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Human coronavirus (HCoV)-NL63 causes respiratory tract infections in humans and uses angiotensin-converting enzyme 2 (ACE2) as a receptor. We sought to establish a mouse model of HCoV-NL63 and determine whether prior rhinovirus (RV)-A1B infection affected HCoV-NL63 replication. HCoV-NL63 was propagated in LLC-MK2 cells expressing human ACE2. RV-A1B was grown in HeLa-H1 cells. C57BL6/J or transgenic mice expressing human ACE2 were infected intranasally with sham LLC-MK2 cell supernatant or 1 × 10 5 tissue culture infectious dose (TCID 50 ) units HCoV-NL63. Wild-type mice were infected with 1 × 10 6 plaque-forming units (PFU) RV-A1B. Lungs were assessed for vRNA, bronchoalveolar lavage (BAL) cells, histology, HCoV-NL63 nonstructural protein 3 (nsp3), and host gene expression by next-generation sequencing and qPCR. To evaluate sequential infections, mice were infected with RV-A1B followed by HCoV-NL63 infection 4 days later. We report that hACE2 mice infected with HCoV-NL63 showed evidence of replicative infection with increased levels of vRNA, BAL neutrophils and lymphocytes, peribronchial and perivascular infiltrates, and expression of nsp3. Viral replication peaked 3 days after infection and inflammation persisted 6 days after infection. HCoV-NL63-infected hACE2 mice showed increased mRNA expression of IFNs, IFN-stimulated proteins, and proinflammatory cytokines. Infection with RV-A1B 4 days before HCoV-NL63 significantly decreased both HCoV-NL63 vRNA levels and airway inflammation. Mice infected with RV-A1B prior to HCoV-NL63 showed increased expression of antiviral proteins compared with sham-treated mice. In conclusion, we established a mouse model of HCoV-NL63 replicative infection characterized by relatively persistent viral replication and inflammation. Prior infection with RV-A1B reduced HCoV-NL63 replication and airway inflammation, indicative of viral interference. NEW & NOTEWORTHY We describe a mouse model of human coronavirus (HCoV) infection. Infection of transgenic mice expressing human angiotensin-converting enzyme 2 (ACE2) with HCoV-NL63 produced a replicative infection with peribronchial inflammation and nonstructural protein 3 expression. Mice infected with RV-A1B 4 days before HCoV-NL63 showed decreased HCoV-NL63 replication and airway inflammation and increased expression of antiviral proteins compared with sham-treated mice. This research may shed light on human coronavirus infections, viral interference, and viral-induced asthma exacerbations.

Infection of immature mice with rhinovirus (RV) induces an asthma-like phenotype consisting of type 2 inflammation, mucous metaplasia, eosinophilic inflammation, and airway hyperresponsiveness that is dependent on IL-25 and type 2 innate lymphoid cells (ILC2s). Doublecortin-like kinase 1-positive (DCLK1+) tuft cells are a major source of IL-25. We sought to determine the requirement of tuft cells for the RV-induced asthma phenotype in wild-type mice and mice deficient in Pou2f3, a transcription factor required for tuft cell development. C57BL/6J mice infected with RV-A1B on day 6 of life and RV-A2 on day 13 of life showed increased DCLK1+ tuft cells in the large airways. Compared with wild-type mice, RV-infected Pou2f3-/- mice showed reductions in IL-25 mRNA and protein expression, ILC2 expansion, type 2 cytokine expression, mucous metaplasia, lung eosinophils, and airway methacholine responsiveness. We conclude that airway tuft cells are required for the asthma phenotype observed in immature mice undergoing repeated RV infections. Furthermore, RV-induced tuft cell development provides a mechanism by which early-life viral infections could potentiate type 2 inflammatory responses to future infections.

Wheezing-associated rhinovirus (RV) infections are associated with asthma development. We have shown that infection of immature mice with RV induces type 2 cytokine production and mucous metaplasia which is dependent on IL-33 and type 2 innate lymphoid cells (ILC2s) and intensified by a second heterologous RV infection. We hypothesize that M2a macrophages are required for the exaggerated inflammation and mucous metaplasia in response to heterologous RV infection. Wild-type C57Bl/6J mice and LysM Cre IL4Rα KO mice lacking M2a macrophages were treated as follows: (1) sham infection on day 6 of life plus sham on day 13 of life, (2) RV-A1B on day 6 plus sham on day 13, (3) sham on day 6 and RV-A2 on day 13, or (4) RV-A1B on day 6 and RV-A2 on day 13. Lungs were harvested one or seven days after the second infection. Wild-type mice infected with RV-A1B at day 6 showed an increased number of Arg1- and Retnla -expressing lung macrophages, indicative of M2a polarization. Compared to wild-type mice infected with RV on day 6 and 13 of life, the lungs of LysM Cre IL4Rα KO mice undergoing heterologous RV infection showed decreased protein abundance of the epithelial-derived innate cytokines IL-33, IL-25 and TSLP, decreased ILC2s, decreased mRNA expression of IL-13 and IL-5, and decreased PAS staining. Finally, mRNA analysis and immunofluorescence microscopy of double-infected LysM Cre IL4Rα KO mice showed reduced airway epithelial cell IL-33 expression, and treatment with IL-33 restored the exaggerated muco-inflammatory phenotype., Conclusion: Early-life RV infection alters the macrophage response to subsequent heterologous infection, permitting enhanced IL-33 expression, ILC2 expansion and intensified airway inflammation and mucous metaplasia., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Han, Breckenridge, Kuo, Singh, Goldsmith, Li, Kreger, Bentley and Hershenson.)

Rhinovirus (RV) infection is linked to early life wheezing and exacerbation of adult asthma. RV infection can be modeled in adult and neonatal mice. This chapter outlines methods for the production of standardized human rhinovirus A1B and mouse infection. The chapter also describes methods to couple infections with allergen (ovalbumin and house dust mite) administrations. The production of the virus involves its amplification, purification, and concentration. In order to standardize the concentrated RV stock, a plaque assay on HeLa cells is outlined as a method of calibrating infectivity. Once the number of plaque-forming units is determined, the standardized virus is used for mouse infection., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)