Influenza A viruses are significant human pathogens causing yearly epidemics and occasional pandemics. Past pandemics have results 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 influenza A virus is central to its surveillance and control. While immune-driven antigenic drift is a key determinant of viral evolution across epidemic seasons, the evolutionary processes shaping influenza virus diversity within seasons are less clear. In this study we showed with a phylogenetic analysis of 413 complete genomes of human H3N2 influenza A viruses collected between 1997 and 2005 from New York State, United States, that genetic diversity is both abundant and largely generated through the seasonal importation of multiple divergent clades of the same subtype (1). These clades co-circulated within New York State, allowing frequent reassortment and generating genome-wide diversity. However, relatively low levels of positive selection and genetic diversity were observed at amino acid sites considered important in antigenic drift. These results indicate that adaptive evolution occurs only sporadically in influenza A virus; rather, the stochastic processes of viral migration and clade reassortment play a vital role in shaping short-term evolutionary dynamics. Thus, predicting future patterns of influenza virus evolution for vaccine strain selection is inherently complex and requires intensive surveillance, whole-genome sequencing, and phenotypic analysis.? ? Avian influenza A virus surveillance:? Surveillance for avian influenza A viruses in wild bird populations is often limited by requirements for a cold chain from time of specimen collection and availability of ultra low temperature specimen storage within a few hours or days and then laborious classical virological procedures. Successful storage of specimens in preservatives at ambient temperature and subsequent detection of RNA by RT-PCR would assist influenza surveillance efforts to become more widespread in remote areas as well as more timely and inexpensive. We evaluated the efficacy of this approach using bird feces spiked with influenza A virus preserved with guanidine and commercial buffers or alcohols at ambient temperature and analyzed by RT-PCR protocols. Virus specific RT-PCR products of at most 206 base pairs for samples were recovered when preserved with alcohols and up to 521 base pairs for samples preserved with guanidine or commercial buffers. This suggests this approach is feasible in the field and that preserved specimens may be better assayed molecularly when preserved in guanidine or commercial buffers (2).? ? We used ethanol-fixed cloacal swabs to allow avian influenza virus surveillance in remote areas of Alaska (3). Alaska was chosen because both Asian/Pacific and North American flyways converge in central Alaska. Our extramural collaborators and NIH staff evaluated avian influenza viruses (AIV) in the Minto Flats State Game Refuge, high-density waterfowl breeding grounds in 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. Differences in the prevalence of AIV infections by sex and by age classes of Northern Pintail and Mallard ducks were detected, but the significance of these differences is undefined. In the 500 paired samples, molecular screening detected positive birds at a higher rate than viral isolation; however, 20 AIV isolates were recovered from PCR-negative ducks. Further research is warranted to compare the two screening protocols potential for estimating true prevalence in wild birds. These results indicate that the Minto Flats region of Alaska will be a valuable study site for a longitudinal research project designed to gain further insight into the natural history, evolution, and ecology of AIV in wild birds.
