Background: Rhinovirus C (RV-C) is one of three species of rhinoviruses (RVs), which cause the common cold, preschool wheezing illnesses and exacerbations of asthma. RV-C types are more virulent, especially in children, but progress in developing treatments is limited by difficulties in generating high-titer virus preparations. The goals of this study were to optimize methods for large-scale production and purification of RV-C to facilitate structure and immune response studies. Methods: We optimized protocols for the propagation and purification of RV-C15a, a clinical isolate adapted to HeLa-E8 cells stably expressing virus receptor CDHR3. We compared virus yields in adherent and suspension cultures, evaluated the effects of calcium supplementation and infection timing, and tested multiple purification strategies, including ultracentrifugation, dialysis, and lipase treatment. Results: RV-C15a yields were significantly lower in suspension vs. adherent cultures despite comparable virus binding and entry, suggesting post-entry replication limitations in suspended cells. In adherent cultures, infecting soon after cell seeding and calcium supplementation reduced the time of virus production and modestly improved virus progeny yields. Surface CDHR3 expression declined over time, potentially restricting viral spread. Among purification methods, lipase treatment of infected cell lysates followed by ultracentrifugation produced highly pure and concentrated virus preparations suitable for structural and immunological applications, with high yields. Conclusions: We present a robust system for large-scale RV-C15a production in adherent HeLa-E8 cells and recommend a lipase-based purification method as a rapid and effective approach for producing high-quality viral preparations. These advances will support structural studies and accelerate the development of RV-C-targeted therapeutics and vaccines.

Of the 3 rhinovirus (RV) species, RV-A and RV-C most frequently cause illnesses, and RV-C is closely associated with childhood wheezing. The large number of RV types (> 160) presents a challenge for vaccine development. Little is known about RV type-specific age predilection. Multicenter data were pooled from 14 cohorts (n = 4344 patients aged 0-18, 10329 samples) including partial sequencing of nasal swab samples collected from 1997-2018. We identified RV of each species that were consistently most prevalent. Mean age of infection for each type was evaluated amongst the top circulating viruses (Tukey's test). The top 5 circulating RV-C types were: C2(4.8%), C11(4.7%), C6(4.0%), C43(3.7%), and C15(3.4%). The top 5 circulating RV-A types were: A78(5.4%), A12(4.4%), A101(4.4%), A21(3.6%), and A36(3.4%). The frequency of these predominant types remained highly stable over the study period. Types C2 and C40 showed the lowest mean ages of infection, 1.54 and 2.31 years respectively. RV types A12, A78, and A56 demonstrated the lowest mean age of infection among RV-A viruses, 1.9, 2.2 and 2.4 years, respectively. This age variation was statistically significant when compared against other commonly circulating RVs. Species of viruses with the lowest mean age of infection were observed to be closely related phylogenetically. We documented remarkable stability of predominant types of all 3 RV species over 20 years. and a lower mean age of infection for certain types. The close phylogenetic relationship between RV with the lowest mean age of infection suggests a possible biologic mechanism for their age-related infectivity patterns.

Rhinoviruses (RVs) can cause severe wheezing illnesses in young children and patients with asthma. Vaccine development has been hampered by the multitude of RV types with little information about cross‐neutralization. We previously showed that neutralizing antibody (nAb) responses to RV‐C are detected twofold to threefold more often than those to RV‐A throughout childhood. Based on those findings, we hypothesized that RV‐C infections are more likely to induce either cross‐neutralizing or longer‐lasting antibody responses compared with RV‐A infections. We pooled RV diagnostic data from multiple studies of children with respiratory illnesses and compared the expected versus observed frequencies of sequential infections with RV‐A or RV‐C types using log‐linear regression models. We tested longitudinally collected plasma samples from children to compare the duration of RV‐A versus RV‐C nAb responses. Our models identified limited reciprocal cross‐neutralizing relationships for RV‐A (A12–A75, A12–A78, A20–A78, and A75–A78) and only one for RV‐C (C2–C40). Serologic analysis using reference mouse sera and banked human plasma samples confirmed that C40 infections induced nAb responses with modest heterotypic activity against RV‐C2. Mixed‐effects regression modeling of longitudinal human plasma samples collected from ages 2 to 18 years demonstrated that RV‐A and RV‐C illnesses induced nAb responses of similar duration. These results indicate that both RV‐A and RV‐C nAb responses have only modest cross‐reactivity that is limited to genetically similar types. Contrary to our initial hypothesis, RV‐C species may include even fewer cross‐neutralizing types than RV‐A, whereas the duration of nAb responses during childhood is similar between the two species. The modest heterotypic responses suggest that RV vaccines must have a broad representation of prevalent types.

