Rhinovirus C replication is associated with the endoplasmic reticulum and triggers cytopathic effects in an in vitro model of human airway epithelium

The clinical impact of rhinovirus C (RV-C) is well-documented; yet, the viral life cycle remains poorly defined. Thus, we characterized RV-C15 replication at the single-cell level and its impact on the human airway epithelium (HAE) using a physiologically-relevant in vitro model. RV-C15 replication was restricted to ciliated cells where viral RNA levels peaked at 12 hours post-infection (hpi), correlating with elevated titers in the apical compartment at 24hpi. Notably, infection was associated with a loss of polarized expression of the RV-C receptor, cadherin-related family member 3. Visualization of double-stranded RNA (dsRNA) during RV-C15 replication revealed two distinct replication complex arrangements within the cell, likely corresponding to different time points in infection. To further define RV-C15 replication sites, we analyzed the expression and colocalization of giantin, phosphatidylinositol-4-phosphate, and calnexin with dsRNA. Despite observing Golgi fragmentation by immunofluorescence during RV-C15 infection as previously reported for other RVs, a high ratio of calnexin-dsRNA colocalization implicated the endoplasmic reticulum as the primary site for RV-C15 replication in HAE. RV-C15 infection was also associated with elevated stimulator of interferon genes (STING) expression and the induction of incomplete autophagy, a mechanism used by other RVs to facilitate non-lytic release of progeny virions. Notably, genetic depletion of STING in HAE attenuated RV-C15 and -A16 (but not -B14) replication, corroborating a previously proposed proviral role for STING in some RV infections. Finally, RV-C15 infection resulted in a temporary loss in epithelial barrier integrity and the translocation of tight junction proteins while a reduction in mucociliary clearance indicated cytopathic effects on epithelial function. Together, our findings identify both shared and unique features of RV-C replication compared to related rhinoviruses and define the impact of RV-C on both epithelial cell organization and tissue functionality–aspects of infection that may contribute to pathogenesis in vivo.

Trends and Intensity of Rhinovirus Invasions in Kilifi, Coastal Kenya Over a Twelve-Year Period, 2007-2018

Background Rhinoviruses (RVs) are ubiquitous pathogens and the principal etiological agents of common cold. Despite the high frequency of RV infections, data describing their long-term epidemiological patterns in a defined population remain limited. Methods Here, we analysed 1,070 VP4/VP2 genomic region sequences sampled at Kilifi County Hospital on the Kenya Coast. The samples were collected between 2007 and 2018 from hospitalised paediatric patients (< 60 months) with acute respiratory illness. Results Of 7,231 children enrolled, RV was detected in 1,497 (20.7%) and VP4/VP2 sequences were recovered from 1,070 samples (71.5%). A total of 144 different RV types were identified (67 Rhinovirus A, 18 Rhinovirus B and 59 Rhinovirus C) and at any month, several types co-circulated with alternating predominance. Within types multiple genetically divergent variants were observed. Ongoing RV infections through time appeared to be a combination of (i) persistent types (observed up to seven consecutive months), (ii) reintroduced genetically distinct variants and (iii) new invasions (average of eight new types, annually). Conclusion Sustained RV presence in the Kilifi community is mainly due to frequent invasion by new types and variants rather than continuous transmission of locally established types/variants.

Comparison of Immune Response To Human Rhinovirus C And Respiratory Syncytial Virus In Highly Differentiated Human Airway Epithelial Cells

Background: Human rhinovirus C (HRV-C) accounts for a large proportion of HRV-related illnesses, but the immune response to HRV-C infection has not been elucidated. Our objective was to assess the effect of HRV-C on cytokine secretion in human bronchial epithelial (HBE) cells grown at air–liquid interface (ALI) and compare it with that of respiratory syncytial virus (RSV). Methods: HBE cells were differentiated at ALI culture and the full-length cDNA clones of HRV-C651 and HRV-C15, clinical isolates of HRV-C79 and HRV-C101, and two RSV isolates were inoculated in the HBE cells. The effect of HRV-C on cytokine secretion were assessed and compared with that of RSV.Results: HRV-Cs infects and propagates in fully differentiated HBE cells and significantly increased the secretion of IFN-λ1, CCL5, IP10, IL-6, IL-8, and MCP-1. The virus load positively correlated with the levels of the cytokines. HRV-C induced lower secretion of CCL5 (P=0.048), IL-6 (P=0.016), MCP-1 (P=0.008), and IL-8 (P=0.032), and similar secretion of IP10 (P=0.214) and IFN-λ1 (P=0.214) when compared with RSV. Conclusion: HBE ALI culture system supported HRV-C infection and propagation and HRV-C induced relatively weaker cytokine expression than RSV.

