Downloaded from www.microbiologyresearch.org by IP: 23.20.70.183 On: Thu, 08 Sep 2016 23:08:32 Prevalence and diversity of avian influenza viruses in environmental reservoirs Andrew S. Lang, 1 Anke Kelly 2 and Jonathan A. Runstadler 2 Correspondence Jonathan A. Runstadler j.runstadler@uaf.edu 1 Department of Biology, Memorial University of Newfoundland, St John’s, NL A1B 3X9, Canada 2 Institute of Arctic Biology, PO Box 757000, University of Alaska Fairbanks, Fairbanks, AK 99775, USA Received 10 August 2007 Accepted 14 September 2007 Little is known about the ecology and evolution of avian influenza in the natural environment, despite how these affect the potential for transmission. Most work has focused on characterizing viruses isolated from hosts such as waterfowl, and there have also been several instances of isolation and detection from abiotic sources such as water and ice. We used RT-PCR to amplify and characterize the influenza virus sequences present in sediments of ponds that are used heavily by waterfowl. The detection rate of influenza virus was high (.50 %). Characterization of the viruses present by sequencing part of the haemagglutinin (HA) gene showed that there is a diverse collection of viruses in these sediments. We sequenced 117 partial HA gene clones from 11 samples and detected four different HA subtypes (H3, H8, H11 and H12), with approximately 65 % of clone sequences being unique. This culture-independent approach was also able to detect a virus subtype that was not found by sampling of birds in the same geographical region in the same year. Viruses were detected readily in the winter when the ponds were frozen, indicating that these sediments could be a year-to-year reservoir of viruses to infect birds using the ponds, although we have not shown that these viruses are viable. We demonstrate that this approach is a feasible and valuable way to assess the prevalence and diversity of viruses present in the environment, and can be a valuable complement to more difficult viral culturing in attempting to understand the ecology of influenza viruses. INTRODUCTION There is increased recent concern about the possible arrival in North America of highly pathogenic strains of avian influenza viruses (Chen et al., 2005, 2006; Ferguson et al., 2005; Liu et al., 2005; Olsen et al., 2006; Savill et al., 2006). Alaska is an intersection of migratory bird routes originating throughout the world, including North and South America, Asia, Australia and Africa. Millions of migratory birds arrive in Alaska to breed each spring, and many of these breeding migrants are waterfowl. There has been previous work to isolate and characterize influenza viruses from waterfowl in Alaska (Ito et al., 1995; Runstadler et al., 2007; Spackman et al., 2005), and these efforts continue. The information gained about influenza viruses in these birds is important for understanding the biology of influenza viruses and their spread amongst birds from different locations. This, in turn, is important for understanding the public health risks associated with these viruses and their potential transmission to agricultural species. Unfortunately, the role of the physical and biogeochemical environment as an integral part of this transmission is poorly understood. We recently described samples collected from an important waterfowl-breeding wetland area, the Minto Flats State Game Refuge in the interior region of Alaska (Runstadler et al., 2007). Cloacal swabs were taken from 880 ducks in the summer of 2005 and screened for the presence of influenza viruses. Over 25 % of the samples were found to be positive for influenza virus (Runstadler et al., 2007). Culturing and subsequent subtyping of viruses from a subset of these samples revealed five different haemagglu- tinin (HA)/neuraminidase (NA) subtypes (H3N6, H3N8, H4N6, H8N4 and H12N5). This work demonstrated an overall high rate of virus infection in birds at this location and showed that a diverse array of subtypes was present. This particular location was also the focus of a previous study between the years 1991 and 1994 (Ito et al., 1995), where six virus subtypes were isolated from 391 waterfowl faecal samples from Minto Lake. Here, we report efforts to characterize the occurrence and diversity of influenza viruses in the sediments of ponds used by a wide variety of migratory waterfowl. Located The GenBank/EMBL/DDBJ accession numbers for the sequences reported in this paper are EU086918–EU087180. A supplementary figure showing sampling locations for this study and supplementary tables identifying viruses included in the H3 and H11 phylogenetic analyses are available with the online version of this paper. Journal of General Virology (2008), 89, 509–519 DOI 10.1099/vir.0.83369-0 0008-3369 G 2008 SGM Printed in Great Britain 509