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The SARS-CoV-2 pandemic continues to spread worldwide, with rapidly increasing numbers of mortalities, placing increasing strain on health care systems. Despite serious public health concerns, no effective vaccines or therapeutics have been approved by regulatory agencies. In this study, we tested the FDA-approved drugs lopinavir-ritonavir, hydroxychloroquine sulfate, and emtricitabine-tenofovir against SARS-CoV-2 infection in a highly susceptible ferret infection model. While most of the drug treatments marginally reduced clinical symptoms, they did not reduce virus titers, with the exception of emtricitabine-tenofovir treatment, which led to diminished virus titers in nasal washes at 8 dpi. Further, the azathioprine-treated immunosuppressed ferrets showed delayed virus clearance and low SN titers, resulting in a prolonged infection. As several FDA-approved or repurposed drugs are being tested as antiviral candidates at clinics without sufficient information, rapid preclinical animal studies should proceed to identify therapeutic drug candidates with strong antiviral potential and high safety prior to a human efficacy trial. Due to the urgent need of a therapeutic treatment for coronavirus (CoV) disease 2019 (COVID-19) patients, a number of FDA-approved/repurposed drugs have been suggested as antiviral candidates at clinics, without sufficient information. Furthermore, there have been extensive debates over antiviral candidates for their effectiveness and safety against severe acute respiratory syndrome CoV 2 (SARS-CoV-2), suggesting that rapid preclinical animal studies are required to identify potential antiviral candidates for human trials. To this end, the antiviral efficacies of lopinavir-ritonavir, hydroxychloroquine sulfate, and emtricitabine-tenofovir for SARS-CoV-2 infection were assessed in the ferret infection model. While the lopinavir-ritonavir-, hydroxychloroquine sulfate-, or emtricitabine-tenofovir-treated group exhibited lower overall clinical scores than the phosphate-buffered saline (PBS)-treated control group, the virus titers in nasal washes, stool specimens, and respiratory tissues were similar between all three antiviral-candidate-treated groups and the PBS-treated control group. Only the emtricitabine-tenofovir-treated group showed lower virus titers in nasal washes at 8 days postinfection (dpi) than the PBS-treated control group. To further explore the effect of immune suppression on viral infection and clinical outcome, ferrets were treated with azathioprine, an immunosuppressive drug. Compared to the PBS-treated control group, azathioprine-immunosuppressed ferrets exhibited a longer period of clinical illness, higher virus titers in nasal turbinate, delayed virus clearance, and significantly lower serum neutralization (SN) antibody titers. Taken together, all antiviral drugs tested marginally reduced the overall clinical scores of infected ferrets but did not significantly affect in vivo virus titers. Despite the potential discrepancy of drug efficacies between animals and humans, these preclinical ferret data should be highly informative to future therapeutic treatment of COVID-19 patients. IMPORTANCE The SARS-CoV-2 pandemic continues to spread worldwide, with rapidly increasing numbers of mortalities, placing increasing strain on health care systems. Despite serious public health concerns, no effective vaccines or therapeutics have been approved by regulatory agencies. In this study, we tested the FDA-approved drugs lopinavir-ritonavir, hydroxychloroquine sulfate, and emtricitabine-tenofovir against SARS-CoV-2 infection in a highly susceptible ferret infection model. While most of the drug treatments marginally reduced clinical symptoms, they did not reduce virus titers, with the exception of emtricitabine-tenofovir treatment, which led to diminished virus titers in nasal washes at 8 dpi. Further, the azathioprine-treated immunosuppressed ferrets showed delayed virus clearance and low SN titers, resulting in a prolonged infection. As several FDA-approved or repurposed drugs are being tested as antiviral candidates at clinics without sufficient information, rapid preclinical animal studies should proceed to identify therapeutic drug candidates with strong antiviral potential and high safety prior to a human efficacy trial.

Few studies have used a longitudinal approach to describe the immune response to SARS-CoV-2 infection. Here, we perform single-cell RNA sequencing of bronchoalveolar lavage fluid cells longitudinally obtained from SARS-CoV-2-infected ferrets. Landscape analysis of the lung immune microenvironment shows distinct changes in cell proportions and characteristics compared to uninfected control, at 2 and 5 days post-infection (dpi). Macrophages are classified into 10 distinct subpopulations with transcriptome changes among monocyte-derived infiltrating macrophages and differentiated M1/M2 macrophages, notably at 2 dpi. Moreover, trajectory analysis reveals gene expression changes from monocyte-derived infiltrating macrophages toward M1 or M2 macrophages and identifies a macrophage subpopulation that has rapidly undergone SARS-CoV-2-mediated activation of inflammatory responses. Finally, we find that M1 or M2 macrophages show distinct patterns of gene modules downregulated by immune-modulatory drugs. Overall, these results elucidate fundamental aspects of the immune response dynamics provoked by SARS-CoV-2 infection. A longitudinal analysis of SARS-CoV-2 infection in humans is challenging. Here the authors show a single cell RNA-sequencing analysis of BAL fluid cells from ferrets and characterise the time dependent recruitment of macrophage subsets to the lungs in response to SARS-CoV-2 infection.

