A novel gamma radiation-inactivated sabin-based polio vaccine.

A concerted action on the part of international agencies and national governments has resulted in the near-eradication of poliomyelitis. However, both the oral polio vaccine (OPV) and the inactivated polio vaccine (IPV) have deficiencies which make them suboptimal for use after global eradication. OPV is composed of attenuated Sabin strains and stimulates robust immunity, but may revert to neurovirulent forms in the intestine which can be shed and infect susceptible contacts. The majority of IPV products are manufactured using pathogenic strains inactivated with formalin. Upon eradication, the production of large quantities of pathogenic virus will present an increased biosecurity hazard. A logical ideal endgame vaccine would be an inactivated form of an attenuated strain that could afford protective immunity while safely producing larger numbers of doses per unit of virus stock than current vaccines. We report here the development of an ionizing radiation (IR)-inactivated Sabin-based vaccine using a reconstituted Mn-decapeptide (MDP) antioxidant complex derived from the radioresistant bacterium Deinococcus radiodurans. In bacteria, Mn2+-peptide antioxidants protect proteins from oxidative damage caused by extreme radiation exposure. Here we show for the first time, that MDP can protect immunogenic neutralizing epitopes in picornaviruses. MDP protects epitopes in Polio Virus 1 and 2 Sabin strains (PV1-S and PV2-S, respectively), but viral genomic RNA is not protected during supralethal irradiation. IR-inactivated Sabin viruses stimulated equivalent or improved neutralizing antibody responses in Wistar rats compared to the commercially used IPV products. Our approach reduces the biosecurity risk of the current PV vaccine production method by utilizing the Sabin strains instead of the wild type neurovirulent strains. Additionally, the IR-inactivation approach could provide a simpler, faster and less costly process for producing a more immunogenic IPV. Gamma-irradiation is a well-known method of virus inactivation and this vaccine approach could be adapted to any pathogen of interest.

Development of a Rhinovirus Inoculum Using a Reverse Genetics Approach.

BACKGROUND: Experimental inoculation is an important tool for common cold and asthma research. Producing rhinovirus (RV) inocula from nasal secretions has required prolonged observation of the virus donor to exclude extraneous pathogens. We produced a RV-A16 inoculum using reverse genetics and determined the dose necessary to cause moderate colds in seronegative volunteers. METHODS: The consensus sequence of RV-A16 from a previous inoculum was cloned, and inoculum virus was produced using reverse genetics techniques. After safety testing, volunteers were inoculated with either RV-A16 (n = 26) or placebo (n = 10), Jackson cold scores were recorded, and nasal secretions were tested for shedding of RV-A16 ribonucleic acid. RESULTS: The reverse genetics process produced infectious virus that was neutralized by specific antisera and had a mutation rate similar to conventional virus growth techniques. The 1000 median tissue culture infectious dose (TCID50) dose produced moderate colds in most individuals with effects similar to that of a previously tested conventional RV-A16 inoculum. CONCLUSIONS: Reverse genetics techniques produced a RV-A16 inoculum that can cause clinical colds in seronegative volunteers, and they also serve as a stable source of virus for laboratory use. The recombinant production procedures eliminate the need to derive seed virus from nasal secretions, thus precluding introduction of extraneous pathogens through this route.

Self-administered acupressure for treating adult psychiatric patients with constipation: a randomized controlled trial.

