Surveillance of avian influenza viruses from 2014 to 2018 in South Korea

Similar to our previous report on avian influenza surveillance from 2009 to 2013 in South Korea¹⁴, percent IAV-positivity of bird fecal samples and prevalent IAV subtypes varied per year and per site over the winters of November 2014 to January 2018. All H5 and H7 IAVs isolated during the surveillance period were LPAIVs and were highly similar to isolates obtained from different locations along the EAAF. As expected, most of the HA and NA genes of the isolates clustered with those from isolates obtained from other countries along the EAAF, including China, Japan, and Mongolia. Supporting recent reports that IAVs from wild birds do not always follow rigid flyway boundaries and that there is intercontinental movement of IAV gene pools carried by wild fowl, some of the HA and NA genes of our isolates also cluster with a number of North American isolates. In addition, as shown by the present study and previous results on the surveillance of avian influenza viruses in South Korea¹⁴, percent IAV-positivity of samples tends to fluctuate across the years; however, we currently have no specific explanation for this interannual variability.

Influenza host species identification using the COI gene amplified from genomic DNA extracted from bird droppings collected during the winter of 2016–2017 indicates that majority of the isolates during the season were carried by migratory geese (Anser spp.). Although we were unable to identify the host at the species level, we speculate that the isolated IAVs came from either the taiga bean goose (A. fabalis) or the greater white-fronted goose (A. albifrons), which are the most dominant goose species that winter in South Korea from breeding sites in Far East Siberia¹⁷. IAVs, including HPAIVs, have been previously isolated from these goose species in other studies in South Korea^13,18,19; migratory geese are therefore presumed to contribute to HPAIV outbreaks in the country.

Similar to our surveillance study in previous years, IAVs of the H6 subtype were among the most frequently isolated during successive winters in South Korea. This is corroborated by studies that suggest that H6-subtype IAVs are the most frequently isolated subtypes from wild waterfowl especially in Asia²⁰. A study from China reported that 11.07% of the isolates from wild birds in 2016–2017 were of the H6 subtype, making H6 the most predominant subtype in that study²¹. In our study, IAVs of the H6 subtype were likewise the most prevalent over the winter of 2016–2017, which may indicate similar distributions of IAV subtypes along the EAAF that year. Another study has reported that 49.44% (44/89) of isolates from wild birds in China along the EAAF over 2015–2019 were of the H6 subtype²². In our study, the H6-subtype IAVs were consistently isolated in all four years, suggesting its persistence in migratory waterfowl populations that traverse the EAAF. Recent studies from China suggest that H6NX viruses from wild birds, including A/Anser fabalis/China/Anhui/L221/2014 (H6N1), which is 99% identical to one of our isolates (A/Kor/CN69/2014 [H6N2]), can infect mice and replicate at high titers in mouse lungs without prior adaptation²³. While H6-subtype IAVs are considered LPAI and are non-reportable, they may still cause damage to the poultry industry (~ 30% drop in egg production)²⁰. Given the potential ability of H6-subtype IAVs to infect mammals, their persistence in the wild, and their effects on poultry, closer investigation of H6-subtype IAVs is warranted.

Meanwhile, in our study, viruses of the H1 subtype were predominant in the winter of 2014–2015, accounting for 50% (18/36) of the isolates that season. This is in line with a previous report of an H1NX outbreak among wild fowl in Anhui, China²⁴ and from another study in bird wintering wetlands in Yangtze River, China²⁵.

Despite H5 HPAI outbreaks in poultry in South Korea over the study period, we found no H5 HPAIV during our surveillance. However, H5-subtype LPAIVs, especially H5N3 LPAIVs, were among the most prevalent subtypes, particularly in the winters of 2014–2015 (27%) and 2016–2017 (12.5%). Another surveillance study in South Korea also reported the prevalence of H5 LPAIVs from wild bird habitats in 2015 (13.1%) and 2017 (14.4%), coinciding with our results²⁶. In contrast to our findings, this previous study reported the isolation of H5-subtype LPAIVs in 2017 in the Jeonnam Province of South Korea, while we obtained no isolate from Gocheonam Reservoir in Jeonnam Province. However, the Jeonnam isolates of this study were obtained in December 2017, while we performed sampling in the same region in November 2016. The differences in isolation rates of H5 and other AIV isolates in this province may be due to sampling bias, as we only sampled a very small portion of a large migratory bird stopover site, and only performed sampling on a single day in a single winter season. Obtaining more avian fecal samples later in the season and on other years may have yielded different results.