While the seroprevalence of SARS-CoV-2 in healthy people does not differ significantly among age groups, those aged 65 years or older exhibit strikingly higher COVID-19 mortality compared to younger individuals. To further understand differing COVID-19 manifestations in patients of different ages, three age groups of ferrets are infected with SARS-CoV-2. Although SARS-CoV-2 is isolated from all ferrets regardless of age, aged ferrets (≥3 years old) show higher viral loads, longer nasal virus shedding, and more severe lung inflammatory cell infiltration, and clinical symptoms compared to juvenile (≤6 months) and young adult (1–2 years) groups. Furthermore, direct contact ferrets co-housed with the virus-infected aged group shed more virus than direct-contact ferrets co-housed with virus-infected juvenile or young adult ferrets. Transcriptome analysis of aged ferret lungs reveals strong enrichment of gene sets related to type I interferon, activated T cells, and M1 macrophage responses, mimicking the gene expression profile of severe COVID-19 patients. Thus, SARS-CoV-2-infected aged ferrets highly recapitulate COVID-19 patients with severe symptoms and are useful for understanding age-associated infection, transmission, and pathogenesis of SARS-CoV-2. Here, Kim et al. characterize SARS-CoV-2 infection in juvenile, young, and old aged ferrets to provide a further understanding of differences in COVID-19 severity in humans at different ages. Aged ferrets have higher viral loads, shed virus longer, and mimic the transcriptomic profile of severely infected patients.

Aging significantly influences host immune responses to viral infections, including Severe Fever with Thrombocytopenia Syndrome Virus (SFTSV), which is associated with high mortality in elderly patients. Despite its high fatality rate and pandemic potential, effective therapies remain unavailable, and the age-dependent mechanisms underlying SFTSV pathogenesis are not fully understood. To address this gap, we employed a ferret model (an immunocompetent animal model that mimics human SFTSV infections) and performed multi-tissue single-cell RNA sequencing and histopathological analyses. Our results reveal that, upon SFTSV infection, aged ferrets experience extensive decrease of critical immune cells (particularly B and T cells) due to infection-induced cell death and excessive hemophagocytosis in hematopoietic organs, whereas young-adult ferrets rapidly clear the virus with minimal lymphocyte changes. Notably, aged ferrets display marked immune dysregulation, characterized by non-specific activation of T-bet  ⁺ age-associated memory B cells ( T-bet + ABCs) and the proliferation of defective plasmablasts ( MKI67  ⁺ PB1), which serve as major viral reservoirs and drive systemic viral dissemination. Comparative analysis further demonstrated that the MKI67  ⁺ PB1 subset dominates SFTSV⁺ cells in both aged ferrets and human fatal cases, exhibiting the highest per-cell viral UMI counts. Moreover, monocytes and macrophages in aged ferrets exhibit heightened inflammatory gene expression, contributing to the hyper-inflammatory state observed during infection. Collectively, these insights underscore the critical role of dysregulated memory B cell responses and hyper-inflammation in age-dependent SFTSV pathogenesis, highlighting potential targets for interventions in elderly populations.

Cases of laboratory-confirmed SARS-CoV-2 reinfection have been reported in a number of countries. Further, the level of natural immunity induced by SARS-CoV-2 infection is not fully clear, nor is it clear if a primary infection is protective against reinfection. To investigate the potential association between serum antibody titres and reinfection of SARS-CoV-2, ferrets with different levels of NAb titres after primary SARS-CoV-2 infection were subjected to reinfection with a heterologous SARS-CoV-2 strain. All heterologous SARS-CoV-2 reinfected ferrets showed active virus replication in the upper respiratory and gastro-intestinal tracts. However, the high NAb titre group showed attenuated viral replication and rapid viral clearance. In addition, direct-contact transmission was observed only from reinfected ferrets with low NAb titres (<20), and not from other groups. Further, lung histopathology demonstrated the presence of limited inflammatory regions in the high NAb titre groups compared with control and low NAb groups. This study demonstrates a close correlation between a low NAb titre and SARS-CoV-2 reinfection in a recovered ferret reinfection model.

