Influenza A viruses continuously evolve through antigenic drift and shift, reducing the effectiveness of vaccines that rely primarily on hemagglutinin (HA). Neuraminidase (NA), a surface antigen with greater sequence conservation, has gained attention as a complementary target for broader influenza vaccine design. Using computational analyses of 707 post-2009 A(H1N1)pdm09 NA sequences, we designed two NA-based mRNA vaccine constructs: NA-D1, derived from contemporary H1N1 isolates, and NA-E2, incorporating conserved features shared between H1N1 and H5N1. Mice received prime-boost immunization followed by homologous H1N1 or heterologous H5N1 challenge. Both NA-D1 and NA-E2 induced NA-specific antibody titers and conferred complete protection against homologous H1N1 infection. In contrast, protection against heterologous H5N1 was partial, consistent with lower predicted antigenic similarity between the vaccine constructs and H5N1 NA. Together, these findings demonstrate that NA-based mRNA vaccination can elicit robust homologous protection but offers limited heterologous protection efficacy. Our results support NA as an important complementary antigen for next-generation influenza vaccines and highlight the potential of computationally guided, dual-antigen (HA + NA) strategies to advance the development of broadly protective mRNA vaccines.

Abstract Highly pathogenic avian influenza (HPAI) viruses from clade 2.3.4.4 have caused recurrent outbreaks in poultry since 2014. In the Republic of Korea, clade 2.3.4.4b viruses have driven five epidemic waves, yet the factors underlying HPAI introduction and farm-to-farm spread remain poorly understood. We compiled hemagglutinin gene sequences of clade 2.3.4.4b viruses from wild birds and poultry in the Republic of Korea (October 2016–March 2024) and reconstructed dispersal dynamics using Bayesian phylogeography. Dispersal patterns suggest that domestic duck farms in the western provinces likely form a key interface for spillover from wild birds into poultry. Mixed-effects generalized linear models showed that both wild-to-poultry and farm-to-farm transition rates were positively associated with the number of poultry farms in the destination province, while wild-to-poultry rates were further associated with higher avian influenza virus infection probability among wild birds. Wild-to-poultry transition rates were lower in 2020–2024 than in 2016–2018, which may reflect strengthened interventions. These findings suggest that poultry farm abundance and introduction pressure from wild birds jointly shape the spatial dynamics of HPAI introduction and spread. More broadly, these factors may provide operational indicators to guide risk-based surveillance and control strategies. Author Summary Highly pathogenic avian influenza (HPAI) H5 viruses continue to cause major losses in poultry and pose recurring risks at the wildlife–livestock interface. Effective control depends on identifying where viruses are most likely to enter poultry populations and how they spread between farms. Using viral genomic data from wild birds and poultry in the Republic of Korea, this study suggests that domestic duck farms in western provinces likely form a key interface for introductions from wild birds into poultry. We also found that regions with more poultry farms were more likely to receive and spread the virus, while introduction risk was further elevated where infection pressure from wild birds was higher. By linking viral genomic patterns with ecological and epidemiological information, our study helps identify where HPAI viruses are most likely to enter poultry populations and spread between farms. These findings can guide targeted surveillance and early control in regions at greatest risk.

[This corrects the article DOI: 10.3389/fmicb.2026.1791088.].

