influenza virus have occurred in Australian poultry production flocks located in the southeastern region of the continent. These poultry production areas are concentrated close to large human population centers (26, 33, 34). Each of the Australian outbreaks has been rapidly controlled by slaughter of infected flocks. All have been caused by avian influenza viruses of the H7 subtype, which appear to have entered production poultry from water birds, possibly wild ducks, via contaminated water supplies used on the poultry farms. Disease has occurred on five occasions: 1976 (H7N7), 1985 (H7N7), 1992 (H7N3), 1994 (H7N3), and 1997 (H7N4) (13, 14, 26, 27, 31, 34). National on-farm biosecurity measures have been focused on reducing the likelihood of future outbreaks. The availability of avian influenza virus isolates from poultry and wild birds associated with these outbreaks, along with a small number of subtype H7 avian influenza viruses isolated from wild ducks during recent national surveillance programs in Australia and New Zealand, provided the opportunity to explore the relationships of Australian and New Zealand subtype H7 avian influenza virus isolates with viruses circulating elsewhere in the world.