BACKGROUND: Constipation has a high prevalence rate (>30 %) in psychiatric patients with psychotropic drugs. Common pharmacological and non-pharmacological interventions for constipation might have longer-term negative and adverse effects that would outweigh their short-term efficacy in symptom reduction. This randomized controlled trial aims to investigate the effect of self-administered acupressure for the management of constipation, in hospitalized psychiatric patients. METHODS: Seventy-eight patients were recruited in matched pairs in terms of gender, age and laxative use from five acute psychiatric wards in Hong Kong. Each of these matched pairs of patients was randomly assigned to either a self-administered acupressure (n = 39) or a sham group (n = 39), using computer-generated random numbers. After baseline measurement, the intervention and sham group received the same training in self-administered acupressure and supervised practice once per day for 10 days, except light pressure on non-acupoints was taught to the sham group. The acupoints chosen for acupressure included Zhongwan (RN12), right and left Tianshu (ST25), right and left Quchi (LI11). Participants' symptoms and quality of life regarding constipation were measured at baseline and immediately and 2 weeks after completion of the interventions with constipation assessment scale and patient assessment of constipation quality of life questionnaire, respectively. RESULTS: After 2 weeks follow-up, participants who had received self-administered acupressure indicated significantly greater improvements in both symptom severity (P = 0.0003) and quality of life (P = 0.0004) when compared with the sham group. CONCLUSION: The psychiatric patients with constipation who practiced self-administered acupressure for 10 days improved their symptom severity and perceived quality of life immediately and 2 weeks after completion of the intervention in comparison with the sham group. TRIAL REGISTRATION: The trial was registered with the ClinicalTrials.gov (Reg. No: NCT02187640).

Molecular identification and quantification of human rhinoviruses in respiratory samples.

PCR-based molecular assays have become standard diagnostic procedures for the identification and quantification of human rhinoviruses (HRVs) and other respiratory pathogens in most, if not all, clinical microbiology laboratories. Molecular assays are significantly more sensitive than traditional culture-based and serological methods. This advantage has led to the recognition that HRV infections are common causes for not only upper airway symptoms but also more severe lower respiratory illnesses. In addition, molecular assays improve turnaround time, can be performed by technicians with ordinary skills, and can easily be automated. This chapter describes two highly sensitive and specific PCR-based methods for identifying and quantifying HRVs. The first is a two-step PCR method for the detection and typing of HRV. The second is a pan-HRV real-time quantitative (q) PCR method for measuring viral loads in respiratory samples.

Growth of human rhinovirus in H1-HeLa cell suspension culture and purification of virions.

HeLa cell culture is the most widely used system for in vitro studies of the basic biology of human rhinovirus (HRV). It is also useful for making sufficient quantities of virus for experiments that require highly concentrated and purified virus. This chapter describes the protocols for producing a large amount of HeLa cells in suspension culture, using these cells to grow a large quantity of virus of HeLa-adapted HRV-A and -B serotypes, and making highly concentrated virus stock and highly purified virions. These purified HRV virions are free of cellular components and suitable for experiments that are sensitive to cellular contaminations.

Infectivity assays of human rhinovirus-A and -B serotypes.

Infectivity is a fundamental property of viral pathogens such as human rhinoviruses (HRVs). This chapter describes two methods for measuring the infectivity of HRV-A and -B serotypes: end point dilution (TCID50) assay and plaque assay. End point dilution assay is a quantal, not quantitative, assay that determines the dilution of the sample at which 50 % of the aliquots have infectious virus. It can be used for all the HRV-A and -B serotypes and related clinical isolates that grow in cell culture and induce cytopathic effect (CPE), degenerative changes in cells that are visible under a microscope. Plaque assay is a quantitative assay that determines the number of infectious units of a virus in a sample. After an infectious unit of virus infects one cell, the infected cell produces progeny viruses that then infect and kill a circle of adjacent cells. This circle of dead cells detaches from the dish and thus leaves a clear hole in a cell monolayer. Plaque assay works only for HeLa-adapted HRV-A and -B serotypes that can make visible plaques on the cell monolayer. Currently the end point dilution assay and plaque assay have not been developed for the newly discovered HRV-C.

Reverse genetics system for studying human rhinovirus infections.

Human rhinovirus (HRV) contains a 7.2 kb messenger-sense RNA genome which is the template for reproducing progeny viruses after it enters the cytoplasm of a host cell. Reverse genetics refers to the regeneration of progeny viruses from an artificial cDNA copy of the RNA genome of an RNA virus. It has been a powerful molecular genetic tool for studying HRV and other RNA viruses because the artificial DNA stage makes it practical to introduce specific mutations into the viral RNA genome. This chapter uses HRV-16 as the model virus to illustrate the strategy and methods for constructing and cloning the artificial cDNA copy of a full-length HRV genome, identifying the infectious cDNA clone isolates, and selecting the most vigorous cDNA clone isolate to serve as the standard parental clone for future molecular genetic study of the virus.