In 2014, enterovirus D68 (EV-D68), previously associated primarily with mild respiratory illness, caused a large outbreak of severe respiratory illness and, in rare instances, paralysis. We compared the viral binding and replication of eight recent EV-D68 clinical isolates collected both before and during the 2014 outbreak and the prototype Fermon strain from 1962 in cultured HeLa cells and differentiated human primary bronchial epithelial cells (BEC) to understand the possible reasons for the change in virus pathogenicity. We selected pairs of closely related isolates from the same phylogenetic clade that were associated with severe vs. asymptomatic infections. We found no significant differences in binding or replication in HeLa cell cultures between the recent clinical isolates. However, in HeLa cells, Fermon had significantly greater binding (2–3 logs) and virus progeny yields (2–4 logs) but a similar level of replication (1.5–2 log increase in viral RNA from 2 h to 24 h post infection) compared to recent isolates. In differentiated BECs, Fermon and the recent EV-D68 isolates had similar levels of binding; however, the recent isolates produced 1.5–2-log higher virus progeny yields than Fermon due to increased replication. Interestingly, no significant differences in replication were identified between the pairs of genetically close recent EV-D68 clinical isolates despite the observed differences in associated disease severity. We then utilized RNA-seq to define the transcriptional responses in BECs infected with four recent EV-D68 isolates, representing major phylogenetic clades, and the Fermon strain. All the tested clinical isolates induced similar responses in BECs; however, numerous upregulated genes in antiviral and pro-inflammatory response pathways were identified when comparing the response to clinical isolates versus Fermon. These results indicate that the recent emergence in severe EV-D68 cases could be explained by an increased replication efficiency and enhanced inflammatory response induced by newly emerged clinical isolates; however, host factors are likely the main determinants of illness severity.

Rationale: Rhinovirus (RV) C can cause asymptomatic infection and respiratory illnesses ranging from the common cold to severe wheezing. Objectives: To identify how age and other individual-level factors are associated with susceptibility to RV-C illnesses. Methods: Longitudinal data from the COAST (Childhood Origins of Asthma) birth cohort study were analyzed to determine relationships between age and RV-C infections. Neutralizing antibodies specific for RV-A and RV-C (three types each) were determined using a novel PCR-based assay. Data were pooled from 14 study cohorts in the United States, Finland, and Australia, and mixed-effects logistic regression was used to identify factors related to the proportion of RV-C versus RV-A detection. Measurements and Main Results: In COAST, RV-A and RV-C infections were similarly common in infancy, whereas RV-C was detected muchless often than RV-A during both respiratory illnesses and scheduled surveillance visits (P < 0.001, chi(2)) in older children. The prevalence of neutralizing antibodies to RV-A or RV-C types was low(5-27%) at the age of 2 years, but by the age of 16 years, RV-C seropositivity was more prevalent (78% vs. 18% for RV-A; P < 0.0001). In the pooled analysis, the RV-C to RV-A detection ratio during illnesses was significantly related to age (P, 0.0001), CDHR3 genotype (P < 0.05), and wheezing illnesses (P, 0.05). Furthermore, certain RV types (e.g., C2, C11, A78, and A12) were consistently more virulent and prevalent over time. Conclusions: Knowledge of prevalent RV types, antibody responses, and populations at risk based on age and genetics may guide the development of vaccines or other novel therapies against this important respiratory pathogen.

