Characterizing the temporal patterns of avian influenza virus introduction into Japan by migratory birds

The objectives of the present study were to observe the temporal pattern of avian influenza virus (AIV) introduction into Japan and to determine which migratory birds play an important role in introducing AIV. In total, 19,407 fecal samples from migratory birds were collected at 52 sites between October 2008 and May 2015. Total nucleic acids extracted from the fecal samples were subjected to reverse transcription loop-mediated isothermal amplification to detect viral RNA. Species identification of host migratory birds was conducted by DNA barcoding for positive fecal samples. The total number of positive samples was 352 (prevalence, 1.8%). The highest prevalence was observed in autumn migration, and a decrease in prevalence was observed. During autumn migration, central to southern Japan showed a prevalence higher than the overall prevalence. Thus, the main AIV entry routes may involve crossing the Sea of Japan and entry through the Korean Peninsula. Species identification was successful in 221 of the 352 positive samples. Two major species sequences were identified: the Mallard/Eastern Spot-billed duck group (115 samples; 52.0%) and the Northern pintail (61 samples; 27.6%). To gain a better understanding of the ecology of AIV in Japan and the introduction pattern of highly pathogenic avian influenza viruses, information regarding AIV prevalence by species, the prevalence of hatch-year migratory birds, migration patterns and viral subtypes in fecal samples using egg inoculation and molecular-based methods in combination is required.