During the 2021/2022 winter season, we isolated highly pathogenic avian influenza (HPAI) H5N1 viruses harbouring an amino acid substitution from Asparagine(N) to Aspartic acid (D) at residue 193 of the hemagglutinin (HA) receptor binding domain (RBD) from migratory birds in South Korea. Herein, we investigated the characteristics of the N193D HA-RBD substitution in the A/CommonTeal/Korea/W811/2021[CT/W811] virus by using recombinant viruses engineered via reverse genetics (RG). A receptor affinity assay revealed that the N193D HA-RBD substitution in CT/W811 increases α2,6 sialic acid receptor binding affinity. The rCT/W811-HA virus caused rapid lethality with high virus titres in chickens compared with the rCT/W811-HA virus, while the rCT/W811-HA virus exhibited enhanced virulence in mammalian hosts with multiple tissue tropism. Surprisingly, a ferret-to-ferret transmission assay revealed that rCT/W811-HA virus replicates well in the respiratory tract, at a rate about 10 times higher than that of rCT/W811-HA , and all rCT/W811-HA direct contact ferrets were seroconverted at 10 days post-contact. Further, competition transmission assay of the two viruses revealed that rCT/W811-HA has enhanced growth kinetics compared with the rCT/W811-HA , eventually becoming the dominant strain in nasal turbinates. Further, rCT/W811-HA exhibits high infectivity in primary human bronchial epithelial (HBE) cells, suggesting the potential for human infection. Taken together, the HA-193D containing HPAI H5N1 virus from migratory birds showed enhanced virulence in mammalian hosts, but not in avian hosts, with multi-organ replication and ferret-to-ferret transmission. Thus, this suggests that HA-193D change increases the probability of HPAI H5N1 infection and transmission in humans.

The threat of severe fever with thrombocytopenia syndrome (SFTS) to public health has been increasing due to the rapid spread of the ticks that carry the causative viral agent. The SFTS virus (SFTSV) was first identified in China and subsequently detected in neighboring countries, including South Korea, Japan, and Vietnam. In addition to the tick-mediated infection, human-to-human transmission has been recently reported with a high mortality rate; however, differential study of the pathogen has been limited by the route of infection. In this study, we investigated the pathogenic potential of SFTSV based on the infection route in aged ferrets, which show clinical signs similar to that of human infections. Ferrets inoculated with SFTSV via the intramuscular and subcutaneous routes show clinical signs comparable to those of severe human infections, with a mortality rate of 100%. Contrastingly, intravascularly infected ferrets exhibit a comparatively lower mortality rate of 25%, although their early clinical signs are similar to those observed following infection via the other routes. These results indicate that the infection route could influence the onset of SFTS symptoms and the pathogenicity of SFTSV. Thus, infection route should be considered in future studies on the pathogenesis of SFTSV infection.

Post-acute sequelae of SARS-CoV-2 (PASC) is characterized by persistent symptoms such as fatigue, respiratory complications and cognitive dysfunction, affecting approximately 13.5% of SARS-CoV-2-infected individuals. Despite its clinical significance, the mechanisms driving PASC remain poorly understood. Here, to address this, we utilized a Phodopus roborovskii hamster model to investigate the long-term effects of SARS-CoV-2 infection compared with influenza A virus. While 46.25–47.50% of hamsters survived SARS-CoV-2 or influenza A virus H1N1 infection, 13.75% of SARS-CoV-2 survivors exhibited impaired weight recovery, severe lung pathology and significant neutrophil accumulation, defining the PASC group. Single-cell RNA sequencing of bronchoalveolar lavage fluid, lung and spleen at 30 days post-infection revealed hallmark PASC gene signatures uniquely upregulated in the PASC group. This was accompanied by elevated neutrophil levels and reduced macrophage populations, indicative of disrupted myeloid cell differentiation. Immunohistochemistry further detected persistent SARS-CoV-2 S1 subunit antigen in the lungs of PASC hamsters at 30 days post-infection, coinciding with marked neutrophil infiltration, which probably drove prolonged inflammatory responses. Indeed, the neutrophils in the PASC group exhibited sustained upregulation of inflammation-related genes, including FPR2 , MMP9 and S100A9 , which are associated with neutrophil degranulation and extracellular trap formation. Importantly, targeting neutrophil-mediated inflammation with small-molecule inhibitors substantially reduced PASC phenotypes. Among these, Sivelestat, a neutrophil elastase inhibitor, demonstrated the most pronounced efficacy, reducing PASC incidence and mortality, and markedly reducing neutrophil levels. These findings underscore the critical role of neutrophil activation in driving lung damage and chronic inflammation during PASC, offering promising therapeutic strategies for mitigating long-term COVID-19 sequelae. Neutrophil activation drives long-term COVID-19 lung damage The COVID-19 pandemic, caused by the SARS-CoV-2 virus, has had a major impact on global health. While vaccines have helped reduce severe cases, new variants and long-term effects, known as long COVID, remain concerns. Long COVID includes symptoms such as fatigue and breathing issues that persist after recovery. This study explores these long-term effects using a hamster model that can induce long COVID symptoms, as observed in patients with COVID-19. Researchers infected hamsters with the virus and studied their tissues using advanced techniques such as single-cell RNA sequencing and histopathological examination. They found that certain immune cells, especially neutrophils, were overly active in long COVID cases. The study also tested drugs targeting these cells and found that Sivelestat, a neutrophil elastase inhibitor, reduced symptoms and mortality. This suggests that targeting neutrophil activity could help manage long COVID. This summary was initially drafted using artificial intelligence, then revised and fact-checked by the author.

