An aggregated mitochondrial distribution in preimplantation embryos disrupts nuclear morphology, function, and developmental potential.

A dispersed cytoplasmic distribution of mitochondria is a hallmark of normal cellular organization. Here, we have utilized the expression of exogenous Trak2 in mouse oocytes and embryos to disrupt the dispersed distribution of mitochondria by driving them into a large cytoplasmic aggregate. Our findings reveal that aggregated mitochondria have minimal impact on asymmetric meiotic cell divisions of the oocyte. In contrast, aggregated mitochondria during the first mitotic division result in daughter cells with unequal sizes and increased micronuclei. Further, in two-cell embryos, microtubule-mediated centering properties of the mitochondrial aggregate prevent nuclear centration, distort nuclear shape, and inhibit DNA synthesis and the onset of embryonic transcription. These findings demonstrate the motor protein-mediated distribution of mitochondria throughout the cytoplasm is highly regulated and is an essential feature of cytoplasmic organization to ensure optimal cell function.

CCR10-mediated Enhancement of T Cell Trafficking for Improved Tumor Immunotherapy.

BACKGROUND/AIM: The effectiveness of adoptive T cell therapy for solid tumors remains suboptimal, partly attributed to insufficient T cell infiltration into the tumor site. A promising strategy involves directing T cells towards the tumor utilizing tumor-specific chemokine receptors. MATERIALS AND METHODS: We analyzed chemokine receptor expression in activated T cells and chemokine expression in breast and lung cancer using The Cancer Genome Atlas (TCGA) data. Subsequently, we generated 1G4 T cell receptor-engineered T (TCR-T) cells with CCR10 and performed in vitro and in vivo efficacy tests. RESULTS: CCR10 exhibited insufficient expression in various human T cells. Analysis of TCGA RNA sequencing data revealed elevated expression of the chemokine CCL28, the corresponding chemokine for CCR10, in breast and lung cancer. Consequently, we generated CCR10-1G4 TCR-T cells. CCR10-1G4 dual expressing TCR-T cells exhibited comparable cellular cytotoxicity but increased mobility compared to 1G4 TCR-T cells in vitro. Furthermore, injecting CCR10-1G4 dual expressing TCR-T cells into a xenograft tumor model demonstrated enhanced in vivo trafficking and a greater reduction of tumor burden. CONCLUSION: This study highlights the potential of CCR10 for developing efficient adoptive T-cell treatments targeting solid tumors.

Miro1 depletion disrupts spatial distribution of mitochondria and leads to oocyte maturation defects.

Mitochondria are dynamic organelles that undergo regulated microtubule- and actin-mediated trafficking to meet local energy and metabolic needs. Mitochondrial trafficking may be particularly critical in large cells such as eggs and early embryos where spindle formation and polar body extrusion occur in specific regions of the cytoplasm. To investigate the role of mitochondrial distribution in oocytes we have targeted the mitochondrial membrane protein, MIRO1, which couples mitochondria to the motor protein-TRAK complex. Oocyte-specific deletion of MIRO1 leads to the formation of large aggregates of mitochondria in perinuclear and cortical compartments. Mitochondria remain capable of long-range trafficking during maturation, indicating redundancy in the mechanisms coupling mitochondria to motor proteins. Polar body extrusion in the absence of MIRO1 was reduced by approximately 20%. In MIRO1-deleted zygotes, mitochondria showed increased accumulation around the pronuclei but this did not affect mitochondrial distribution to daughter blastomeres. In vitro development of parthenogenetic embryos was also reduced, although no differences were found in the fertility of oocyte-specific Miro1 KO mice. These findings demonstrate MIRO1 acts as a mitochondrial adaptor, setting mitochondrial distribution in oocytes and early embryos, and disrupting this process compromises in vitro oocyte maturation and embryo development.

Depletion of oocyte dynamin-related protein 1 shows maternal-effect abnormalities in embryonic development.

Eggs contain about 200,000 mitochondria that generate adenosine triphosphate and metabolites essential for oocyte development. Mitochondria also integrate metabolism and transcription via metabolites that regulate epigenetic modifiers, but there is no direct evidence linking oocyte mitochondrial function to the maternal epigenome and subsequent embryo development. Here, we have disrupted oocyte mitochondrial function via deletion of the mitochondrial fission factor Drp1. Fission-deficient oocytes exhibit a high frequency of failure in peri- and postimplantation development. This is associated with altered mitochondrial function, changes in the oocyte transcriptome and proteome, altered subcortical maternal complex, and a decrease in oocyte DNA methylation and H3K27me3. Transplanting pronuclei of fertilized Drp1 knockout oocytes to normal ooplasm fails to rescue embryonic lethality. We conclude that mitochondrial function plays a role in establishing the maternal epigenome, with serious consequences for embryo development.

