Human rhinovirus (HRV) infections are associated with the common cold, occasionally with more serious lower respiratory tract illnesses, and frequently with asthma exacerbations. The clinical features of HRV infection and its association with asthma exacerbation suggest that some HRV disease results from virus-induced host immune responses to infection. To study the HRV-infection-induced host responses and the contribution of these responses to disease, we have developed an in vitro model of HRV infection of human airway epithelial cells (Calu-3 cells) and subsequent exposure of human peripheral blood mononuclear cells (PBMCs) to these infected cells in a two-chamber trans-well tissue culture system. Using this model, we studied HRV 14 (species B) and HRV 16 (species A) induced cytokine and chemokine responses with PBMCs from four healthy adults. Infection of Calu-3 cells with either virus induced HRV-associated increases in FGF-Basic, IL-15, IL-6, IL-28A, ENA-78 and IP-10. The addition of PBMCs to HRV 14-infected cells gave significant increases in MIP-1β, IL-28A, MCP-2, and IFN-α as compared with mock-infected cells. Interestingly, ENA-78 levels were reduced in HRV 14 infected cells that were exposed to PBMCs. Addition of PBMCs to HRV 16-infected cells did not induce MIP-1β, IL-28A and IFN-α efficiently nor did it decrease ENA-78 levels. Our results demonstrate a clear difference between HRV 14 and HRV 16 and the source of PBMCs, in up or down regulation of several cytokines including those that are linked to airway inflammation. Such differences might be one of the reasons for variation in disease associated with different HRV species including variation in their link to asthma exacerbations as suggested by other studies. Further study of immune responses associated with different HRVs and PBMCs from different patient groups, and the mechanisms leading to these differences, should help characterize pathogenesis of HRV disease and generate novel approaches to its treatment.

Nanoparticle vaccines were produced using layer-by-layer fabrication and incorporating respiratory syncytial virus (RSV) G protein polypeptides comprising the CX3C chemokine motif. BALB/c mice immunized with G protein nanoparticle vaccines produced a neutralizing antibody response that inhibited RSV replication in the lungs following RSV challenge. ELISPOT analysis showed that G nanoparticle vaccinated mice had increased levels of RSV G protein-specific IL-4 and IFN-γ secreting cells compared to controls following RSV challenge. Remarkably, RSV challenge of G protein nanoparticle vaccinated mice resulted in increased RSV M2-specific IL-4 and IFN-γ secreting T cells, and increased M2-specific H-2Kd-tetramer positive CD8(+) T cells in the lungs compared to controls. Cell type analysis showed vaccination was not associated with increased pulmonary eosinophilia following RSV challenge. These results demonstrate that vaccination of mice with the RSV G protein nanoparticle vaccines induces a potent neutralizing antibody response, increased G protein- and M2-specific T cell responses, and a reduction in RSV disease pathogenesis.

Rates of Sudden Unexplained Infant Death (SUID), bronchiolitis, and central apnea increase in winter in temperate climates. Though associations between these three conditions are suggested, more work is required to establish if there is a causal pathway linking bronchiolitis to SUID through inducing central apnea. Utilizing a large population-based cohort of infants studied over a 20-year period (n = 834,595, from birth years 1989-2009)), we analyzed ecological associations between timing of SUID cases, bronchiolitis, and apnea healthcare visits. Data were analyzed between 2013 and 2015. We used a Cox Proportional Hazards model to analyze possible interactions between maternal smoking and maternal asthma with infant bronchiolitis on time to SUID. SUID and bronchiolitis both occurred more frequently in winter. An increase in bronchiolitis clinical visits occurred within a few days prior to apnea visits. We found a temporal relationship between infant bronchiolitis and apnea. In contrast, no peak in SUID cases was seen during peaks of bronchiolitis. Among those without any bronchiolitis visits, maternal smoking was associated with an increased risk of SUID: Hazard Ratio (HR) of 2.38 (95% CI: 2.11, 2.67, p-value <0.001). Maternal asthma was associated with an increased risk of SUID among infants with at least one bronchiolitis visit: HR of 2.40 (95% CI: 1.04, 5.54, p-value = 0.04). Consistent trends between bronchiolitis, apnea, and SUID were not established due to small numbers of SUID cases. However, interaction analysis revealed potential differential associations of bronchiolitis and SUID by maternal smoking, maternal asthma status.

