Male Sex and Age Biases Viral Burden, Viral Shedding, and Type 1 and 2 Interferon Responses During SARS-CoV-2 Infection in Ferrets

Background: The SARS-CoV-2 (Severe Acute Respiratory Syndrome coronavirus 2) has led to more than 73 million COVID-19 cases and ~1.6 million deaths worldwide. Epidemiological analysis of COVID-19 severity has revealed a male sex bias which increases with age. Understanding the mechanisms underlying this disparity will not only be beneficial to host-specific treatment but will give insight into the pathogenesis of the virus in general. Here we investigated the male sex and age bias as well as the corresponding immunological regulation contributing to COVID-19 using ferrets as a model of SARS-CoV-2 infection. Methods: We investigated infected adult male, aged male, and adult female ferrets with SARS-CoV-2 at 106 TCID50 intranasally. Ferrets were monitored for clinical signs post-inoculation and in-life samples and end-point tissues were collected. The samples were analyzed for viral dynamics by qRT-PCR and live viral load by TCID 50 assays. Respiratory tissue was investigated for tissue destruction and viral antigen presence by histopathology and immunohistochemistry. RNA sequencing of isolated RNA from the upper and lower respiratory tract was performed using the NovaSeq 6000 50 base-pair, paired-end run for a minimum of 30 million reads per sample. Sequence analysis was used to determine differential gene expression.    Findings: Clinically, all groups of ferrets developed mild disease although all males had consistent temperature decreases while females developed a fever. Viral load analysis revealed that aged male ferrets shed virus from their nares for longer and had higher viral loads in the upper respiratory tract associated with increased pathology. Host response analysis indicated that female ferrets had significant increases in antiviral and interferon response genes as soon as day 2 post-infection characterized by the upregulation of OASL, MX1, and ISG15 as well as others, whereas upregulation of antiviral genes was delayed until day 7 in aged males. In the later time points, males had significant increases in genes associated with neurological signalling and sensory detection (taste and smell) such as RTP1, CHGA, and CHGA1. Interpretation: Older male ferrets had increased viral shedding and burden compared to females which may have been due to the dysregulation of the male antiviral responses. Increases in olfactory-associated genes during the infection time course may suggest mechanisms regulating anosmia, which has been associated with COVID-19. These results provide insight into COVID-19 epidemiological findings and suggest that the male sex bias in hospitalizations has a biological component and is not solely due to cultural and behavioural factors. Considering the threat of SARS-CoV-2 mutations in hosts that have prolonged viral replication and shedding, understanding why older males have increased viral durability is of public health importance. Funding Statement: Canadian Institutes of Health Research (CIHR), NIH/NIAID, Genome Atlantic, and Canadian Foundation for Innovation through the Major Science Initiates Fund and by Innovation Saskatchewan. Declaration of Interests: Authors declare no competing interests. Ethics Approval Statement: All work was conducted in accordance with the Canadian Council of Animal Care (CCAC) guidelines, AUP number 20200016 by the University Animal Care Committee (UACC) Animal Research Ethics Board (AREB) from the University of Saskatchewan.