IMPORTANCE:Understanding the molecular mechanisms driving H5N1 clade 2.3.4.4b is critical for pandemic preparedness. OBJECTIVE:To characterize the molecular drivers of viral fitness and mammalian adaptability in recent H5N1 viruses by integrating evolutionary dynamics with structural simulations. METHODS:This study analyzed 2,398 H5Nx genomes (2000-2024) through phylogenetic and selective pressure analyses. HA/NA structures were predicted with AlphaFold 3 and evaluated by AutoDock4 docking, whereas polymerase-ANP32A/B complexes were modeled using template-based methods and their binding free energies were estimated using MM/GBSA. Polymerase-ANP32E complexes were predicted with AlphaFold 3 and similarly evaluated by MM/GBSA. The binding affinities (ΔG) for the sialic acid (SA) receptors and human ANP32 proteins were quantified through molecular mechanics/generalized born surface area calculations. RESULTS:Clade 2.3.4.4b showed significant antigenic drift in the HA receptor binding site, reducing affinity for α2,3-SA and α2,6-SA receptors. On the other hand, the emergence of a full-length stalk N1 NA with second sialic acid-binding site mutations (e.g., N366S) compensated for reduced HA affinity by enhancing the NA binding stability. In the polymerase complex, both the PB2-627E/631L variant (-144.00 kcal/mol; unadjusted p = 0.0058) and the known mammalian-adaptive 627K/631M variant (-144.67 kcal/mol; unadjusted p = 0.0165) showed more favorable predicted human ANP32B binding free energies than the ancestral 627E/631M state (-136.46 kcal/mol). CONCLUSIONS AND RELEVANCE:The co-occurrence of HA, NA, PB1, and PB2 signatures was associated with clade expansion and produced structural predictions consistent with altered receptor or ANP32 interactions; experimental validation is required before inferring effects on fitness or zoonotic risk.

The phylogeographic inference approach aims to connect genomic data with epidemiology to understand the spread and evolution of pathogens using visualization of spatiotemporal reconstructions. Orthohantavirus hantanense (HTNV), the causative agent of hemorrhagic fever with renal syndrome (HFRS), represents a significant global public health concern. Here, we introduce a localized Nextstrain platform for HTNV, offering a comprehensive resource for facilitating spatiotemporal genomic surveillance and the study of evolutionary dynamics of viral genomes. Nextstrain enables web-based visualization and simple sharing of graphic and numeric data through unique web addresses. The Nextstrain build for HTNV stands out for its user-friendly interface and is readily accessible online at https://nextstrain.org/community/KU-MV/Hantavirus. This study provides valuable insights into genomic surveillance, viral phylodynamics, and the evolutionary history of orthohantaviruses for the development of public health policies against endemic HFRS outbreaks.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the global coronavirus disease 2019 (COVID-19) pandemic and continues to drive successive waves of infection through the emergence of novel variants. Consequently, accurately predicting the next clade roots through global surveillance is crucial for effective prevention, control, and timely updates of vaccine antigen updates. This study evaluated the evolutionary dynamics of SARS-CoV-2 using phylogeny-informed genetic distances based on 394 complete genomes and spike (S) gene sequences. Furthermore, we introduced a forecasting framework to estimate the potential of emerging variants leading to clade replacement by analyzing non-synonymous and synonymous genetic distances from clade roots, which reflect global herd immune pressure. Non-synonymous and synonymous genetic distances from both Wuhan and clade root strains were assessed to predict whether a clade would become dominant or extinct within 3 months before the clade replacement. Through five observed clade replacements up to January 2024, we captured the quantifiable heterogeneity in non-synonymous and synonymous genetic distances of the S gene from clade roots between dominant and extinct variants, as measured by the extent of novelty, whether through gradual or drastic change. Our framework demonstrated high predictability for identifying the next clade root before replacement in both training and test datasets (area under the receiver operating characteristic curve [AUROC] > 0.90) by incorporating differential weighting of non-synonymous and synonymous genetic distances. Additionally, the framework solely using spike gene data demonstrated similar accuracy to those using the complete genome. Overall, our approach establishes quantifiable molecular criteria for identifying potential updates to the SARS-CoV-2 vaccine, contributing to proactive pandemic preparedness.

