VI by egg inoculation is considered the gold standard method for the diagnosis of AI viruses. Hence, Korea is likely exposed to an inflow of AI virus via wild birds. The Korea peninsula is situated within the East Asia-Australian flyway of migratory wild birds during the fall to winter seasons. Indeed, wild birds may play a critical role in the introduction of the Avian influenza (AI) virus from foreign virus reservoirs through migration. In 2006, HPAI virus was first detected in the feces of wild birds, and many studies were subsequently conducted to reveal the nature of the correlation between wild birds and HPAI virus outbreaks. Before 2018, HPAI outbreaks have occurred seven times, causing great economic losses in the poultry industry. The highly pathogenic avian influenza (HPAI) virus was first reported in 2003 at a layer farm in Korea. The isolation of LPAI virus from migratory wild birds is also consistently reported. Keywords: Avian influenza, conventional method, next generation sequencing, Korea, wild birdĮver since the low-pathogenic avian influenza (LPAI) virus was first reported in Korea in 1996, isolation of several subtypes of LPAI viruses originating from live-bird markets and backyard bird stalls has been reported. Therefore, different methods should be combined for optimal surveillance, and further studies are needed in aspect of the introduction and application of new methods such as NGS. However, conventional methods of detection have some limitations.
Prevention of the occurrence of AI virus requires a workflow for rapid and accurate virus detection and verification. NGS analysis confirmed that 12% of the identified viruses were mixed-subtypes which were not detected by cRT-PCR. The cRT-PCR subtyping results mostly coincided with the NGS results, although NGS detected the presence of 11 HA genes and four NA genes that were not detected by cRT-PCR. Fifty AI virus specimens were isolated from fresh fecal samples and typed. In this study, 124 out of 11,145 fresh samples of wild migratory birds tested were rRT-PCR positive only 52.0% of VI positive samples were determined as positive by rRT-PCR from fecal supernatant. To identify and characterize the AI virus, we employed commonly used methods, namely, virus isolation (VI) via egg inoculation, real-time reverse transcription-polymerase chain reaction (rRT-PCR), conventional RT-PCR (cRT-PCR) and a newly developed next generation sequencing (NGS) approach. In the current study, we surveyed wild bird feces for the presence of AI virus that had been introduced to Korea between September 2017 and February 2018. Consequently, the likelihood of introduction of numerous subtypes and pathotypes of the Avian influenza (AI) virus to Korea has been thought to be very high. Korea is located within the East Asian-Australian flyway of wild migratory birds during the fall and winter seasons.