SARS-CoV-2 variants have demonstrated distinct epidemiological patterns and clinical presentations throughout the COVID-19 pandemic. Understanding variant-specific differences at the respiratory epithelium is crucial for understanding their pathogenesis. Here, we utilized human nasal organoid air–liquid interface (HNO-ALI) cell cultures to compare the viral replication kinetics, innate immune response, and epithelial damage of six different strains of SARS-CoV-2 (B.1.2, WA, Alpha, Beta, Delta, and Omicron). All variants replicated efficiently in HNO-ALIs, but with distinct replication kinetic patterns. The Delta variant exhibited delayed replication kinetics, achieving a steady state at 6 days post-infection compared to 3 days for other variants. Cytokine analysis revealed robust pro-inflammatory and chemoattractant responses (IL-6, IL-8, IP-10, CXCL9, and CXCL11) in WA1, Alpha, Beta, and Omicron infections, while Delta significantly dampened the innate immune response, with no significant induction of IL-6, IP-10, CXCL9, or CXCL11. Immunofluorescence and H&E analysis showed that all variants caused significant ciliary damage, though WA1 and Delta demonstrated less destruction at early time points (3 days post-infection). Together, these data show that, in our HNO-ALI model, the Delta variant employs a distinct “stealth” strategy characterized by delayed replication kinetics and epithelial cell innate immune evasion when compared to other variants of SARS-CoV-2, potentially explaining a mechanism that the Delta variant can use for its enhanced transmissibility and virulence observed clinically. Our findings demonstrate that variant-specific differences at the respiratory epithelium could explain some of the distinct clinical presentations and highlight the utility of the HNO-ALI system for the rapid assessment of emerging variants.

Coinfection with respiratory syncytial virus (RSV) and rhinovirus (RV) is the most frequently detected coinfection in infants, yet mechanisms governing viral interference and disease outcome remain poorly understood. Although infection order is a key determinant of coinfection outcome involving RSV, influenza, rhinovirus, and SARS-CoV-2, its role in RSV/RV coinfections has not been defined. Using infant-derived human nasal organoids at air-liquid interface, we demonstrate that coinfection order fundamentally determines the outcome of RSV/RV interactions. An initial RSV infection almost completely inhibits secondary RV coinfection, whereas primary RV infection only partially impairs a subsequent RSV coinfection. These distinct outcomes are accompanied by divergent temporal antiviral and pro-inflammatory responses. Mechanistically, we identify type III interferons as a sufficient mediator of RSV-induced restriction of RV, establishing a host-driven mechanism of viral interference. In contrast, partial impairment of RSV following RV infection occurs through an interferon-independent mechanism. Despite marked differences, RSV/RV coinfections do not exacerbate epithelial injury or innate immune responses beyond that induced by RSV alone, indicating RSV is the dominant driver of pathology in coinfections. Collectively, our data establishes coinfection order as a critical determinant of RSV/RV outcome and reveals novel and mechanistic insight into critical viral-host interactions in the infant nasal epithelium. Importance: This study uncovers fundamental differences in infant epithelial control of two major pediatric viral pathogens. We demonstrate for the first time that the order of coinfection fundamentally determines the outcome of RSV-RV interactions within infant respiratory epithelium. By identifying type III interferon as an important regulator of these interactions, our study suggests that differences in interferon susceptibility among respiratory viruses can heavily influence coinfection outcomes. Importantly, despite clear differences in viral interference, RSV/RV coinfections did not produce greater epithelial injury or pro-inflammatory responses than RSV infection alone. Instead, RSV consistently emerged as the dominant driver of epithelial damage and inflammation, providing a mechanistic explanation for clinical observations that RSV/RV coinfections are often not more severe than RSV infection alone.

Respiratory syncytial virus (RSV) causes millions of lower respiratory tract infections (LRTIs) in young children, older adults, and immunocompromised populations every year. RSV infection initiates in the upper respiratory tract and can progress to the lower airways, resulting in bronchiolitis, pneumonia, and even death. RSV primarily infects epithelial cells apically, but we hypothesized that basolateral exposure of the respiratory epithelium could provide an alternative mechanism of infection that contributes to LRTI development. Using a human nose organoid-air-liquid interface (HNO-ALI) model, we performed apical and basolateral inoculations with contemporaneous RSV strains (RSV/A/Ontario [RSV/A/ON] and RSV/B/Buenos Aires [RSV/B/BA]) representing the two RSV subgroups (A and B) in both adult- and infant-derived HNO-ALIs. Basolateral RSV exposure resulted in delayed viral replication and apical release compared to apical infection. A statistically significant difference in basolateral infection frequency was observed between RSV/B/BA and RSV/A/ON (81.3% versus 25%). Basolateral infection selectively targeted a rare basal cell population, while preserving epithelial integrity. Using undifferentiated HNO-ALIs, we determined for the first time that Krt23+ activated basal cells are uniquely susceptible to RSV infection, a finding we confirmed in fully differentiated HNO-ALIs. Together, our findings show that RSV can infect the respiratory epithelium from the basolateral side by initially targeting a rare subset of basal cells before spreading apically to ciliated cells. Moreover, RSV/B/BA may have an advantage over RSV/A/ON in utilizing the basolateral infection route. These findings highlight an alternative RSV infection pathway and could be a potential mechanism for RSV spread to the lower airways. IMPORTANCE: Understanding the pathogenesis of respiratory syncytial virus (RSV) is essential to understanding and preventing acute and long-term sequelae from infection. The canonical understanding of RSV infection is that the virus infects and is restricted to the apical ciliated cells upon inhalation or fomite exposure. We demonstrate that an alternative route of infection—the basolateral route—can be utilized by RSV to infect the apical ciliated cells of the respiratory epithelium. We also show for the first time a novel difference in infectivity between the two contemporaneous RSV strains (RSV/A/Ontario and RSV/B/Buenos Aires). In addition, we describe a rare basal subset—the Krt23+ activated basal cells—that are uniquely susceptible to RSV, expanding the known cellular tropism of RSV. Infection of basal cells can impact airway differentiation, homeostasis, and remodeling. Overall, our findings expand on RSV pathogenesis and indicate there are alternative mechanisms of infection and cell populations that are susceptible to RSV.