Seoul (SEOV) and Hantaan (HTNV) orthohantaviruses are significant zoonotic pathogens responsible for hemorrhagic fever with renal syndrome. Here, we investigated the molecular evolution of SEOV and HTNV through phylogenetic and bioinformatic analyses using complete genome sequences of their large (L), medium (M), and small (S) gene segments. Despite similar epizootic cycles and clinical symptoms, SEOV and HTNV exhibited distinct genetic and evolutionary dynamics. The phylogenetic trees of each segment consistently showed major genetic clades associated with the geographical distribution of both viruses. Remarkably, SEOV M and S segments exhibit higher evolutionary rates, rapidly increasing genetic diversity, and a more recent origin in contrast to HTNV. Reassortment events were infrequent, but both viruses appear to utilize the M gene segment in genetic exchanges. SEOV favors the L or M segment reassortment, while HTNV prefers the M or S segment exchange. Purifying selection dominates in all three gene segments of both viruses, yet SEOV experiences an elevated positive selection in its glycoprotein Gc ectodomain. Key amino acid differences, including a positive 'lysine fence' (through residues K77, K82, K231, K307, and K310) located at the tip of the Gn, alongside the physical stability around an RGD-like motif through M108-F334 interaction, may contribute to the unique antigenic properties of SEOV. With the increasing global dispersion and potential implications of SEOV for the global public health landscape, this study highlights the unique evolutionary dynamics and antigenic properties of SEOV and HTNV in informing vaccine design and public health preparedness.

The evolution of SARS-CoV-2, which limits public control and treatment, seems to have occurred through multiple mechanisms, including recombination of cocirculating strains in hosts. However, insufficient experimental data have been obtained after coinfection. Therefore, we investigated the emergence of variants after coinfection with parental SARS-CoV-2 and the SARS-CoV-2 Delta. We found that fewer (approximately 50%) mutations accumulated in Calu-3 cells than in other cells after serial passaging. Previously, we established a long-term replication mouse model by infecting Calu-3 cell-derived xenograft tumors with SARS-CoV-2. Here, we utilized our model to investigate the outcome after coinfection. More diverse viral mutations, along with multiple high-frequency simultaneous mutations, were discovered in the tumors than during cell passaging. Viral isolates from the tumors showed no cytopathic effects and formed much smaller plaques. Phylogenetic analysis suggested that the genetic makeup of the variants remained largely the same as that of parental SARS-CoV-2 rather than the SARS-CoV-2 Delta. Viral challenge revealed that the isolates were less lethal than the parental SARS-CoV-2 and SARS-CoV-2 Delta strains. These findings suggest that parental SARS-CoV-2 predominates over the SARS-CoV-2 Delta when coinfected, but the SARS-CoV-2 Delta contributes to the evolution of parental SARS-CoV-2 variants toward better host adaptation without recombination.

We recently established a long-term SARS-CoV-2 infection model using lung-cancer xenograft mice and identified mutations that arose in the SARS-CoV-2 genome during long-term propagation. Here, we applied our model to the SARS-CoV-2 Delta variant, which has increased transmissibility and immune escape compared with ancestral SARS-CoV-2. We observed limited mutations in SARS-CoV-2 Delta during long-term propagation, including two predominant mutations: R682W in the spike protein and L330W in the nucleocapsid protein. We analyzed two representative isolates, Delta-10 and Delta-12, with both predominant mutations and some additional mutations. Delta-10 and Delta-12 showed lower replication capacity compared with SARS-CoV-2 Delta in cultured cells; however, Delta-12 was more lethal in K18-hACE2 mice compared with SARS-CoV-2 Delta and Delta-10. Mice infected with Delta-12 had higher viral titers, more severe histopathology in the lungs, higher chemokine expression, increased astrocyte and microglia activation, and extensive neutrophil infiltration in the brain. Brain tissue hemorrhage and mild vacuolation were also observed, suggesting that the high lethality of Delta-12 was associated with lung and brain pathology. Our long-term infection model can provide mutant viruses derived from SARS-CoV-2 Delta and knowledge about the possible contributions of emergent mutations to the properties of new variants.

