Assessing the severity of influenza infection in ferrets with a history of COVID-19

In a recent study published on bioRxiv* preprint server, researchers studied the effect of exposure to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) on the severity of influenza in ferrets.

Study: Influenza infection in ferrets with a history of SARS-CoV-2 infection. Image Credit: Harald Schmidt/Shutterstock


Over the past two years since the onset of coronavirus disease 2019 (COVID-19), non-pharmaceutical interventions (NPIs) such as mandatory use of face masks, social distancing and travel restrictions have reduced interactions close social relationships in the world. Thus, the circulation of all respiratory pathogens, including the ‘H1N1’ influenza virus, has been significantly reduced.

Previously, the number of hospitalizations due to H1N1 cases was around three to five million per year worldwide. From March 2020, the number of weekly cases dropped significantly, from over 40,000 to zero in the following months. With reduced NPI measures in place now, there is growing concern about the increase in flu cases, especially among people with a history of COVID-19, especially those experiencing the long-term effects of SARS-CoV-2 infection.

Early in the pandemic, when influenza virus co-circulated with SARS-CoV-2, studies demonstrated a higher risk of serious outcomes in cases of COVID-19 with influenza co-infection; however, it is unclear whether recent SARS-CoV-2 infection could negatively impact sequential influenza infection.

Nevertheless, interrupted seasonal exposure due to strict implementation of NPI measures weakened immunity, raising the possibility that residual effects of COVID-19 may worsen clinical manifestations of sequential respiratory infection, such as influenza.

So far, the ferret model has emerged as the most suitable for studying the effects of influenza A virus (IAV) infection following resolved SARS-CoV-2 infection or during a Post-acute COVID-19. Initially, ferrets develop mild or non-clinical COVID-19, and 21 days after experimental SARS-CoV-2 infection has cleared, they exhibit follicular hyperplasia in the upper airways of the lung and inflammation of the nasal cavity. These observations reflect long-term or post-acute COVID-19, characterized by persistent respiratory problems and fatigue.

About the study

In the current study, researchers investigated the impact of prior exposure to SARS-CoV-2 on the severity of sequential influenza (H1N1) infection, including the effect on respiratory organs.

Test animals with an average weight of 1.3 kg ± 0.1 kg were semi-randomly divided into three matched groups. The research team inoculated intranasally (in) 12 male ferrets with 107 tissue culture median infectious dose (TCID50) of SARS-CoV-2 Variant Concern (VOC) Beta (B.1.351) at day 0. They were followed up to four weeks post-infection (pi) for onset of post-acute symptoms of COVID-19. Six of twelve ferrets were euthanized at day 28 pi during the post-acute phase of SARS-CoV-2 Beta VOC infection.

The other six ferrets infected with SARS-CoV-2 were infected with 106 TCID50 of H1N1. The control and simulation groups included five ferrets infected only with H1N1 and three ferrets given 1 ml of phosphate-buffered saline (PBS), respectively.

The team collected nose and throat swabs for virology testing on days 0, 3, 5 and 9 pi from SARS-CoV-2 infected animals and H1N1 infected animals on days 0, 2, 4 and 5 ft. In addition, they took blood samples from the cranial vena cava on days 0, 14, 21 and 29 of the study and by cardiac puncture on days 28 and 35 after euthanasia.

They monitored all clinical manifestations, including behavioral activity, breathing, nasal discharge and sneezing, daily for nine days after the onset of SARS-CoV-2 infection and five days after infection. H1N1 infection. Similarly, they monitored their body temperature via the implanted temperature transponder every 30 minutes starting five days before infection. They determined changes in body temperature and body weight from baseline values ​​noted on day 0 of each infection.

Additionally, the researchers used quantitative reverse transcription-polymerase chain reaction (RT-qPCR) to quantify viral load in swabs and tissue samples from ferrets. Additionally, they recorded the cycle threshold (Cyou) values ​​for genomic and subgenomic ribonucleic acid (RNA) to determine viral load and infectious viral particles, respectively. In addition, the team took tissue from the trachea, bronchi and left lung for histopathological examinations under light microscopy.

They measured antibody concentrations by optical density (OD) at an absorbance of 450 nm and presented them at a serum dilution of 1:100 or 1:200 for spike protein (S) and binding domain to the receiver (RBD), respectively.

Study results

Although histological evidence is insignificant, after four weeks of infection with SARS-CoV-2, H1N1-infected ferrets showed an increase in clinical symptoms of influenza. This finding demonstrated that mild COVID-19 had a small negative impact on clinical influenza symptoms; however, the effect of robust SARS-CoV-2 infection is still unknown.

Beta COV reproduced only at low levels in male ferrets despite administration of a high infectious dose of 107 TCID50/mL, although it induced cellular and humoral responses. Notably, these immune responses were associated with protective immunity against COVID-19 in humans.

Sequential H1N1 influenza virus infection in ferrets recovering from mild COVID-19 induced moderate inflammation in the nasal turbinates only. Additionally, these ferrets had insignificant histopathological changes in the lower respiratory tract. This indicated inefficient Beta VOC replication in the ferret model; however, previous studies have shown a prolonged effect of SARS-CoV-2 infection in ferrets infected with the original strain of SARS-CoV-2.

The authors also noted a trend for more severe bronchitis and type II pneumocyte hyperplasia in ferrets sequentially infected with SARS-CoV-2 and H1N1 influenza virus compared to those infected with H1N1 virus only. , although the difference is statistically insignificant.


The ferret model could be beneficial for testing sequential infections of SARS-CoV-2 and H1N1 influenza in controlled environments and its results could be transposed to humans. The observed acute lung injury due to influenza appeared to have worsened in ferrets previously infected with SARS-CoV-2.

Therefore, further studies are warranted to confirm the impact of more virulent SARS-CoV-2 COVs and the consequent development of long-COVID. Additionally, the authors recommended including long-COVID patients in the high-risk group for influenza vaccination.

*Important Notice

bioRxiv publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be considered conclusive, guide clinical practice/health-related behaviors, or treated as established information.


  • Caroline Vilas Boas de Melo, Florence Peters, Harry van Dijken, Stefanie Lenz, Koen van de Ven, Lisa Wijsman, Angéla Gommersbach, Tanja Schouten, Puck B van Kasteren, Judith MA van den Brand, Jorgen de Jonge. (2022). Influenza infection in ferrets with a history of SARS-CoV-2 infection. bioRxiv. do I:

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