Detection of pathogenic bacteria during rhinovirus infection is associated with increased respiratory symptoms and asthma exacerbations.

BACKGROUND: Detection of either viral or bacterial pathogens is associated with wheezing in children; however, the influence of both bacteria and viruses on illness symptoms has not been described. OBJECTIVE: We evaluated bacterial detection during the peak rhinovirus season in children with and without asthma to determine whether an association exists between bacterial infection and the severity of rhinovirus-induced illnesses. METHODS: Three hundred eight children (166 with asthma and 142 without asthma) aged 4 to 12 years provided 5 consecutive weekly nasal samples during September and scored cold and asthma symptoms daily. Viral diagnostics and quantitative PCR for Streptococcus pneumoniae, Haemophilus influenzae, and Moraxella catarrhalis were performed on all nasal samples. RESULTS: Detection rates were 53%, 17%, and 11% for H influenzae, S pneumoniae, and M catarrhalis, respectively, with detection of rhinovirus increasing the risk of detecting bacteria within the same sample (odds ratio [OR], 2.0; 95% CI, 1.4-2.7; P < .0001) or the following week (OR, 1.6; 95% CI, 1.1-2.4; P = .02). In the absence of rhinovirus, S pneumoniae was associated with increased cold symptoms (mean, 2.7 [95% CI, 2.0-3.5] vs 1.8 [95% CI, 1.5-2.2]; P = .006) and moderate asthma exacerbations (18% [95% CI, 12% to 27%] vs 9.2% [95% CI, 6.7% to 12%]; P = .006). In the presence of rhinovirus, S pneumoniae was associated with increased moderate asthma exacerbations (22% [95% CI, 16% to 29%] vs 15% [95% CI, 11% to 20%]; P = .01). Furthermore, M catarrhalis detected alongside rhinovirus increased the likelihood of experiencing cold symptoms, asthma symptoms, or both compared with isolated detection of rhinovirus (OR, 2.0 [95% CI, 1.0-4.1]; P = .04). Regardless of rhinovirus status, H influenzae was not associated with respiratory symptoms. CONCLUSION: Rhinovirus infection enhances detection of specific bacterial pathogens in children with and without asthma. Furthermore, these findings suggest that M catarrhalis and S pneumoniae contribute to the severity of respiratory tract illnesses, including asthma exacerbations.

Comparison of viral load in individuals with and without asthma during infections with rhinovirus.

RATIONALE: Most virus-induced attacks of asthma are caused by rhinoviruses (RVs). OBJECTIVES: To determine whether people with asthma are susceptible to an increased viral load during RV infection. METHODS: Seventy-four children (4-18 yr old) were enrolled; 28 with wheezing, 32 with acute rhinitis, and 14 without respiratory tract symptoms. Nasal washes were evaluated using quantitative polymerase chain reaction for RV to judge viral load along with gene sequencing to identify strains of RV. Soluble intercellular adhesion molecule-1, IFN-λ1, and eosinophil cationic protein in nasal washes, along with blood eosinophil counts and total and allergen-specific IgE in sera, were also evaluated. Similar assessments were done in 24 young adults (16 with asthma, 8 without) who participated in an experimental challenge with RV (serotype 16). MEASUREMENTS AND MAIN RESULTS: Fifty-seven percent of wheezing children and 56% with acute rhinitis had nasal washes testing positive for RV. The geometric mean of viral loads by quantitative polymerase chain reaction in washes from wheezing children was 2.8-fold lower, but did not differ significantly from children with rhinitis (7,718 and 21,612 copies of viral RNA per microliter nasal wash, respectively; P = 0.48). The odds for wheezing were increased if children who tested positive for RV were sensitized to one or more allergens (odds ratio, 3.9; P = 0.02). Similarly, neither peak nor cumulative viral loads differed significantly in washes from adults with asthma compared with those without asthma during the experimental RV challenge. CONCLUSIONS: During acute symptoms, children infected with RV enrolled for wheezing or acute rhinitis had similar viral loads in their nasal washes, as did adults with and without asthma infected with RV-16 experimentally.

Human rhinovirus species C infection in young children with acute wheeze is associated with increased acute respiratory hospital admissions.

