Development of a Mouse Model of Human Coronavirus NL63 (HCoV-NL63) Infection

Abstract

Introduction: The SARS CoV2 pandemic has produced morbidity and mortality worldwide which depends on age, body mass index, cardiovascular disease and other factors. Of the human coronaviruses, only SARS-CoV2 and HCoV-NL63 bind the ACE2 protein. HCoV-NL63 causes upper respiratory tract infections in children but has also been reported to cause pneumonia and even Kawasaki disease. Hypothesis: HCoV-NL63 produces lung inflammation in mice expressing the human ACE2 protein. Methods: HCoV-NL63 (BEI Resources) was propagated in Caco-2 cells. Cells were harvested and concentrated to produce a working virus stock. Virus cytopathic effect in Vero cells at 48 h infection was determined by TCID50. B6.Cg-Tg(K18-ACE2)2Prlmn mice (Jackson Laboratories) express the human ACE2 protein in epithelial cells under the direction of the keratin 18 promoter. Eight-10 week-old K18-hACE2 or C57BL6/J mice were infected with 50 μ L (100,000 TCID50 units) HCoV-NL63 or sham Caco-2 extract intranasally and euthanized at 2, 4, and 7 days post-treatment. Mouse lungs were assessed for histology, IFN and cytokine mRNAs, ACE2, viral RNA and HCoV-NL63 proteins (nucleoprotein, Nsp3 and Nsp4). Primers designed from HCoV-NL63 sequence (NC-005831.2) were used to measure vRNA by qPCR. Polyclonal antibodies to Nsp3 and Nsp4 were generated (Chen et al, J Virol 2007). Antibodies to human ACE2 (InVitrogen), HCoV-NL63 nucleoprotein (clone 2D5, Eurofins), Nsp3 and Nsp4 were conjugated with AlexaFluor dyes. Results: HCoV-NL63 produced cytopathic effects in Vero cells colocalized with viral nucleoprotein. Virus propagated in K18-hACE2 but not C57BL6/J mice. vRNA levels plateaued 4 days after infection and were detectable 7 days after infection. Lung mRNA expression of IFN-α, IFN-β, IFN-λ, CXCL1 and IL-6 were significantly increased 48 h after infection in HCoV-NL63-treated K18-hACE2 but not C57BL6/J mice (N=3, p<0.05, Kruskal-Wallis test). Sections of mouse lung demonstrated bronchovascular and peribronchial inflammation that were most evident 7 days after infection. Nucleoprotein was found on the apical surface of airway epithelial cells of HCoV-NL63- but not sham-infected K18-hACE2 mice, and Nsp3 and Nsp4 were found in the basal and perinuclear cytoplasm of airway epithelial cells. Conclusion: Human coronavirus NL63 infects K18-ACE2 mice inducing an airway inflammatory response. Viral replication is evidenced by vRNA levels, interferon mRNA expression and production non-structural viral proteins. This model may be used to study HCoV-NL63-induced exacerbation of allergic airways disease;the effect of allergy, obesity and aging on human coronavirus infection, and possible cross-immunity between HCoV-NL63 and other respiratory viruses.