All our H5 isolates carried markers of increased transmission to mammals. These markers include the N171and T172 variations which, when present together, have been reported to increase binding of the A/Viet Nam/1203/2004 (H5N1) HA to the human ⍺2,6-linked sialic acid without loss of binding to the avian ⍺2,3-linked sialic acid¹⁵. Another variation found in most of our isolates was P251, which has been reported to slightly increase the binding of the A/duck/Egypt/D1Br12/2007 (H5N1) HA binding to ⍺2,6-linked sialic acid¹⁶. The L226 variation was not present in any of our H5 isolates. These findings suggest that H5 genotypes circulating in wild birds passing through South Korea and adjacent countries continue to gain potential to infect mammals. The persistence of these H5 sequences in the wild increases their chances of reassortment with other IAV genes that may lead to eventual crossing to the mammalian species barrier and perhaps to sustained transmission among mammals including humans. Considering that H5 LPAIVs have been reported as precursors of HPAIVs, continued monitoring of H5 LPAIVs is needed to guide mitigation and control measures for H5 IAV infections in both wild and domestic fowl.

Only 2 fecal samples (2.6% of total) yielded H7-subtype isolates, both of which were H7N7 LPAIVs. Interestingly, both the HA and NA genes of the isolates were highly identical to those of an H7N7 isolate from a red-crowned crane (A/red-crowned crane/Korea/H1026/2017) in a zoo in South Korea isolated in March 2017²⁷. Our H7N7 samples were collected in January 2017, placing this particular H7N7 gene pool in South Korea earlier than closely related H7N7 isolates collected from migratory mallards in February to March of 2017. Notably, H7 isolation was more frequent in the other study (43.6% of all isolates in early 2017) than in ours (8.3%), possibly owing to differences in sample timing, host species, and sampling method²⁸. Regardless, transmission of H7 IAVs from migratory fowl to other bird species was highlighted by the isolation of the H7N7 isolate in the captive crane, which further emphasizes the likelihood of transmission of H7 IAVs from migratory wild birds to domestic birds.

As in previous years of our study, the IAV isolation rate was variable per year. This may be a result of changes in seasonal temperatures across different years. Migration of fowl, including bean geese and white-fronted geese, is highly affected by temperatures. The timing of bird migration to wintering sites may change based on temperatures in both the breeding and wintering sites²⁹. This would then affect the composition of migratory fowl populations at stopover sites and, in turn, affect observed IAV prevalence. Environmental temperatures also affect the persistence of IAVs in aquatic environments and may affect the maintenance of IAVs in wild birds³⁰; changes in temperatures across the years would then alter the prevalence of IAV infection in wild birds. Thus, the timing of IAV surveillance studies such as this should take into account potential variabilities in environmental temperatures and timing of bird migration to be able to obtain a more comprehensive view of IAV prevalence in breeding, stopover, or wintering sites.

One limitation of our study is that, at the time of writing, the sequences we report are already at least five years old. Several studies have already reported more recent data (2019 to 2022) on circulating avian influenza viruses among wildfowl that visit South Korea, although most have focused on H5-, H7-, and H9-subtype influenza viruses^19,31,32,33. Given the continued threat of AIVs to both animal and human health, large amounts of genetic data are important to allow us to track the movement of avian influenza virus gene pools across time and geographic locations. Ensuring that sequences of influenza viruses over large periods of time are reported will fill in potential gaps of information in tracing back the evolution of future avian-origin influenza virus pandemics. This has been highlighted by the recent coronavirus disease (COVID-19) pandemic, where, owing to very few surveillance studies in the past and very little existing genetic information on severe acute respiratory syndrome (SARS)-related viruses prior to the pandemic, scientists have difficulty in fully understanding the emergence of SARS coronavirus 2 (SARS-CoV-2). Global public health will undoubtedly benefit from all available genetic information on AIVs especially within the past couple of decades, as these would be the first resources to investigate should an avian-origin pandemic arise. It would also allow us to further understand the dynamics of the continued spread and circulation of avian influenza viruses in the wild in the hope of preparing for large outbreaks in both wild and domestic birds.

Additionally, most AIV surveillance studies in South Korea focus on H5-, H7-, and H9-subtype influenza viruses. While these subtypes pose the highest and most immediate risk to veterinary and human health, other subtypes of AIVs may contribute genetic material to future threats, and, in rare cases, cause disease and death in humans³⁴. Thus, we believe that long-term surveillance studies on IAVs that include LPAIVs and HPAIVs and the reportable and non-reportable subtypes can provide the scientific community a wealth of genetic information that can be utilized when the need arises.

All in all, in this study, we were able to collect a total of 6758 bird fecal samples from five migratory bird stopover sites in South Korea during the winters of 2014 to 2018 (November 2014 to January 2018). Of these, 75 (1.11%) tested positive for IAVs based on egg isolation and RT-PCR. The most common HA subtypes were H1, H6, and H5, and the most common NA subtypes were N1, N3, and N2. Based on sequence analysis, all isolates, including the H5- and H7-subtypes, were LPAIVs and were most similar to isolates collected along the EAAF. As in previous years, isolation rates and predominant subtypes varied per site and per year. Although we were unable to isolate HPAIVs from the bird fecal samples despite HPAIVs outbreaks in South Korea during the study period, this surveillance study provides insight into the dynamics of IAVs carried by migratory birds into South Korea.