Severe fever with thrombocytopenia syndrome (SFTS) is an emerging tick‐borne disease with high mortality in Eastern Asia. The disease is caused by the SFTS virus (SFTSV), also known as Dabie bandavirus, which has a segmented RNA genome consisting of L, M, and S segments. Previous studies have suggested differential viral virulence depending on the genotypes of SFTSV; however, the critical viral factor involved in the differential viral virulence is unknown. Here, we found a significant difference in viral replication in vitro and virulence in vivo between two Korean isolates belonging to the F and B genotypes, respectively. By generating viral reassortants using the two viral strains, we demonstrated that the L segment, which encodes viral RNA‐dependent RNA polymerase (RdRp), is responsible for the enhanced viral replication and virulence. Comparison of amino acid sequences and viral replication rates revealed a point variation, E251K, on the surface of RdRp to be the most significant determinant for the enhanced viral replication rate and in vivo virulence. The effect of the variation was further confirmed using recombinant SFTSV generated by reverse genetic engineering. Therefore, our results indicate that natural variations affecting the viral replicase activity could significantly contribute to the viral virulence of SFTSV.

SARS-CoV-2 variants have continuously emerged globally, including in South Korea. To characterize the molecular evolution of SARS-CoV-2 in South Korea, we performed phylogenetic and genomic recombination analyses using more than 12,000 complete genome sequences collected until October 2022. The variants in South Korea originated from globally identified variants of concern and harbored genetic cladecommon and clade-specific amino acid mutations mainly around the N-terminal domain (NTD) or receptor binding domain (RBD) in the spike protein. Several point mutation residues in key antigenic sites were under positive selection persistently with changing genetic clades of SARS-CoV-2. Furthermore, we detected 17 potential genomic recombinants and 76.4% (13/17) retained the mosaic NTD or RBD genome. Our results suggest that point mutations and genomic recombination in the spike contributed to the molecular evolution of SARS-CoV-2 in South Korea, which will form an integral part of global prevention and control measures against SARS-CoV-2.

Background Severe fever with thrombocytopenia syndrome virus (SFTSV) is a viral pathogen causing significant clinical signs from mild fever with thrombocytopenia to severe hemorrhages. World Health Organization has paid special attention to the dramatic increase in human SFTS cases in China, Japan, and South Korea since the 2010s. The present study investigated the molecular evolution and genetic reassortment of SFTSVs using complete genomic sequences. Methods/Principal finding We collected the complete genome sequences of SFTSVs globally isolated until 2019 (L segment, n = 307; M segment, n = 326; and S segment, n = 564) and evaluated the evolutionary profiles of SFTSVs based on phylogenetic and molecular selection pressure analyses. By employing a time-scaled Bayesian inference method, we found the geographical heterogeneity of dominant SFTSV genotypes in China, Japan, and South Korea around several centuries before and locally spread by tick-born spillover with infrequent long-distance transmission. Purifying selection predominated the molecular evolution of SFTSVs with limited gene reassortment and fixed substitution, but almost all three gene segments appeared to harbor at least one amino acid residue under positive selection. Specifically, the nonstructural protein and glycoprotein (Gn/Gc) genes were preferential selective targets, and the Gn region retained the highest number of positively selected residues. Conclusion/Significance Here, the large-scale genomic analyses of SFTSVs improved prior knowledge of how this virus emerged and evolved in China, Japan, and South Korea. Our results highlight the importance of SFTSV surveillance in both human and non-human reservoirs at the molecular level to fight against fatal human infection with the virus.

