The relationship between respiratory viruses and chronic lung disease has long been a concern for many researchers worldwide. Since the early days of my undergraduate training, the correlation of asthma and chronic obstructive pulmonary disease (COPD) development with infection by respiratory viruses was something already established, but the phenomenon still intrigued me. Transitioning to Dr. Carolina B. López’s Lab, I joined as a postdoc at a challenging time—during the early days of the COVID-19 pandemic. Even by then the scientific community was already interested in understanding the long-term implications for COVID-19 survivors. Dr. López’s group had previously discovered that non-segmented negative-sense RNA viruses can establish persistent infections in vitro through the accumulation of non-standard viral genomes (nsVGs), which subsequently trigger cell pro-survival mechanisms¹. Suddenly, I saw myself in a lab that not only had the tools and know-how to study persistent respiratory virus infections but also had significantly contributed to the field. The group further explored the long-term impacts of murine parainfluenza virus (Sendai virus – SeV) infection in immunocompetent mice, which results in a robust post-viral lung disease. SeV-infected mice carry unresolved pathogenic lung alterations for months after the initial infection compared to uninfected littermate controls². These were strong indicators that the murine infection by SeV was a good model for studying respiratory viral infections and their effects on chronic lung disease. 

Friend or foe? Immune cells are sources of persistent viral subproducts in the lung

The first thing to do would be to search for long-term virus presence in SeV-infected mouse lungs. Using a combination of RT-qPCR, immunofluorescence, in situ hybridization, and spectral flow cytometry, we found viral nucleoprotein (NP) and RNA in mouse lungs for months after the acute infection was considered cleared (no detectable infectious particles in whole lung homogenates). In comparison with acutely infected lungs, we noticed that cells expressing persistent viral protein and RNA were not in the epithelial layer of the large airways, the primary site of viral replication for SeV. In fact, persistent viral proteins were detected in infiltrating cells from the alveolar compartment, characterized as macrophages, and type 2 innate lymphoid cells (ILC2s). The detection of persistent viral products in macrophages was something already described³ and expected, due to the phagocytic activity displayed by these cells. However, the fact that ILC2s harbored persistent viral products was very surprising to us. ILC2s lack phagocytic activity and the presence of viral proteins could indicate direct viral infection. The implications of these findings could be significant, especially considering the pivotal role played by macrophages and ILC2s in the SeV-driven chronic lung disease⁴. 

Long-term immune activation has been defined as one of the most important factors governing chronic post-viral lung disease development. In this context, what is the impact of persistent expression of viral subproducts by immune cells? We found out that macrophages and ILC2s isolated from long-term SeV-infected lungs exhibited transcriptomes with a strong type 2 inflammatory profile in comparison with cells isolated from uninfected controls. Based on that, we hypothesized that viral persistence and long-term expression of viral subproducts can sustain the activation state of immune cells and directly impact the establishment of chronic lung pathology.

Back to the drawing board: Designing a reporter system to simultaneously track virus replication and cell fate

 Due to the relatively low frequency of persistently infected cells, it was difficult to perform more granular analysis regarding virus-cell interactions. To solve that, we developed a double-reporter recombinant Sendai virus by inserting eGFP and Cre recombinase coding sequences into the virus genome (rSeV-C^eGFP-Cre). Combining the rSeV-C^eGFP-Cre virus with loxP-STOP-loxP tdTomato (tdTom) reporter mice resulted in a robust system where we could track actively infected cells (eGFP⁺tdTom⁺), cells that were infected at some point (tdTom⁺), cells that cleared the infection in a non-lytic way (tdTom⁺), and cells derived from infected cells (tdTom⁺). Using this system, we found first, that within the same infected lung, persistent virus-imprinted innate immune cells had higher inflammation status than its bystander cell counterparts (Figure 1). Second, as demonstrated for influenza⁵ but with no detectable viral RNA or protein, a considerable fraction of cells that were infected with SeV remain in the lungs, including cells without any viral subproducts (virus survivor cells and daughter cells) and by so can go undetected by most standard screening methods. Third, both survivor and persistently infected cells exhibited long-lasting activated transcriptomic footprints (Figure 1). Finally, we were able to specifically deplete cells that interacted with SeV and survived the acute infection clearance. Mice that had persistently infected/survivor cells depleted showed significantly improved lung pathology scores, highlighting the impact of virus-imprinting in the establishment and maintenance of chronic lung disease.

Figure 1. The landscape of long-term paramyxovirus infections in the lower respiratory tract.

Conclusions

Overall, our study expanded the understanding that respiratory viral infections typically considered acute in fact can leave long-term molecular footprints with direct impact on disease outcome. Using a murine paramyxovirus model of severe respiratory viral infection, we demonstrated that innate immune cells are sources of persistent virus subproducts, maintaining a long-lasting type 2 inflammatory profile. In addition, our data strongly suggests that survivor cells that cleared the infection in a non-cytolytic manner also contribute to the chronic disease, by maintaining abnormal transcriptomic signatures involved with keratinization and tissue remodeling. This further confirms that even in immunocompetent hosts, the clearance of infectious virus during the acute phase may not be as complete as imagined. With the recent advances in antiviral therapies and vaccination strategies, our ability to fight viral infections significantly increased. Consequently, we expect an increase in the number of individuals who overcame severe viral infections, and our study highlights the utmost necessity to monitor these cases, as persisting viruses could lead to an increase in long-term post-viral disease.

References

1          Xu, J. et al. Replication defective viral genomes exploit a cellular pro-survival mechanism to establish paramyxovirus persistence. Nature Communications 8 (2017). https://doi.org/10.1038/s41467-017-00909-6

2          Garcia, G. L., Valenzuela, A., Manzoni, T., Vaughan, A. E. & López, C. B. Distinct Chronic Post-Viral Lung Diseases upon Infection with Influenza or Parainfluenza Viruses Differentially Impact Superinfection Outcome. American Journal of Pathology 190, 543-553 (2020). https://doi.org/10.1016/j.ajpath.2019.11.003

3          Huot, N. et al. SARS-CoV-2 viral persistence in lung alveolar macrophages is controlled by IFN-γ and NK cells. Nature Immunology (2023). https://doi.org/10.1038/s41590-023-01661-4

4          Wu, K. et al. Group 2 Innate Lymphoid Cells Must Partner with the Myeloid–Macrophage Lineage for Long-Term Postviral Lung Disease. The Journal of Immunology 205, 1084-1101 (2020). https://doi.org/10.4049/jimmunol.2000181

5          Hamilton, J. R. et al. Club cells surviving influenza A virus infection induce temporary nonspecific antiviral immunity. Proc Natl Acad Sci U S A 113, 3861-3866 (2016). https://doi.org/10.1073/pnas.1522376113