Functional Characterization of Endothelial Cells Differentiated from Porcine Epiblast Stem Cells.

Endothelial cells (ECs), lining blood vessels' lumen, play an essential role in regulating vascular functions. As multifunctional components of vascular structures, pluripotent stem cells (PSCs) are the promising source for potential therapeutic applications in various vascular diseases. Our laboratory has previously established an approach for differentiating porcine epiblast stem cells (pEpiSCs) into ECs, representing an alternative and potentially superior cell source. However, the condition of pEpiSCs-derived ECs growth has yet to be determined, and whether pEpiSCs differentiate into functional ECs remained unclear. Changes in morphology, proliferation and functional endothelial marker were assessed in pEpiSCs-derived ECs in vitro. pEpiSCs-derived ECs were subjected to magnetic-activated cell sorting (MACS) to collect CD-31+ of ECs. We found that sorted ECs showed the highest proliferation rate in differentiation media in primary culture and M199 media in the subculture. Next, sorted ECs were examined for their ability to act as typical vascular ECs through capillary-like structure formation assay, Dil-acetylated low-density lipoprotein (Dil-Ac-LDL) uptake, and three-dimensional spheroid sprouting. Consequently, pEpiSCs-derived ECs function as typical vascular ECs, indicating that pEpiSC-derived ECs might be used to develop cell therapeutics for vascular disease.

Oocyte mitochondria-key regulators of oocyte function and potential therapeutic targets for improving fertility.

The development of oocytes and early embryos is dependent on mitochondrial ATP production. This reliance on mitochondrial activity, together with the exclusively maternal inheritance of mitochondria in development, places mitochondria as central regulators of both fertility and transgenerational inheritance mechanisms. Mitochondrial mass and mtDNA content massively increase during oocyte growth. They are highly dynamic organelles and oocyte maturation is accompanied by mitochondrial trafficking around subcellular compartments. Due to their key roles in generation of ATP and reactive oxygen species (ROS), oocyte mitochondrial defects have largely been linked with energy deficiency and oxidative stress. Pharmacological treatments and mitochondrial supplementation have been proposed to improve oocyte quality and fertility by enhancing ATP generation and reducing ROS levels. More recently, the role of mitochondria-derived metabolites in controlling epigenetic modifiers has provided a mechanistic basis for mitochondria-nuclear crosstalk, allowing adaptation of gene expression to specific metabolic states. Here, we discuss the multi-faceted mechanisms by which mitochondrial function influence oocyte quality, as well as longer-term developmental events within and across generations.

Endothelial Cells Differentiated from Porcine Epiblast Stem Cells.

Pluripotent stem cells (PSCs) have the ability of self-renewal that can retain the characteristics of the mother cell, and of pluripotency that can differentiate into several body types. PSCs typically include embryonic stem cells (ESCs) derived from the inner cell mass of the preimplantation embryo, and epiblast stem cells (EpiSCs) derived from the epiblast of postimplantation embryo. Although PSCs are able to be used by differentiation into endothelial cells as a potential treatment for vascular diseases, human ESCs and induced PSCs (iPSCs) are followed by ethical and safety issues. Pigs are anatomically and physiologically similar to humans. Therefore, the goal of this study was to establish an efficient protocol that differentiates porcine EpiSCs (pEpiSCs) into the endothelial cells for applying the treatment of human vascular diseases. As a result, alkaline phosphatase (AP)-negative (-) pEpiSCs cultured in endothelial cell growth basal medium-2 (EBM-2) differentiation medium in association with 50 ng/mL of vascular endothelial growth factor (VEGF) for 8 days were changed morphologically like the feature of endothelial cells, and expression of pluripotency-associated markers (OCT-3/4, NANOG, SOX2, and C-MYC) in porcine differentiated cells was significantly decreased (p < 0.05). Additionally, when pEpiSCs were cultured in EBM-2 + 50 ng/mL of VEGF, porcine differentiated cells represented a common endothelial cell marker positive (CD31+) but monocytes and lymphocytes marker negative (CD45-). Therefore, these results indicated that pEpiSCs cultured in EBM-2 + 50 ng/mL of VEGF culture condition were efficiently differentiated into endothelial cells for the treatment of blood vessel diseases.