Respiratory syncytial virus (RSV) causes lower respiratory tract illness frequently. No effective antivirals or vaccines for RSV are approved for use in the United States; however, there are at least 50 vaccines and monoclonal antibody products in development, with those targeting older adults and pregnant women (to protect young infants) in phase 2 and 3 clinical trials. Unanswered questions regarding RSV epidemiology need to be identified and addressed prior to RSV vaccine introduction to guide the measurement of impact and future recommendations. The Centers for Disease Control and Prevention (CDC) convened a technical consultation to gather input from external subject matter experts on their individual perspectives regarding evidence gaps in current RSV epidemiology in the United States, potential studies and surveillance platforms needed to fill these gaps, and prioritizing efforts. Participants articulated their individual views, and CDC staff synthesized individuals' input into this report.

Human respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract disease in young children. With repeat infections throughout life, it can also cause substantial disease in the elderly and in adults with compromised cardiac, pulmonary and immune systems. RSV is a pleomorphic enveloped RNA virus in the Pneumoviridae family. Recently, the three-dimensional (3D) structure of purified RSV particles has been elucidated, revealing three distinct morphological categories: spherical, asymmetric, and filamentous. However, the native 3D structure of RSV particles associated with or released from infected cells has yet to be investigated. In this study, we have established an optimized system for studying RSV structure by imaging RSV-infected cells on transmission electron microscopy (TEM) grids by cryo-electron tomography (cryo-ET). Our results demonstrate that RSV is filamentous across several virus strains and cell lines by cryo-ET, cryo-immuno EM, and thin section TEM techniques. The viral filament length varies from 0.5 to 12 μm and the average filament diameter is approximately 130 nm. Taking advantage of the whole cell tomography technique, we have resolved various stages of RSV assembly. Collectively, our results can facilitate the understanding of viral morphogenesis in RSV and other pleomorphic enveloped viruses.

Human respiratory syncytial virus (RSV) is themost significant cause of acute lower respiratory infection in children. However, there is no licensed vaccine available. Here, we investigated the effect of five or 20 copies of C-Class of CpG ODN (CpG-C) motif incorporated into a plasmid DNA vaccine encoding RSV fusion (F) glycoprotein on the vaccine-induced immune response. The addition of CpG-C motif enhanced serum binding and virus-neutralizing antibody responses in BALB/c mice immunized with the DNA vaccines. Moreover, mice vaccinated with CpG-modified vaccines, especially with the higher 20 copies, resulted in an enhanced shift toward a Th1-biased antibody and T-cell response, a decrease in pulmonary pathology and virus replication, and a decrease in weight loss after RSV challenge. This study suggests that CpG-C motif, cloned into the backbone of DNA vaccine encoding RSV F glycoprotein, functions as a built-in adjuvant capable of improving the efficacy of DNA vaccine against RSV infection.

Enzyme immunoassays (EIAs) to detect and quantify antibodies against respiratory syncytial virus (RSV) and RSV proteins in human plasma or sera are described. The first EIA uses RSV lysate antigens produced in HEp-2 cell line. The second EIA uses RSV F or G gene-expressed antigen in HEp-2 cells. The third EIA uses 30-amino acid synthetic peptides from central conserved region of G protein of RSV A2 or RSV B1 virus and a peptide from the SARS CoV nucleoprotein as a negative control peptide. All three EIAs have been evaluated for detecting and quantifying the respective antibodies in human sera or plasma.