Rhinovirus C Infection Induces Type 2 Innate Lymphoid Cell Expansion and Eosinophilic Airway Inflammation

Rhinovirus C (RV-C) infection is associated with severe asthma exacerbations. Since type 2 inflammation is an important disease mechanism in asthma, we hypothesized that RV-C infection, in contrast to RV-A, preferentially stimulates type 2 inflammation, leading to exacerbated eosinophilic inflammation. To test this, we developed a mouse model of RV-C15 airways disease. RV-C15 was generated from the full-length cDNA clone and grown in HeLa-E8 cells expressing human CDHR3. BALB/c mice were inoculated intranasally with 5 x 106 ePFU RV-C15, RV-A1B or sham. Mice inoculated with RV-C15 showed lung viral titers of 1 x 105 TCID50 units 24 h after infection, with levels declining thereafter. IFN-α, β, γ and λ2 mRNAs peaked 24-72 hrs post-infection. Immunofluorescence verified colocalization of RV-C15, CDHR3 and acetyl-α-tubulin in mouse ciliated airway epithelial cells. Compared to RV-A1B, mice infected with RV-C15 demonstrated higher bronchoalveolar eosinophils, mRNA expression of IL-5, IL-13, IL-25, Muc5ac and Gob5/Clca, protein production of IL-5, IL-13, IL-25, IL-33 and TSLP, and expansion of type 2 innate lymphoid cells. Analogous results were found in mice treated with house dust mite before infection, including increased airway responsiveness. In contrast to Rorafl/fl littermates, RV-C-infected Rorafl/flIl7rcre mice deficient in ILC2s failed to show eosinophilic inflammation or mRNA expression of IL-13, Muc5ac and Muc5b. We conclude that, compared to RV-A1B, RV-C15 infection induces ILC2-dependent type 2 airway inflammation, providing insight into the mechanism of RV-C-induced asthma exacerbations.

Rhinovirus C replication is associated with the endoplasmic reticulum and triggers cytopathic effects in an in vitro model of human airway epithelium

The clinical impact of rhinovirus C (RV-C) is well-documented; yet the viral life cycle remains poorly defined. Thus, we characterized RV-C15 replication at the single-cell level and its impact on the human airway epithelium (HAE) using a physiologically-relevant in vitro model. RV-C15 replication was restricted to ciliated cells where viral RNA levels peaked at 12 hours post-infection (hpi), correlating with elevated titers in the apical compartment at 24 hpi. Notably, infection was associated with a loss of polarized expression of the RV-C receptor, cadherin-related family member 3. Visualization of double-stranded RNA (dsRNA) during RV-C15 replication revealed two distinct replication complex arrangements within the cell, likely corresponding to different time points in infection and correlating with the formation of large intracellular vesicles. To further define RV-C15 replication sites, we analyzed the expression of giantin, phosphatidylinositol-4-phosphate, and calnexin, as well as the colocalization of these markers with dsRNA. Fluorescence levels of all three cellular markers were elevated during infection and altered giantin distribution further indicated Golgi fragmentation. However, unlike previously characterized RVs, the high ratio of calnexin-dsRNA colocalization implicated the endoplasmic reticulum as the primary site for RV-C15 replication in HAE. RV-C15 infection was also associated with elevated stimulator of interferon genes (STING) expression, facilitating replication, and the induction of incomplete autophagy, a mechanism used by other RVs to promote non-lytic release of progeny virions. Finally, RV-C15 infection resulted in a temporary loss in epithelial barrier integrity and the translocation of tight junction proteins while a reduction in mucociliary clearance indicated cytopathic effects on epithelial function. Together, our findings identify both shared and unique features of RV-C replication compared to related rhinoviruses and define the impact of RV-C on both epithelial cell organization and tissue functionality - aspects of infection that may contribute to pathogenesis in vivo.

Solution NMR Determination of the CDHR3 Rhinovirus-C Binding Domain, EC1

Cadherin Related Family Member 3 (CDHR3) is the identified and required cellular receptor for all virus isolates in the rhinovirus-C species (RV-C). Cryo-EM determinations recently resolved the atomic structure of RV-C15a, and subsequently, a complex of this virus bound to CDHR3 extracellular domain 1 (EC1), the N-terminal portion of this receptor responsible for virus interactions. The EC1 binds to a hypervariable sequence footprint on the virus surface, near the 3-fold axis of icosahedral symmetry. The key contacts involve discontinuous residues from 3 viral proteins, VP1, VP2 and VP3. That single cryo-EM EC1 structure, however, could not resolve whether the virus-receptor interface was structurally adaptable to accommodate multiple virus sequences. We now report the solution NMR determination of CDHR3 EC1, showing that this protein, in fact, is mostly inflexible, particularly in the virus-binding face. The new, higher resolution dataset identifies 3 cis-Pro residues in important loop regions, where they can influence both rigidity and overall protein conformation. The data also provide clarification about the residues involved in essential calcium ion binding, and a potential CDHR3 surface groove feature that may be involved in native protein interactions with cellular partners.