Genetic characterization of viruses isolated in the environment can provide interesting information regarding gene prevalence and circulation in habitats used by waterfowl. Such information is critical to assess the possibility of inter-annual persistence in water, long-term circulation of environmentally-isolated virus strains in wild duck populations, and identify XAV939 potential genetic basis for the persistence of IA viruses in water. Previous studies have demonstrated differences in the ability of IA viruses to remain infective in water and have suggested that subtyperelated variations in persistence may exist, especially at low temperatures. Both isolates tested in our study persisted for several months in distilled water at constant temperatures, below 17uC and at neutral pH. Overall, the estimated persistence was higher than reported for 12 different waterfowl derived viruses. Limited data precludes conclusions related to increased environmental persistence associated with viruses recovered from the environment. Future experiments should however consider such a possibility as the demonstration of long-time persistence of water-isolated viruses would suggest the potential for inter-annual persistence in aquatic habitats. Inversely, habitats characterized by deep water may prevent freezing and strong temperature changes affecting viral persistence, but are less likely to be used by dabbling ducks. Viral persistence in frozen pond and lake water has been suggested to be an potential mechanisms for inter-annual persistence and source of contamination for waterfowl in breeding grounds. In high latitude habitats, shallow lakes and ponds may freeze relatively quickly during fall and winter, potentially reducing effects of such temperature changes. The effects of latitudinal-dependent temperature regimes on surface water viruses warrant future experimental work. Because diurnal surface water temperature fluctuations could represent a constraint on viral persistence, we also investigated the effects of temperature variations between 17uC and 23uC. Compared to results from constant temperature, no effects on viral persistence were observed. This suggests that diurnal surface water temperature fluctuations are not likely to significantly affect virus persistence, although as for freeze-thaw cycles additional environmental simulation studies would be required to confirm this trend. Experimental infections provided evidence that both waterisolated IA viruses replicated efficiently in Mallards and, in both cases, shedding patterns were consistent with a previous study performed with duck-isolated LP IA viruses. These results support that viruses isolated from surface water replicate in ducks as well as LP virus isolates that have a wild duck-origin. In this study, we highlighted that water-isolated IA viruses can provide complementary information regarding circulating viruses and viral genes in wild waterfowl populations. With current methodological limitations the recovery of viruses from aquatic habitats should not be considered as a replacement of traditional surveillance approaches utilizing wild bird or fecal sampling.