RATIONALE: Human rhinovirus species C (HRV-C) is the most common cause of acute wheezing exacerbations in young children presenting to hospital, but its impact on subsequent respiratory illnesses has not been defined. OBJECTIVES: To determine whether acute wheezing exacerbations due to HRV-C are associated with increased hospital attendances due to acute respiratory illnesses (ARIs). METHODS: Clinical information and nasal samples were collected prospectively from 197 children less than 5 years of age, presenting to hospital with an acute wheezing episode. Information on hospital attendances with an ARI before and after recruitment was subsequently obtained. MEASUREMENTS AND MAIN RESULTS: HRV was the most common virus identified at recruitment (n = 135 [68.5%]). From the 120 (88.9%) samples that underwent typing, HRV-C was the most common HRV species identified, present in 81 (67.5%) samples. Children with an HRV-related wheezing illness had an increased risk of readmission with an ARI (relative risk, 3.44; 95% confidence interval, 1.17-10.17; P = 0.03) compared with those infected with any other virus. HRV-C, compared with any other virus, was associated with an increased risk of a respiratory hospital admission before (49.4% vs. 27.3%, respectively; P = 0.004) and within 12 months (34.6% vs. 17.0%; P = 0.01) of recruitment. Risk for subsequent ARI admissions was further increased in atopic subjects (relative risk, 6.82; 95% confidence interval, 2.16-21.55; P = 0.001). Admission risks were not increased for other HRV species. CONCLUSIONS: HRV-C-related wheezing illnesses were associated with an increased risk of prior and subsequent hospital respiratory admissions. These associations are consistent with HRV-C causing recurrent severe wheezing illnesses in children who are more susceptible to ARIs.

Interaction between allergy and innate immunity: model for eosinophil regulation of epithelial cell interferon expression.

BACKGROUND: Eosinophils in asthmatic airways are associated with risk of exacerbations. The most common cause of asthma exacerbations is viral respiratory infections, particularly human rhinovirus (HRV). OBJECTIVE: To determine the mechanism by which eosinophils may influence virus-induced responses. METHODS: We used an in vitro coculture model of primary human eosinophils and the BEAS-2B epithelial cell line either stimulated with HRV1A infection or polyinosinic-polycytidylic acid (poly[I:C]). The messenger RNA (mRNA) expression of interferon (IFN) ß1 and IFN-λ1 was assessed by quantitative reverse-transcriptase polymerase chain reaction and the protein level of IFN- λ1 by enzyme-linked immunosorbent assay. RESULTS: Both poly(I:C) and HRV1A infection induced BEAS-2B expression of IFN-ß1 and IFN-λ1 mRNA. Coculture of eosinophils resulted in suppression of poly(I:C)-stimulated IFN-ß1 and IFN-λ1 mRNA expression (2.5-fold and 3.6-fold less, respectively). Separation of cells did not block eosinophil regulatory activity. Coculture of eosinophils with HRV1A-infected BEAS-2B cells also suppressed IFN-ß1 and IFN-λ1 mRNA (5.7-fold and 5.0-fold less, respectively) and reduced IFN-λ1 protein secretion (1.6-fold decrease). This corresponded to a 34% increase in the quantity of HRV1A virus RNA on coculture with eosinophils. Recombinant transforming growth factor ß suppressed IFN-λ1 from HRV1A-infected BEAS-2B cells. Coculture of eosinophils and BEAS-2B cells induced transforming growth factor ß secretion, which may mediate suppression of HRV-induced interferon expression. CONCLUSION: Eosinophils suppressed HRV-induced expression of interferons from epithelial cells, resulting in increased quantity of HRV. This represents one mechanism for interaction between allergic inflammation and innate immunity.

Detection of viruses in young children with fever without an apparent source.

