Conclusion
Since 1996, reassortment events involving H5N1 HPAI viruses have, as far as detected, only rarely led to the generation of new H5Nx subtypes. The 2008 generation of an H5N5 reassortant virus (prototype A/duck/Guangdong/wy/24/2008) represents the creation of a new HPAI virus that has led to the generation of a range of novel H5Nx reassortants that acquired novel NA proteins (H5N2, H5N6, and H5N8). The H5N6 reassortant became of particular concern after spreading over a wide geographic area in Southeast Asia and causing a fatal human infection in China. Meanwhile, the H5N8 subtype spread to Europe in November 2014, resulting in large economic losses in the poultry industry. On the basis of reports from the World Organisation for Animal Health, H5N8 and H5N2 viruses were detected in Canada and the United States in December 2014.
In this study, we exclusively focused on the unique occurrence of new HA–NA combinations. Recent publications have already described the reassortment events of the internal gene segments of several of the viruses mentioned above. In contrast to novel HA–NA combinations, novel constellations of internal gene segments are far from unique and have frequently been observed for HPAI H5N1 viruses. Our analysis indicates that new HPAI viruses have emerged that carry H5 proteins capable of matching with multiple NA subtypes. Whether the formation of new HA–NA combinations confers a selective advantage that contributed to the emergence of these novel subtypes is not known and requires elaborate research. However, the balance between HA (receptor binding) and NA (receptor cleavage) protein activities is known to be critical to cell entry and host tropism and may be an important factor that lead to the emergence of new HA–NA combinations. In contrast to HPAI H5N1, the novel clade 2.3.4.4 viruses, excluding H5N6 viruses, have not caused human infections. However, it is unknown to what extent the repeated acquisition of a new NA proteins could enhance the rate of evolution of the HA protein. Obviously such changes could further affect host and tissue specificity, potentially having serious consequences. Therefore, surveillance is required to monitor further spread, evolution, and potential changes in host range.