Acentriolar microtubule organization centers and Ran-mediated microtubule formation pathways are both required in porcine oocytes.

Mammalian oocytes lack centrioles but can generate bipolar spindles using several different mechanisms. For example, mouse oocytes have acentriolar microtubule organization centers (MTOCs) that contain many components of the centrosome, and which initiate microtubule polymerization. On the contrary, human oocytes lack MTOCs and the Ran-mediated mechanisms may be responsible for spindle assembly. Complete knowledge of the different mechanisms of spindle assembly is lacking in various mammalian oocytes. In this study, we demonstrate that both MTOC- and Ran-mediated microtubule nucleation are required for functional meiotic metaphase I spindle generation in porcine oocytes. Acentriolar MTOC components, including Cep192 and pericentrin, were absent in the germinal vesicle and germinal vesicle breakdown stages. However, they start to colocalize to the spindle microtubules, but are absent in the meiotic spindle poles. Knockdown of Cep192 or inhibition of Polo-like kinase 1 activity impaired the recruitment of Cep192 and pericentrin to the spindles, impaired microtubule assembly, and decreased the polar body extrusion rate. When the RanGTP gradient was perturbed by the expression of dominant negative or constitutively active Ran mutants, severe defects in microtubule nucleation and cytokinesis were observed, and the localization of MTOC materials in the spindles was abolished. These results demonstrate that the stepwise involvement of MTOC- and Ran-mediated microtubule assembly is crucial for the formation of meiotic spindles in porcine oocytes, indicating the diversity of spindle formation mechanisms among mammalian oocytes.

Spire localization via zinc finger-containing domain is crucial for the asymmetric division of mouse oocyte.

Zinc plays an essential role in mammalian oocyte maturation, fertilization, and early embryogenesis, and depletion of zinc impairs cell cycle control, asymmetric division, and cytokinesis in oocyte. We report that zinc, via the actin nucleator Spire, acts as an essential regulator of the actin cytoskeleton remodeling during mouse oocyte maturation and fertilization. Depletion of zinc in the mouse oocyte impaired cortical and cytoplasmic actin formation. Spire is colocalized with zinc-containing vesicles via its zinc finger-containing Fab1, YOTB, Vac 1, EEA1 (FYVE) domain. Improper localization of Spire by zinc depletion or mutations in the FYVE domain impair cytoplasmic actin mesh formations and asymmetric division and cytokinesis of oocyte. All 3 major domains of the Spire are required for its proper localization and activity. After fertilization or parthenogenetic activation, Spire localization was dramatically altered following zinc release from the oocyte. Collectively, our data reveal novel roles for zinc in the regulation of the actin nucleator Spire by controlling its localization in mammalian oocyte.-Jo, Y.-J., Lee, I.-W., Jung, S.-M., Kwon, J., Kim, N.-H., Namgoong, S. Spire localization via zinc finger-containing domain is crucial for the asymmetric division of mouse oocyte.

Generation of a High-Growth Influenza Vaccine Strain in MDCK Cells for Vaccine Preparedness.

As shown during the 2009 pandemic H1N1 (A(H1N1)pdm09) outbreak, egg-based influenza vaccine production technology is insufficient to meet global demands during an influenza pandemic. Therefore, there is a need to adapt cell culture-derived vaccine technology using suspended cell lines for more rapid and larger-scale vaccine production. In this study, we attempted to generate a high-growth influenza vaccine strain in MDCK cells using an A/Puerto/8/1934 (H1N1) vaccine seed strain. Following 48 serial passages with four rounds of virus plaque purification in MDCK cells, we were able to select several MDCK-adapted plaques that could grow over 108 PFU/ml. Genetic characterization revealed that these viruses mainly had amino acid substitutions in internal genes and exhibited higher polymerase activities. By using a series of Rg viruses, we demonstrated the essential residues of each gene and identified a set of high-growth strains in MDCK cells (PB1D153N, M1A137T, and NS1N176S). In addition, we confirmed that in the context of the high-growth A/PR/8/34 backbone, A/California/7/2009 (H1N1), A/Perth/16/2009 (H3N2), and A/environment/Korea/deltaW150/2006 (H5N1) also showed significantly enhanced growth properties (more than 107 PFU/ml) in both attached- and suspended-MDCK cells compared with each representative virus and the original PR8 vaccine strain. Taken together, this study demonstrates the feasibility of a cell culture-derived approach to produce seed viruses for influenza vaccines that are cheap and can be grown promptly and vigorously as a substitute for egg-based vaccines. Thus, our results suggest that MDCK cell-based vaccine production is a feasible option for producing large-scale vaccines in case of pandemic outbreaks.