OBJECTIVE: Fever without an apparent source is common in young children. Currently in the United States, serious bacterial infection is unusual. Our objective was to determine specific viruses that might be responsible. METHODS: We enrolled children aged 2 to 36 months with temperature of 38°C or greater without an apparent source or with definite or probable bacterial infection being evaluated in the St Louis Children's Hospital Emergency Department and afebrile children having ambulatory surgery. Blood and nasopharyngeal swab samples were tested with an extensive battery of virus-specific polymerase chain reaction assays. RESULTS: One or more viruses were detected in 76% of 75 children with fever without an apparent source, 40% of 15 children with fever and a definite or probable bacterial infection, and 35% of 116 afebrile children (P < .001). Four viruses (adenovirus, human herpesvirus 6, enterovirus, and parechovirus) were predominant, being detected in 57% of children with fever without a source, 13% of children with fever and definite or probable bacterial infection, and 7% of afebrile children (P < .001). Thirty-four percent of 146 viral infections were detected only by polymerase chain reaction performed on blood. Fifty-one percent of children with viral infections and no evidence of bacterial infection were treated with antibiotics. CONCLUSIONS: Viral infections are frequent in children with fever without an apparent source. Testing of blood in addition to nasopharyngeal secretions expanded the range of viruses detected. Future studies should explore the utility of testing for the implicated viruses. Better recognition of viruses that cause undifferentiated fever in young children may help limit unnecessary antibiotic use.

Comparison of the etiology of viral respiratory illnesses in inner-city and suburban infants.

BACKGROUND: The risk of developing childhood asthma has been linked to the severity and etiology of viral respiratory illnesses in early childhood. Since inner-city infants have unique environmental exposures, we hypothesized that patterns of respiratory viral infections would also be distinct. METHODS: We compared the viral etiology of respiratory illnesses in 2 groups: a cohort of 515 infants from 4 inner-city areas and a cohort of 285 infants from mainly suburban Madison, Wisconsin. Nasal secretions were sampled during periods of respiratory illness and at 1 year of age and were analyzed for viral pathogens by multiplex polymerase chain reaction. RESULTS: Overall, inner-city infants had lower rates of viral detection. Considering specific viruses, sick urban infants had lower rates of detectable rhinovirus or respiratory syncytial virus infection and higher rates of adenovirus infection. Every urban site had a higher proportion of adenovirus-positive samples associated with illnesses (10%-21%), compared with Madison (6%). CONCLUSIONS: These findings provide evidence that inner-city babies have different patterns of viral respiratory illnesses than babies who grow up in a more suburban location. These findings raise important questions about the etiology of virus-negative illnesses in urban infants and the possibility of long-term consequences of early life infections with adenovirus in this population.

Human rhinovirus species and season of infection determine illness severity.

RATIONALE: Human rhinoviruses (HRVs) consist of approximately 160 types that cause a wide range of clinical outcomes, including asymptomatic infections, common colds, and severe lower respiratory illnesses. OBJECTIVES: To identify factors that influence the severity of HRV illnesses. METHODS: HRV species and types were determined in 1,445 nasal lavages that were prospectively collected from 209 infants participating in a birth cohort who had at least one HRV infection. Questionnaires were used during each illness to identify moderate to severe illnesses (MSI). MEASUREMENTS AND MAIN RESULTS: Altogether, 670 HRV infections were identified, and 519 of them were solitary infections (only one HRV type). These 519 viruses belonged to 93 different types of three species: 49 A, 9 B, and 35 C types. HRV-A (odds ratio, 8.2) and HRV-C (odds ratio, 7.6) were more likely to cause MSI compared with HRV-B. In addition, HRV infections were 5- to 10-fold more likely to cause MSI in the winter months (P < 0.0001) compared with summer, in contrast to peak seasonal prevalence in spring and fall. When significant differences in host susceptibility to MSI (P = 0.004) were considered, strain-specific rates of HRV MSI ranged from less than 1% to more than 20%. CONCLUSIONS: Factors related to HRV species and type, season, and host susceptibility determine the risk of more severe HRV illness in infancy. These findings suggest that anti-HRV strategies should focus on HRV-A and -C species and identify the need for additional studies to determine mechanisms for seasonal increases of HRV severity, independent of viral prevalence, in cold weather months.

Prevalence of and risk factors for human rhinovirus infection in healthy aboriginal and non-aboriginal Western Australian children.