Human rhinoviruses (RV) are a common cause of asthma exacerbations in children. RV-C causes more severe respiratory illnesses than RV-A or RV-B. However, the mechanism(s) underlying this observation remain unclear. Prior studies have shown that children with allergic asthma have impaired antiviral responses in peripheral blood mononuclear cells (PBMCs). We hypothesize that RV-C would induce differential antiviral responses in PBMCs when compared to RV-A and RV-B. PBMCs were isolated from peripheral blood samples of 40 children ages 13-14 years enrolled in the Childhood Origins of ASThma (COAST study). PBMCs were incubated with RV-A36, RV-B52, RV-C15 or medium alone for 22 hours. Supernatants were frozen at -80 and interferons (IFN-alpha2, IFN-gamma, IFN-lambda1) were measured using multiplex ELISA. Interferon levels induced by the different RV species were compared. Stimulation with RV-A led to significantly greater production of IFN-alpha2 and IFN-lambda1 compared to RV-B, RV-C, or control [(median IFN-alpha2: RV-A 1000 pg/ml, RV-B 4 pg/ml, RV-C <3.2 pg/ml, control <3.2 pg/ml), p<0.0001; (median IFN-lambda1: RV-A 142 pg/ml, RV-B <12.2 pg/ml, RV-C <12.2 pg/ml, control <12.2 pg/ml, p<0.0001]. All RV species increased production of IFN-gamma compared to control, with RV-C leading to the least IFN-gamma [(median IFN-gamma: RV-A 55 pg/ml, RV-B 15 pg/ml, RV-C 6 pg/ml, control <3.2 pg/ml), p<0.0001)]. PBMCs stimulated with RV-C15 secreted significantly less interferon compared to cells stimulated with other RV species. RV-C may evade the antiviral response of mononuclear cells in the airway, which could contribute to greater illness severity.

Members of rhinovirus C (RV-C) species are more likely to cause wheezing illnesses and asthma exacerbations compared with other rhinoviruses. The cellular receptor for these viruses was heretofore unknown. We report here that expression of human cadherin-related family member 3 (CDHR3) enables the cells normally unsusceptible to RV-C infection to support both virus binding and replication. A coding single nucleotide polymorphism (rs6967330, C529Y) was previously linked to greater cell-surface expression of CDHR3 protein, and an increased risk of wheezing illnesses and hospitalizations for childhood asthma. Compared with wild-type CDHR3, cells transfected with the CDHR3-Y-529 variant had about 10-fold increases in RV-C binding and progeny yields. We developed a transduced HeLa cell line (HeLa-E8) stably expressing CDHR3-Y-529 that supports RV-C propagation in vitro. Modeling of CDHR3 structure identified potential binding sites that could impact the virus surface in regions that are highly conserved among all RV-C types. Our findings identify that the asthma susceptibility gene product CDHR3 mediates RV-C entry into host cells, and suggest that rs6967330 mutation could be a risk factor for RV-C wheezing illnesses.

Previous studies have demonstrated increased numbers of circulating endothelial progenitor cells (EPCs), defined as peripheral blood mononuclear cells (PBMCs) co-expressing CD34 and CD133, in adults with asthma. However, it is unknown whether EPCs are differentially expressed developmentally based upon gender, puberty, and/or asthma diagnosis. A subset (n=42) of children in the Childhood Origins of Asthma (COAST) study were selected for this pilot study based upon pubertal status, defined as tanner stage 4 for males, and menarche for females. Pre- and post-pubertal PBMC samples from 26 females, and 16 males fulfilling these criteria were assessed using flow cytometry to identify the percentage of EPCs (CD34+/CD133+). Relationships among EPC numbers, gender, puberty, and asthma diagnosis were assessed. Both pre- and post-puberty, females had significantly higher percentages of circulating EPCs (CD34+/CD133+) compared to males (pre-puberty: 0.060% vs 0.041%, p=0.01; post-puberty: 0.062% vs. 0.031%, p=0.0001). EPC percentages were not significantly higher in children with asthma vs. no asthma (pre-puberty: 0.060% vs. 0.049%, p=0.13; post-puberty: 0.052% vs. 0.048%, p=0.63). The percentage of circulating EPCs did not differ by asthma severity or the presence of aeroallergen sensitization. Circulating EPCs were increased in females compared to males both pre- and post-puberty. In contrast to prior studies in adults, no differences in EPCs were seen in children with asthma. Prospective pubertal follow up in the COAST cohort will help determine if these differences persist or change with the expression and remission of asthma, as well as changes in severity of asthma based on gender and age.