Background Omicron variants have rapidly diversified into sublineages with mutations that enhance immune evasion, posing challenges for vaccination and antibody responses. This study aimed to compare serum cross‐neutralizing antibody responses against various SARS‐CoV‐2 Omicron sublineages (BA.1, BA.5, XBB.1.17.1, FK.1.1, and JN.1) in recipients of monovalent COVID‐19 boosters, bivalent booster recipients, and individuals who had recovered from Omicron BA.5 infections. Methods We conducted a micro‐neutralization assay on serum samples from monovalent BNT162b2 booster recipients (N = 54), bivalent BNT162b2 booster recipients (N = 24), and SARS‐CoV‐2 Omicron BA.5‐recovered individuals (N = 13). The history of SARS‐CoV‐2 Omicron infection was assessed using ELISA against the SARS‐CoV‐2 NP protein. Results Bivalent booster recipients exhibited significantly enhanced neutralization efficacy against Omicron sublineages compared to those who had received monovalent booster vaccinations. Omicron BA.5‐recovered individuals displayed similar neutralizing antibodies (NAbs) to the bivalent booster recipients. Despite the improved neutralization in bivalent recipients and BA.5‐recovered individuals, there were limitations in neutralization against the recently emerged Omicron subvariants: XBB.1.17.1 FK.1.1, and JN.1. In both monovalent and bivalent booster recipients, a history of Omicron breakthrough infection was associated with relatively higher geometric mean titers of NAbs against Omicron BA.1, BA.5, and XBB.1.17.1 variants. Conclusion This study underscores the intricate interplay between vaccination strategies, immune imprinting, and the dynamic landscape of SARS‐CoV‐2 variants. Although bivalent boosters enhance neutralization, addressing the challenge of emerging sublineages like XBB.1.17.1, FK.1.1, and JN.1 may necessitate the development of tailored vaccines, underscoring the need for ongoing adaptation to effectively combat this highly mutable virus.

Abstract Severe fever with thrombocytopenia syndrome virus (SFTSV) poses a significant public health challenge in East Asia, necessitating a deeper understanding of its evolutionary dynamics to effectively manage its spread and pathogenicity. This study provides a comprehensive analysis of the genetic diversity, recombination patterns, and selection pressures across the SFTSV genome, utilizing an extensive dataset of 2041 sequences from various hosts and regions up to November 2023. Employing maximum likelihood and Bayesian evolutionary analysis by sampling trees (BEAST), we elucidated the phylogenetic relationships among nine distinct SFTSV genotypes (A, B1, B2, B3, B4, C, D, E, and F), revealing intricate patterns of viral evolution and genotype distribution across China, South Korea, and Japan. Furthermore, our analysis identified 34 potential reassortments, underscoring a dynamic genetic interplay among SFTSV strains. Genetic recombination was observed most frequently in the large segment and least in the small segment, with notable recombination hotspots characterized by stem-loop hairpin structures, indicative of a structural propensity for genetic recombination. Additionally, selection pressure analysis on critical viral genes indicated a predominant trend of negative selection, with specific sites within the RNA-dependent RNA polymerase and glycoprotein genes showing positive selection. These sites suggest evolutionary adaptations to host immune responses and environmental pressures. This study sheds light on the intricate evolutionary mechanisms shaping SFTSV, offering insights into its adaptive strategies and potential implications for vaccine development and therapeutic interventions.