Comparison of the pathogenic potential of highly pathogenic avian influenza (HPAI) H5N6, and H5N8 viruses isolated in South Korea during the 2016-2017 winter season.

Highly pathogenic avian influenza (HPAI) A(H5N6) and A(H5N8) virus infections resulted in the culling of more than 37 million poultry in the Republic of Korea during the 2016/17 winter season. Here we characterize two representative viruses, A/Environment/Korea/W541/2016 [Em/W541(H5N6)] and A/Common Teal/Korea/W555/2017 [CT/W555(H5N8)], and evaluate their zoonotic potential in various animal models. Both Em/W541(H5N6) and CT /W555(H5N8) are novel reassortants derived from various gene pools of wild bird viruses present in migratory waterfowl arising from eastern China. Despite strong preferential binding to avian virus-type receptors, the viruses were able to grow in human respiratory tract tissues. Em/W541(H5N6) was found to be highly pathogenic in both chickens and ducks, while CT/W555(H5N8) caused lethal infections in chickens but did not induce remarkable clinical illness in ducks. In mice, both viruses appeared to be moderately pathogenic and displayed limited tissue tropism relative to HPAI H5N1 viruses. Em/W541(H5N6) replicated to moderate levels in the upper respiratory tract of ferrets and was detected in the lungs, brain, spleen, liver, and colon. Unexpectedly, two of three ferrets in direct contact with Em/W541(H5N6)-infected animals shed virus and seroconverted at 14 dpi. CT/W555(H5N8) was less pathogenic than the H5N6 virus in ferrets and no transmission was detected. Given the co-circulation of different, phenotypically distinct, subtypes of HPAI H5Nx viruses for the first time in South Korea, detailed virologic investigations are imperative given the capacity of these viruses to evolve and cause human infections.

Comparison of the virulence and transmissibility of canine H3N2 influenza viruses and characterization of their canine adaptation factors.

Recent canine influenza outbreaks have raised concerns about the generation of pathogenic variants that may pose a threat to public health. Here, we examine avian-like H3N2 canine influenza viruses (CIVs) isolated from 2009 to 2013 in South Korea from dogs. Phylogenetic analysis revealed that these viruses are closely related to strains previously isolated from dogs in Korea and China. However, molecular characterization demonstrated non-synonymous mutations between the canine viruses, particularly in the putative H3 antigenic sites, NA stalk regions, and in the internal genes of the 2012-2013 isolates compared with the 2009 isolate. Animal experiments showed that three representative isolates (A/canine/Korea/AS-01/2009(AS-01/09), A/canine/Korea/AS-05/2012(AS-05/12) and A/canine/Korea/AS-11/2013(AS-11/13), were readily droplet transmitted between dogs, whereas AS-05/12 induced more severe clinical disease and was lethal in dogs compared with AS-01/09. Although all viruses were able to infect ferrets, AS-05/12 consistently yielded higher nasal wash titers and was transmissible to ferrets via airborne droplets. Using reverse genetics, we show that the NA, NP, and M genes of CIV are critical for the adaptation of avian H3N2 viruses, and the resulting reassortant genotypes promote viral growth in dogs in a manner similar to that of the wild-type AS-01/09 virus. Taken together, these results demonstrate that CIVs continuously evolve in dogs thereby allowing them to gain a foothold in mammalian hosts. Importantly, we elucidated the genetic contributions of the NA, NP, and M genes to the adaptability of CIVs derived from the avian H3N2 virus.

The Effect of Precursor Composition on the Structural Properties of Nanocrystalline Diamond Films.