Background: Human rhinoviruses (HRVs) are the most common cause of asthma exacerbations. In airway epithelial cells, the primary site of HRV infection, decreased production of interferons (IFNs) may result in greater susceptibility to HRV and worsened symptoms. Thus, exogenous IFN could supplement the innate immune response and provide a treatment for virus-induced asthma exacerbations. Furthermore, the effects of exogenous IFN could be type specific in part because of the cellular distribution of type 1 and type 2 IFN receptors.Objective: To investigate the effects of exogenous IFNs on HRV replication in bronchial epithelial cells.Methods: Frozen stocks of primary human bronchial epithelial cells from healthy donors were cultured in monolayers; pretreated (24 hours) with 0.1-ng/mL, 1-ng/mL, or 10-ng/mL doses of IFN-alpha, -beta, -lambda 1, or -lambda 2; and infected with HRV-1A. Viral replication was quantified using real-time reverse transcription-polymerase chain reaction, and cytokine and chemokine secretion 24 hours after infection was measured by multiplex enzyme-linked immunosorbent assay.Results: Compared with untreated samples, IFN-alpha, IFN-beta, IFN-lambda 1, and IFN-lambda 2 (0.1 ng/mL) significantly reduced HRV replication after high-(P <. 02) and low-dose inoculation (P <. 05). Similar effects were seen in 1-ng/mL and 10-ng/mL doses of IFN, where HRV replication was significantly decreased in both high( P < .001) and low-dose inoculation (P < .001). Treatment with IFNs also enhanced HRV-induced IFN-ge induced protein 10 secretion (P < .001). Finally, treatment with either IFN-lambda 1 or IFN-lambda 2 significantly increased HRV-induced secretion of RANTES (regulated on activation, normal T-expressed, and presumably secreted) (P < .05) but not IL-1 beta or vascular endothelial growth factor.Conclusion: These findings suggest that exogenous IFNs, IFN-lambda 1 in particular, warrant further study as a potential therapy for virus-induced asthma exacerbations. (C) 2013 American College of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.

Acute exacerbations are the leading cause of morbidity and mortality associated with asthma, frequently leading to hospitalization, and accounting for approximately half of the total health care costs associated with asthma.1 Asthma exacerbations have been linked to progressive loss of lung function,2, 3 which may lead to increased asthma severity later in life. Upper respiratory tract viral infections (URIs) are the most significant risk factor for asthma exacerbations. In the northern hemisphere, there is clear correlation between URI outbreaks in September and concurrent hospitalizations for asthma exacerbations.4 Nearly 85% of acute asthma exacerbations in children and approximately 60% in adults have been linked to the presence of a viral infection through the use of reverse transcription-polymerase chain reaction (RT-PCR),5, 6 with human rhinoviruses (HRVs) as the most common cause.7, 8 While current therapies are effective in controlling day-to-day asthma symptoms, they are less effective at preventing exacerbations,9 suggesting that new strategies are needed for exacerbation prevention.\r\n\r\nAirway epithelial cells are recognized a s the primary site of HRV infection and play an important role in the intrinsic antiviral response.10 Interferons (IFNs) are critical to host defense against HRV replication. Increased viral replication, decreased epithelial production of IFN-β and IFN-λ, and reduced induction of apoptosis in response to HRV-16 infection have been reported in asthmatic individuals, suggesting a deficient innate immune response in these individuals that may contribute to the risk of viral exacerbations.11, 12 While not all groups have identified differences in epithelial cell IFN expression between asthmatic subjects and healthy controls,13, 14 it is important to note that deficiencies in peripheral blood IFN-α and -λ1 responses to viruses have been identified in allergic asthmatic children as well.15, 16\r\n\r\nInterestingly, treatment with exogenous IFN-β restored induction of apoptosis and inhibited HRV replication in bronchial epithelial cells (BECs) from asthmatic individuals, suggesting potential therapeutic benefit.11, 17 In an animal model of rotavirus infection, treating suckling mice systemically with IFN-λ1 led to decreased replication of rotavirus in the gut and less severe viral illness,18 further supporting the potential utility of exogenous supplementation of IFN as a clinical treatment for viral infections.\r\n\r\nAlthough IFN-α and -β (type I) and IFN-λ (type III) activate similar intracellular signaling pathways and biological activities, type I and type III IFNs use distinct receptor complexes on the cell surface. Unlike the type I high-affinity receptor component, which is broadly expressed, the type III high-affinity receptor component is restricted to dendritic cells and cells of epithelial origin.19 Collectively, these findings suggest that the impact on HRV replication and epithelial antiviral and inflammatory responses may be IFN-type-dependent.\r\n\r\nTaken together, these data suggest that IFNs may be useful in treatment of respiratory viral infections and associated asthma exacerbations. In this study, we aimed to determine whether exogenous interferons, at physiologic concentrations, inhibit HRV replication and alter inflammatory responses in primary BECs. Further, we sought to determine whether there were differences in the effects of IFN-α, -β, -λ1, and -λ2. Some of the findings in this manuscript were previously reported in an abstract.20

Background\r\nHuman rhinoviruses (HRVs) are the most common cause of asthma exacerbations. In airway epithelial cells, the primary site of HRV infection, decreased production of interferons (IFNs) may result in greater susceptibility to HRV and worsened symptoms. Thus, exogenous IFN could supplement the innate immune response and provide a treatment for virus-induced asthma exacerbations. Furthermore, the effects of exogenous IFN could be type specific in part because of the cellular distribution of type 1 and type 2 IFN receptors.