Highly pathogenic avian influenza (HPAI) H5 viruses have circulated globally causing incidental human infection with a substantial pandemic threat. The present study investigated the molecular evolution and phylodynamics of hemagglutinin (HA) in avian and human-isolated H5Nx viruses globally circulating since 2000. We investigated the dynamics of amino acid substitution in the HA sequences of avian and human H5Nx viruses and performed a phylogenetic analysis. Our study found that the H5Nx lineages dominantly expanded since 2000 and diverged into multiple sublineages with unique genetic mutations. P185S mutation in HA became a molecular characteristic of dominant H5Nx viruses throughout clades 2.3.4.1 to 2.3.4.4 (2.3.4.1–4). The key mutations, ΔE130 and I155T, and potential N-linked glycosylation at residues 128, 144, and 159 in the HA gene of human-isolated viruses possibly contributed to both the individual and population levels of the H5 evolution and the host adaptation. Our analysis detected heterogeneity in amino acid sites under positive selection in the HA gene of clades 2.3.4.1–4. Accumulated mutations in the HA protein may potentially affect not only the genetic and antigenic diversity of HPAI H5Nx viruses but also increase the functional compatibility with NA subtypes. Given the global spread and incessantly occurring HA mutations of H5Nx viruses, our results emphasize the importance of early identification of HA mutations as well as the need for a comprehensive assessment of H5Nx variants in terms of pandemic preparedness.

Novel coronavirus (SARS-CoV-2) has caused more than 100 million confirmed cases of human infectious disease (COVID-19) since December 2019 to paralyze our global community. However, only limited access has been allowed to COVID-19 vaccines and antiviral treatment options. Here, we report the efficacy of the anticancer drug pralatrexate against SARS-CoV-2. In Vero and human lung epithelial Calu-3 cells, pralatrexate reduced viral RNA copies of SARS-CoV-2 without detectable cytotoxicity, and viral replication was successfully inhibited in a dose-dependent manner. In a time-to-addition assay, pralatrexate treatment at almost half a day after infection also exhibited inhibitory effects on the replication of SARS-CoV-2 in Calu-3 cells. Taken together, these results suggest the potential of pralatrexate as a drug repurposing COVID-19 remedy.

The most effective way to control newly emerging infectious disease, such as the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic, is to strengthen preventative or therapeutic public health strategies before the infection spreads worldwide. However, global health systems remain at the early stages in anticipating effective therapeutics or vaccines to combat the SARS-CoV-2 pandemic. While maintaining social distance is the most crucial metric to avoid spreading the virus, symptomatic therapy given to patients on the clinical manifestations helps save lives. The molecular properties of SARS-CoV-2 infection have been quickly elucidated, paving the way to therapeutics, vaccine development, and other medical interventions. Despite this progress, the detailed biomolecular mechanism of SARS-CoV-2 infection remains elusive. Given virus invasion of cells is a determining factor for virulence, understanding the viral entry process can be a mainstay in controlling newly emerged viruses. Since viral entry is mediated by selective cellular proteases or proteins associated with receptors, identification and functional analysis of these proteins could provide a way to disrupt virus propagation. This review comprehensively discusses cellular machinery necessary for SARS-CoV-2 infection. Understanding multifactorial traits of the virus entry will provide a substantial guide to facilitate antiviral drug development.

Cross-species transmission of viral diseases alarms our global community for its potential of novel pandemic events. Of various viral pathogens noted recently, parvoviruses have posed public health threats not only to humans but also to wild animals. To investigate the prevalence of parvoviruses in wild Manchurian chipmunks, here we detected genetic fragments of the nonstructural protein of parvovirus by polymerase chain reaction in wild Manchurian chipmunk specimens captured in the central and southern regions of South Korea and compared their sequence homology with references. Of a total of 348 specimens examined, chipmunk parvovirus (ChpPV)-specific gene fragments were detected with a 31.32% rate (109 chipmunks of 348) in their kidney, liver, lung, and spleen samples, and the chipmunks captured in Gangwon Province exhibited the highest positive rate (45.37%), followed by Gyeongsang (35.29%), Gyeonggi (31.03%), Chungcheong (20.00%), and Jeolla (19.70%). When compared with the reference sequences, a partial ChpPV sequence showed 97.70% identity to the previously reported Korean strain at the nucleic acid level. In the phylogenetic analysis, ChpPV exhibited closer relationship to primate parvoviruses, erythroviruses, and bovine parvovirus than to adeno-associated viruses. Despite limited sample size and genetic sequences examined in this study, our results underline the prevalence of ChpPV in Korea and emphasize the need of close surveillance of parvoviruses in wild animals.