Background: Alaska Native infants from the Yukon-Kuskokwim Delta (YKD) experienced respiratory syncytial virus (RSV) hospitalization rates 5 times higher and an RSV season twice as long as the general US infant population. We describe trends in hospitalization rates and seasonality during 18 years of continuous RSV surveillance in this population and explore contributions of climate and sociodemographic factors. Methods: We abstracted clinical and RSV test information from computerized medical records at YKD Regional Hospital and Alaska Native Medical Center from 1994 to 2012 to determine hospitalization rates and RSV season timing. Descriptive village and weather data were acquired through the US Census and Alaska Climate Research Center, University of Alaska, Fairbanks, respectively. Results: During 1994-2012, YKD infant RSV hospitalization rates declined nearly 3-fold, from 177 to 65 per 1000 infants/yr. RSV season onset shifted later, from mid October to late December, contributing to a significantly decreased season duration, from 30 to 11 weeks. In a multivariate analysis, children from villages with more crowded households and lacking plumbed water had higher rates of RSV hospitalizations (relative rate, 1.17; P = 0.0005 and relative rate, 1.45; P = 0.0003). No association of temperature or dew point was found with the timing or severity of RSV season. Conclusions: Although the RSV hospitalization rate decreased 3-fold, YKD infants still experience a hospitalization rate 3-fold higher than the general US infant population. Overcrowding and lack of plumbed water were associated with RSV hospitalization. Dramatic changes occurred in RSV seasonality, not explained by changes in climate.

Background: Previous studies support a strong association between viral respiratory tract infections and asthma exacerbations. The effect of newly discovered viruses on asthma control is less well defined. Objective: We sought to determine the contribution of respiratory viruses to asthma exacerbations in children with a panel of PCR assays for common and newly discovered respiratory viruses. Methods: Respiratory specimens from children aged 2 to 17 years with asthma exacerbations (case patients, n = 65) and with well-controlled asthma (control subjects, n = 77), frequency matched by age and season of enrollment, were tested for rhinoviruses, enteroviruses, respiratory syncytial virus, human metapneumovirus, coronaviruses 229E and OC43, parainfluenza viruses 1 to 3, influenza viruses, adenoviruses, and human bocavirus. Results: Infection with respiratory viruses was associated with asthma exacerbations (63.1% in case patients vs 23.4% in control subjects; odds ratio, 5.6; 95% CI, 2.7- 11.6). Rhinovirus was by far the most prevalent virus (60% among case patients vs 18.2% among control subjects) and the only virus significantly associated with exacerbations (odds ratio, 6.8; 95% CI, 3.2-14.5). However, in children without clinically manifested viral respiratory tract illness, the prevalence of rhinovirus infection was similar in case patients (29.2%) versus control subjects (23.4%, P > .05). Other viruses detected included human metapneumovirus (4.6% in patients with acute asthma vs 2.6% in control subjects), enteroviruses (4.6% vs 0%), coronavirus 229E (0% vs 1.3%), and respiratory syncytial virus (1.5% vs 0%). Conclusion: Symptomatic rhinovirus infections are an important contributor to asthma exacerbations in children. Clinical implications: These results support the need for therapies effective against rhinovirus as a means to decrease asthma exacerbations.

Respiratory syncytial virus (RSV) replication is primarily limited to the upper respiratory tract epithelium and primary, differentiated normal human bronchial epithelial cells (NHBE) have, therefore, been considered a good system for in vitro analysis of lung tissue response to respiratory virus infection and virus-host interactions. However, NHBE cells are expensive, difficult to culture, and vary with the source patient. An alternate approach is to use a continuous cell line that has features of bronchial epithelial cells such as Calu-3, an epithelial cell line derived from human lung adenocarcinoma, as an in vitro model of respiratory virus infection. The results show that Calu-3 fully polarize when grown on permeable supports as liquid-covered cultures. Polarized Calu-3 are susceptible to RSV infection and release infectious virus primarily from the apical surface, consistent with studies in NHBE cells. The data demonstrate that polarized Calu-3 may serve as a useful in vitro model to study host responses to RSV infection.