Background: Children with allergic asthma have more frequent and severe human rhinovirus (HRV)-induced wheezing and asthma exacerbations through unclear mechanisms.Objective: We sought to determine whether increased high-affinity IgE receptor (Fc epsilon RI) expression and cross-linking impairs innate immune responses to HRV, particularly in allergic asthmatic children.Methods: PBMCs were obtained from 44 children, and surface expression of Fc epsilon RI on plasmacytoid dendritic cells (pDCs), myeloid dendritic cells, monocytes, and basophils was assessed by using flow cytometry. Cells were also incubated with rabbit anti-human IgE to cross-link Fc epsilon RI, followed by stimulation with HRV-16, and IFN-alpha and IFN-lambda 1 production was measured by Luminex. The relationships among Fc epsilon RI expression and cross-linking, HRV-induced IFN-alpha and IFN-lambda 1 production, and childhood allergy and asthma were subsequently analyzed.Results: Fc epsilon RI alpha expression on pDCs was inversely associated with HRV-induced IFN-alpha and IFN-lambda 1 production. Cross-linking Fc epsilon RI before HRV stimulation further reduced PBMC IFN-alpha (47% relative reduction; 95% CI, 32% to 62%; P < .0001) and IFN-lambda 1 ( 81% relative reduction; 95% CI, 69% to 93%; P < .0001) secretion. Allergic asthmatic children had higher surface expression of Fc epsilon RIa on pDCs and myeloid dendritic cells when compared with that seen in nonallergic nonasthmatic children. Furthermore, after Fc epsilon RI cross-linking, allergic asthmatic children had significantly lower HRV-induced IFN responses than allergic nonasthmatic children (IFN-alpha, P = .004; IFN-lambda 1, P = .02) and nonallergic nonasthmatic children ( IFN-alpha, P = .002; IFN-lambda 1, P = .01).Conclusions: Allergic asthmatic children have impaired innate immune responses to HRV that correlate with increased Fc epsilon RI expression on pDCs and are reduced by Fc epsilon RI cross-linking. These effects likely increase susceptibility to HRV-induced wheezing and asthma exacerbations. (J Allergy Clin Immunol 2012; 130:489-95.)

SIRT6 is a member of the evolutionarily conserved sirtuin family of NAD+-dependent protein deacetylases and functions in genomic stability and transcriptional control of glucose metabolism. Early reports suggested that SIRT6 performs ADP-ribosylation, whereas more recent studies have suggested that SIRT6 functions mainly as a histone deacetylase. Thus, the molecular functions of SIRT6 remain uncertain. Here, we perform biochemical, kinetic, and structural studies to provide new mechanistic insight into the functions of SIRT6. Utilizing three different assays, we provide biochemical and kinetic evidence that SIRT6-dependent histone deacetylation produces O-acetyl-ADP-ribose but at a rate ∼1,000 times slower than other highly active sirtuins. To understand the molecular basis for such low deacetylase activity, we solved the first crystal structures of this class IV sirtuin in complex with ADP-ribose and the non-hydrolyzable analog of O-acetyl-ADP-ribose, 2′-N-acetyl-ADP-ribose. The structures revealed unique features of human SIRT6, including a splayed zinc-binding domain and the absence of a helix bundle that in other sirtuin structures connects the zinc-binding motif and Rossmann fold domain. SIRT6 also lacks the conserved, highly flexible, NAD+-binding loop and instead contains a stable single helix. These differences led us to hypothesize that SIRT6, unlike all other studied sirtuins, would be able to bind NAD+ in the absence of an acetylated substrate. Indeed, we found that SIRT6 binds NAD+ with relatively high affinity (Kd = 27 ± 1 μm) in the absence of an acetylated substrate. Isothermal titration calorimetry and tryptophan fluorescence binding assays suggested that ADP-ribose and NAD+ induce different structural perturbations and that NADH does not bind to SIRT6. Collectively, these new insights imply a unique activating mechanism and/or the possibility that SIRT6 could act as an NAD+ metabolite sensor.