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

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


In the past two years since the onset of coronavirus disease 2019 (COVID-19), non-pharmaceutical interventions (NPIs) such as the mandatory use of face masks, social distancing, and travel restrictions decreased close social interactions globally. Thus, the circulation of all respiratory pathogens, including influenza virus ‘H1N1’ was reduced considerably.

Earlier, the number of hospitalizations due to H1N1 cases was approximately three to five million per year globally. From March 2020, the weekly case numbers dramatically decreased from over 40,000 to zero in the following months. With reduced NPI measures in place now, there is a growing concern for increased influenza cases, particularly in individuals with a history of COVID-19, especially those experiencing long-term effects of SARS-CoV-2 infection.

At the beginning of the pandemic, when the influenza virus was co-circulating with SARS-CoV-2, studies demonstrated a higher risk for severe outcomes in COVID-19 cases with influenza co-infection; however, it is unknown whether a recent SARS-CoV-2 infection could negatively impact a sequential influenza infection.

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

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

About the study

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

The test animals with an average weight of 1.3 kg ± 0.1 kg were semi-randomly allocated in three matched groups. The research team intranasally (i.n.) inoculated 12 male ferrets with 107 median tissue culture infectious dose (TCID50) of SARS-CoV-2 variant of concern (VOC) Beta (B.1.351) on day 0. They were followed up to four weeks post-infection (p.i.) for post-acute COVID-19 symptoms to appear. Six out of twelve ferrets were euthanized on day 28 p.i. during the post-acute phase of SARS-CoV-2 Beta VOC infection.

The remaining six SARS-CoV-2-infected ferrets were infected i.n. with 106 TCID50 of H1N1. The control and mock groups comprised of five ferrets infected only with H1N1, and three ferrets receiving 1 mL of phosphate buffer saline (PBS) i.n., respectively.

The team collected the nose and throat swabs for virology assays on days 0, 3, 5, and 9 p.i. from SARS-CoV-2-infected animals and H1N1-infected animals on days 0, 2, 4, and 5 p.i. Additionally, they collected blood samples from the cranial vena cava on days 0, 14, 21, and 29 during the study and via heart puncture on days 28 and 35 after euthanasia.

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

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

They measured antibody concentrations by optical density (O.D.) at 450 nm absorbance and presented these at a 1:100 or 1:200 sera dilution for spike (S) protein and receptor-binding domain (RBD), respectively.

Study findings

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

The Beta VOC replicated only to low levels in male ferrets despite receiving a high infectious dose of 107 TCID50/mL, albeit it induced cellular and humoral responses. Notably, these immune responses were associated with protective immunity against COVID-19 in humans.

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

The authors also noted a tendency 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 was statistically insignificant.


The ferret model could be beneficial to test sequential infections from SARS-CoV-2 and influenza H1N1 under controlled settings and its findings could be translated to humans. The observed acute lung injury due to influenza appeared to be worsened in ferrets with previous SARS-CoV-2 infection.

Therefore, further studies are warranted to confirm the impact of more virulent SARS-CoV-2 VOCs and the consequent development of long-COVID. Furthermore, 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 regarded as conclusive, guide clinical practice/health-related behavior, or treated as established information.



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