Influenza A viruses (IAV) are significant human pathogens causing yearly epidemics and occasional pandemics. Past pandemics have resulted in significant morbidity and mortality. The 1918 influenza pandemic was thought to have resulted in the death of at least 675,000 people in the U.S., and 40 million people worldwide. Annual influenza A virus epidemics are also very significant, resulting in approximately 30,000 deaths in the U.S. per year. Pandemic strains of influenza emerge periodically and are thought to be derived ultimately from avian influenza A viruses. The natural reservoir of influenza A viruses is thought to be wild waterfowl. Genetically and antigenically diverse influenza A viruses circulate in wild birds and viral strains from this pool can adapt to new hosts, including humans and domestic animals. Influenza A viruses are also significant pathogens for agriculturally important animals like poultry, swine, and horses. Understanding the mechanisms of host switching are very important for surveillance and pandemic preparedness. Understanding the molecular basis underlying the annual evolution of human influenza will aid in vaccine strain selection. ? ? Human influenza A virus evolution:? Understanding the evolutionary dynamics of human influenza A virus (IAV) is central to its surveillance and control. It was demonstrated that intrasubtypic reassortment is commonly observed in human H1N1 viruses, using a data set of 71 representative complete genome sequences sampled between 1918 and 2006. Specifically, we demonstrated that the severe 1947 epidemic strain acquired novel PB2 and HA genes through intrasubtypic reassortment, which may explain the abrupt antigenic evolution of this virus. Similarly, the severe 1951 influenza epidemic may also have been associated with reassortant H1N1 viruses.? Another analysis of human IAV evolution examined the genome-scale evolutionary dynamics of IAV. How genomic processes relate to global influenza epidemiology, in which the H3N2 and H1N1 subtypes co-circulate, is still poorly understood. Through an analysis of 1,302 complete viral genomes sampled from temperate populations in both hemispheres, we showed that the genomic evolution of IAV is characterized by a complex interplay between frequent reassortment and periodic selective sweeps. The H3N2 and H1N1 subtypes exhibit different evolutionary dynamics, with diverse lineages circulating in H1N1, indicative of weaker antigenic drift. These results suggest a source-sink model of viral ecology in which new lineages are seeded from a persistent influenza reservoir source, which we hypothesize to be located in the tropics, to sink populations in temperate regions. ? These analyses demonstrated that, at any given time, individual IAV gene segments can differ substantially in their relative genetic diversity and hence in phylogenetic history. To determine the causes of these differences in genetic diversity, we estimated the time to the most recent common ancestor (TMRCA) of each segment for each influenza season. Most TMRCAs fall well before the start of the season from which they were sampled, such that multiple lineages persist across multiple epidemic troughs, consistent with our source-sink model. TMRCAs also vary among segments and among years, reflecting the interacting processes of genomic reassortment, natural selection and gene flow. Reassortment places antigenically novel hemagglutinin variants into different genomic backgrounds, a fraction of which may restore, or even increase, the viral replicative fitness that may have been lost as a result of the change in hemagglutinin. ? In addition to genome-wide interactions, it is essential to consider the complex spatial epidemiological dynamics of influenza if we are to fully understand antigenic evolution. We observed consistent dynamical patterns in two populations illustrative of temperate regions in the Northern and Southern Hemispheres, together with the persistence of viral lineages across multiple epidemics. To resolve these apparently contradictory observations, we proposed the existence of a continuous reservoir or source population, within which the strong selection for antigenic change takes place. Such complexity necessarily means that the long-term success of any individual lineage of influenza virus is dependent not only on its antigenic properties but also on its replicative capacity, its transmissibility and the environmental factors that perhaps underlie the seasonality of influenza in temperate regions. ? Whether homologous recombination is another mechanism employed in IAV evolution was unclear. To determine the extent of homologous recombination in human influenza A virus, we assembled a data set of 13,852 sequences representing all eight segments and both major circulating subtypes. We concluded that, if it occurs at all, homologous recombination plays only a very minor role in the evolution of human IAV.? ? Avian influenza A virus surveillance:? We used ethanol-fixed cloacal swabs to allow avian influenza virus surveillance in remote areas of Alaska. Five hundred paired cloacal samples from dabbling ducks (Northern Pintail, Mallard, Green Wing Teal, and Widgeon) were placed into ethanol and viral transport medium. Additional ethanol-preserved samples were taken. Of the ethanol-preserved samples, 25.6% were AIV RNA-positive by real-time RT-PCR. The hemagglutinin and neuraminidase subtypes were determined for 38 of the first-passage isolates. Five influenza A virus HA-NA combinations were identified: H3N6, H3N8, H4N6, H8N4, and H12N5. In the 500 paired samples, molecular screening detected positive birds at a higher rate than viral isolation. ? We developed a molecular method for hemagglutinin (HA) subtyping that could be used with fixed samples. We developed a novel method for molecular subtyping of AIV HA genes using degenerate primers designed to amplify all known hemagglutinin subtypes. This method was used to perform subtyping RT-PCR on 191 influenza RNA-positive ethanol-fixed cloacal swabs obtained from 880 wild ducks in central Alaska in 2005. Seven different co-circulating HA subtypes were identified in this study set, including H1, H3, H4, H5, H6, H8, and H12. In addition, 16% of original cloacal samples showed evidence of mixed infection, with samples yielding from two-to-five different HA subtypes. This study further demonstrates the complex ecobiology of avian IAV in wild birds.? We determined the complete genomic sequences of 167 wild bird avian IAV isolates from 14 bird species sampled in four locations across the United States representing 29 HA and neuraminidase (NA) subtype combinations, with up to 26% of isolates showing evidence of mixed subtype infection. Through a phylogenetic analysis of the largest data set of avian IAV genomes compiled to date, we observed a remarkably high rate of genome reassortment but little evidence of gene segment association. Evidence for occasional inter-hemisphere gene segment migration and reassortment was obtained. From this, we proposed that avian IAV in wild birds forms transient genome constellations, continually reshuffled by reassortment, in contrast to the spread of a limited number of stable genome constellations that characterizes the evolution of mammalian-adapted IAV.Based on these analyses, we hypothesize that avian IAV in wild birds exists as a large pool of functionally equivalent, and often inter-changeable gene segments that form transient genome constellations, without the strong selective pressure to be maintained as linked genomes.