Nanocrystalline diamond (NCD) films were grown by hot filament CVD and the precursor composition dependence of the structural properties was examined. Films grown at 1 and 2 CH4 Vol% were found to be NCD layers with grain sizes of ~23-25 nm while films grown at 3-5 Vol% were identified as the mixtures of microcrystalline diamond and graphitic phase. The sp2/sp3 bonded carbon ratio in the grown films increased as the CH4 content increased up to 3 Vol% and then decreased beyond 4 Vol%. Microstructure and deposition rate were also found to be affected by the precursor composition and the NCD film grown at 1 CH4 Vol% showed a very dense microstructure and the highest deposition rate of ~3 nm/min.

Altered virulence of Highly Pathogenic Avian Influenza (HPAI) H5N8 reassortant viruses in mammalian models.

Recently identified highly pathogenic avian influenza (HPAI) H5N8 viruses (clade 2.3.4.4) are relatively low to moderately pathogenic in mammalian hosts compared with HPAI H5N1 viruses. In this study, we generated reassortant viruses comprised of A/MD/Korea/W452/2014(H5N8) with substitution of individual genes from A/EM/Korea/W149/2006(H5N1) to understand the contribution of each viral gene to virulence in mammals. Substituting the PB2 gene segment or the NA gene segment of the H5N8 virus by that from the H5N1 virus resulted in significantly enhanced pathogenicity compared with the parental H5N8 virus in mice. Of note, substitution of the PB2 gene segment of the H5N8 virus by that from the H5N1 virus resulted in a 1000-fold increase in virulence for mice compared with the parental virus (MLD50 decreased from 105.8 to 102.5 EID50). Further, the W452W149PB2 virus also induced the highest virus titers in lungs at all time points and the highest levels of inflammatory cytokine responses among all viruses tested. This high virulence phenotype was also confirmed by high viral titers in the respiratory tracts of infected ferrets. Further, a mini-genome assay revealed that W452W149PB2 has significantly increased polymerase activity (p < 0.001). Taken together, our study demonstrates that a single gene substitution from other avian influenza viruses can alter the pathogenicity of recent H5N8 viruses, and therefore emphasizes the need for intensive monitoring of reassortment events among co-circulating avian and mammalian viruses.

Distinct roles of Cep192 and Cep152 in acentriolar MTOCs and spindle formation during mouse oocyte maturation.

Mammalian oocytes lack a centriole that acts as a microtubule organization center (MTOC) in most somatic cells. During oocyte maturation, MTOCs undergo remodeling processes, including decondensation, fragmentation, and self-organization. However, the underlying mechanisms of MTOC remodeling in mouse oocytes are not well understood. We showed that two pericentriolar proteins, Cep192 and Cep152, play crucial roles during MTOC remodeling in mouse oocytes. Cep192 is present in MTOCs at all stages of oocyte maturation, and its depletion induces ablation of MTOCs, delay in spindle formation, and abnormal chromosomal alignment in spindles. In the case of Cep152, its localization on MTOCs is limited at the germinal vesicle stage and then disappears from the MTOCs after the germinal vesicle breakdown stage. Cep152 exclusion from MTOCs is involved in the fragmentation of MTOCs, and it is regulated by cyclin-dependent kinase 1 activity. Our results demonstrate the different roles of Cep192 and Cep152 in MTOC remodeling and a novel regulatory mechanism during meiotic spindle formation in mouse oocytes.-Lee, I.-W., Jo, Y.-J., Jung, S.-M., Wang, H.-Y., Kim, N.-H., Namgoong, S. Distinct roles of Cep192 and Cep152 in acentriolar MTOCs and spindle formation during mouse oocyte maturation.

CIP2A acts as a scaffold for CEP192-mediated microtubule organizing center assembly by recruiting Plk1 and aurora A during meiotic maturation.

In somatic cells spindle microtubules are nucleated from centrosomes that act as major microtubule organizing centers (MTOCs), whereas oocytes form meiotic spindles by assembling multiple acentriolar MTOCs without canonical centrosomes. Aurora A and Plk1 are required for these events, but the underlying mechanisms remain largely unknown. Here we show that CIP2A regulates MTOC organization by recruiting aurora A and Plk1 at spindle poles during meiotic maturation. CIP2A colocalized with pericentrin at spindle poles with a few distinct cytoplasmic foci. Although CIP2A has been identified as an endogenous inhibitor of protein phosphatase 2A (PP2A), overexpression of CIP2A had no effect on meiotic maturation. Depletion of CIP2A perturbed normal spindle organization and chromosome alignment by impairing MTOC organization. Importantly, CIP2A was reciprocally associated with CEP192, promoting recruitment of aurora A and Plk1 at MTOCs. CIP2A was phosphorylated by Plk1 at S904, which targets CIP2A to MTOCs and facilitates MTOC organization with CEP192. Our results suggest that CIP2A acts as a scaffold for CEP192-mediated MTOC assembly by recruiting Plk1 and aurora A during meiotic maturation in mouse oocytes.

