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Influenza remains one of the most significant public health threats due to its ability to cause high morbidity and mortality worldwide. Although understanding of influenza viruses has greatly increased in recent years, shortcomings remain. Additionally, the continuous mutation of influenza viruses through genetic reassortment and selection of variants that escape host immune responses can render current influenza vaccines ineffective at controlling seasonal epidemics and potential pandemics. Thus, there is a knowledge gap in the understanding of influenza viruses and a corresponding need to develop novel universal vaccines and therapeutic treatments. Investigation of viral pathogenesis, transmission mechanisms, and efficacy of influenza vaccine candidates requires animal models that can recapitulate the disease. Furthermore, the choice of animal model for each research question is crucial in order for researchers to acquire a better knowledge of influenza viruses. Herein, we reviewed the advantages and limitations of each animal model—including mice, ferrets, guinea pigs, swine, felines, canines, and non-human primates—for elucidating influenza viral pathogenesis and transmission and for evaluating therapeutic agents and vaccine efficacy.

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.

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.

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.

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.

With the convergent global emergence of SARS-CoV-2 variants of concern (VOC), a precise comparison study of viral fitness and transmission characteristics is necessary for the prediction of dominant VOCs and the development of suitable countermeasures. While airway temperature plays important roles in the fitness and transmissibility of respiratory tract viruses, it has not been well studied with SARS-CoV-2. Here we demonstrate that natural temperature differences between the upper (33℃) and lower (37℃) respiratory tract have profound effects on SARS-CoV-2 replication and transmission. Specifically, SARS-COV-2 variants containing the P323L or P323L/G671S mutation in the NSP12 RNA-dependent RNA polymerase (RdRp) exhibited enhanced RdRp enzymatic activity at 33℃ compared to 37℃ and high transmissibility in ferrets. MicroScale Thermophoresis demonstrated that the NSP12 P323L or P323L/G671S mutation stabilized the NSP12-NSP7-NSP8 complex interaction. Furthermore, reverse genetics-derived SARS-CoV-2 variants containing the NSP12 P323L or P323L/G671S mutation displayed enhanced replication at 33℃, and high transmission in ferrets. This suggests that the evolutionarily forced NSP12 P323L and P323L/G671S mutations of recent SARS-CoV-2 VOC strains are associated with increases of the RdRp complex stability and enzymatic activity, promoting the high transmissibility. Competing Interest Statement The authors have declared no competing interest. Footnotes * author name updated