Of various rodent-borne hantaviruses, Seoul orthohantavirus (SEOV) causes haemorrhagic fever with renal syndrome (HFRS), as does Hantaan orthohantavirus (HTNV). Given global-scale of cases of human infection with SEOV, it is of great clinical importance to distinguish SEOV from other HFRS-causing hantaviruses. In May 2019, a middle-aged patient who had lived in a suburban area of Chungcheong Province, Republic of Korea and enjoyed outdoor activities was transferred to Asan Medical Center in Seoul, Republic of Korea with HFRS; his symptoms included high fever and generalized myalgia. The rapid diagnostic test performed immediately after his transfer detected HTNV-specific antibodies, and the patient was treated accordingly. However, two consecutive IFAs performed at ten-day intervals showed no HTNV-specific immunoglobulin (Ig) G. During continuous supportive care, next-generation sequencing successfully identified viral genomic sequences in the patient's serum, which were SEOV and not HTNV. Phylogenetic analysis grouped the L, M, and S genes of this SEOV strain together with those of rat- or human-isolated Korean strains reported previously. Given global outbreaks and public health threats of zoonotic hantaviruses, a causative pathogen of hantavirus HFRS should be identified correctly at the time of diagnosis and by point-of-care testing.

Zoonotic transmission of orthohantaviruses from rodent reservoirs to humans has been the cause of severe fatalities. Human infections are reported worldwide, but vaccines have been approved only in China and Korea. Orthohantavirus vaccine development has been pursued with no sense of urgency due to the relative paucity of cases in countries outside China and Korea. However, the orthohantaviruses continuously evolve in hosts and thus the current vaccine may not work as well against some variants. Therefore, a more effective vaccine should be prepared against the orthohantaviruses. In this review, we discuss the issues caused by the orthohantavirus vaccine. Given the pros and cons of the orthohantavirus vaccine, we suggest strategies for the development of better vaccines in terms of pandemic preparedness.

Since the first human case was reported in Wuhan Province, China in December 2019, SARS-CoV-2 has caused millions of human infections in more than 200 countries worldwide with an approximately 4.01% case-fatality rate (as of 27 July, 2020; based on a WHO situation report), and COVID-19 pandemic has paralyzed our global community. Even though a few candidate drugs, such as remdesivir (a broad antiviral prodrug) and hydroxychloroquine, have been investigated in human clinical trials, their therapeutic efficacy needs to be clarified further to be used to treat COVID-19 patients. Here we show that pyronaridine and artesunate, which are the chemical components of anti-malarial drug Pyramax ® , exhibit antiviral activity against SARS-CoV-2 and influenza viruses. In human lung epithelial (Calu-3) cells, pyronaridine and artesunate were highly effective against SARS-CoV-2 while hydroxychloroquine did not show any effect at concentrations of less than 100 μM. In viral growth kinetics, both pyronaridine and artesunate inhibited the growth of SARS-CoV-2 and seasonal influenza A virus in Calu-3 cells. Taken together, we suggest that artesunate and pyronaridine might be effective drug candidates for use in human patients with COVID-19 and/or influenza, which may co-circulate during this coming winter season.

Pandemics affect human lives severely and globally. Experience predicts that there will be a pandemic for sure although the time is unknown. When a viral epidemic breaks out, assessing its pandemic risk is an important part of the process that characterizes genomic property, viral pathogenicity, transmission in animal model, and so forth. In this review, we intend to figure out how a pandemic may occur by looking into the past influenza pandemic events. We discuss interpretations of the experimental evidences resulted from animal model studies and extend implications of viral pandemic potentials and ingredients to emerging viral epidemics. Focusing on the pandemic potential of viral infectious diseases, we suggest what should be assessed to prevent global catastrophes from influenza virus, Middle East respiratory syndrome coronavirus, dengue and Zika viruses.