Genetic and phylogenetic characterizations of a novel genotype of highly pathogenic avian influenza (HPAI) H5N8 viruses in 2016/2017 in South Korea.

During the outbreaks of highly pathogenic avian influenza (HPAI) H5N6 viruses in 2016 in South Korea, novel H5N8 viruses were also isolated from migratory birds. Phylogenetic analysis revealed that the HA gene of these H5N8 viruses belonged to clade 2.3.4.4, similarly to recent H5Nx viruses, and originated from A/Brk/Korea/Gochang1/14(H5N8), a minor lineage of H5N8 that appeared in 2014 and then disappeared. At least four reassortment events occurred with different subtypes (H5N8, H7N7, H3N8 and H10N7) and a chicken challenge study revealed that they were classified as HPAI viruses according to OIE criteria.

Evaluation of the Immune Responses to and Cross-Protective Efficacy of Eurasian H7 Avian Influenza Viruses.

Due to increasing concerns about human infection by various H7 influenza viruses, including recent H7N9 viruses, we evaluated the genetic relationships and cross-protective efficacies of three different Eurasian H7 avian influenza viruses. Phylogenic and molecular analyses revealed that recent Eurasian H7 viruses can be separated into two different lineages, with relatively high amino acid identities within groups (94.8 to 98.8%) and low amino acid identities between groups (90.3 to 92.6%). In vivo immunization with representatives of each group revealed that while group-specific cross-reactivity was induced, cross-reactive hemagglutination inhibition (HI) titers were approximately 4-fold lower against heterologous group viruses than against homologous group viruses. Moreover, the group I (RgW109/06) vaccine protected 100% of immunized mice from various group I viruses, while only 20 to 40% of immunized mice survived lethal challenge with heterologous group II viruses and exhibited high viral titers in the lung. Moreover, while the group II (RgW478/14) vaccine also protected mice from lethal challenge with group II viruses, it failed to elicit cross-protection against group I viruses. However, it is noteworthy that vaccination with RgAnhui1/13, a virus of a sublineage of group I, cross-protected immunized mice against lethal challenge with both group I and II viruses and significantly attenuated lung viral titers. Interestingly, immune sera from RgAnhui1/13-vaccinated mice showed a broad neutralizing spectrum rather than the group-specific pattern observed with the other viruses. These results suggest that the recent human-infective H7N9 strain may be a candidate broad cross-protective vaccine for Eurasian H7 viruses.IMPORTANCE Genetic and phylogenic analyses have demonstrated that the Eurasian H7 viruses can be separated into at least two different lineages, both of which contain human-infective fatal H7 viruses, including the recent novel H7N9 viruses isolated in China since 2013. Due to the increasing concerns regarding the global public health risk posed by H7 viruses, we evaluated the genetic relationships between Eurasian H7 avian influenza viruses and the cross-protective efficacies of three different H7 viruses: W109/06 (group I), W478/14 (group II), and Anhui1/13 (a sublineage of group I). While each vaccine induced group-specific antibody responses and cross-protective efficacy, only Anhui1/13 was able to cross-protect immunized hosts against lethal challenge across groups. In fact, the Anhui1/13 virus induced not only cross-protection but also broad serum neutralizing antibody responses against both groups of viruses. This suggests that Anhui1/13-like H7N9 viruses may be viable vaccine candidates for broad protection against Eurasian H7 viruses.

Genetic characterisation of novel, highly pathogenic avian influenza (HPAI) H5N6 viruses isolated in birds, South Korea, November 2016.

A novel genotype of H5N6 influenza viruses was isolated from migratory birds in South Korea during November 2016. Domestic outbreaks of this virus were associated with die-offs of wild birds near reported poultry cases in Chungbuk province, central South Korea. Genetic analysis and animal studies demonstrated that the Korean H5N6 viruses are highly pathogenic avian influenza (HPAI) viruses and that these viruses are novel reassortants of at least three different subtypes (H5N6, H4N2 and H1N1).