BACKGROUND: Human rhinovirus (HRV) species C (HRV-C) have been associated with frequent and severe acute lower respiratory infections and asthma in hospitalized children. The prevalence of HRV-C among healthy children and whether this varies with ethnicity is unknown. OBJECTIVE: To describe the prevalence of HRV species and their associations with demographic, environmental and socioeconomic factors in healthy Aboriginal and non-Aboriginal children. METHODS: Respiratory viruses and bacteria were identified in 1006 nasopharyngeal aspirates collected from a cohort of 79 Aboriginal and 88 non-Aboriginal Western Australian children before 2 years of age. HRV-positive nasopharyngeal aspirates were typed for HRV species and genotypes. Longitudinal growth models incorporating generalized estimating equations were used to investigate associations between HRV species and potential risk factors. RESULTS: Of the 159 typed specimens, we identified 83 (52.2%) human rhinovirus species A (HRV-A), 26 (16.4%), human rhinovirus species B and 50 (31.4%) HRV-C. HRV-C was associated with upper respiratory symptoms in Aboriginal (odds ratio, 3.77; 95% confidence interval:1.05-13.55) and non-Aboriginal children (odds ratio, 5.85; 95% confidence interval: 2.33-14.66). HRV-A and HRV-C were associated with carriage of respiratory bacteria. In Aboriginal children, HRV-A was more common in the summer and in those whose mothers were employed prior to delivery. In non-Aboriginal children, day-care attendance and exclusive breast-feeding at age 6-8 weeks were associated with detection of HRV-A, and gestational smoking with detection of HRV-C. CONCLUSIONS: Factors associated with the presence of HRV differ between Aboriginal and non-Aboriginal children. In contrast to HRV-A, HRV-C is associated with upper respiratory symptoms suggesting that HRV-C is likely to be implicated in respiratory illness.

Increased H1N1 infection rate in children with asthma.

RATIONALE: The 2009 H1N1 flu appeared to cause more severe cold symptoms during the 2009-2010 flu season. OBJECTIVES: We evaluated H1N1 infections during peak viral season in children with and without asthma to determine whether the H1N1 infectivity rate and illness severity were greater in subjects with asthma. METHODS: One hundred and eighty children, 4-12 years of age, provided eight consecutive weekly nasal mucus samples from September 5 through October 24, 2009, and scored cold and asthma symptoms daily. Viral diagnostics were performed for all nasal samples. MEASUREMENTS AND MAIN RESULTS: One hundred and sixty-one children (95 with asthma, 66 without asthma) completed at least 6 of the 8 nasal samples. The incidence of H1N1 infection was significantly higher in children with asthma (41%) than in children without asthma (24%; odds ratio, 4; 95% confidence interval, 1.8-9; P < 0.001), but rates of human rhinovirus infection (90% each) and other viral infections (47 vs. 41%) were similar. In children with asthma, there was a nonsignificant trend for increased loss of asthma control during H1N1 infections compared with human rhinovirus infections (38 vs. 21%; odds ratio, 2.6; 95% confidence interval, 0.9-7.2; P = 0.07). CONCLUSIONS: During peak 2009 H1N1 flu season, children with asthma were infected almost twice as often with H1N1 compared with other respiratory viruses. H1N1 infection also caused increased severity of cold symptoms compared with other viral infections. Given the increased susceptibility of children with asthma to infection, these findings reinforce the need for yearly influenza vaccination to prevent infection, and raise new questions about the mechanism for enhanced susceptibility to influenza infection in asthma.

Negative control of TLR3 signaling by TICAM1 down-regulation.

Toll-IL-1 receptor (TIR) domain-containing adaptor molecule-1 (TICAM1, also called TRIF) is an important adaptor protein in TLR3 and TLR4 signaling pathways that mediate proinflammatory cytokine and IFN responses. Negative regulation of TICAM1 by exogenous viral protease or by endogenous caspase and proteasome have been reported to shut down TICAM1-mediated signaling. In this study, we discovered that down-regulation of TICAM1, but not other components in this signaling pathway, occurred in a natural process of TLR3 activation induced by double-stranded RNA or human rhinovirus (RV) infection in airway epithelial cells and various other cell types. TICAM1 was essential for IFN expression, and the loss of TICAM1 significantly elevated RV production. The low level of TICAM1 protein expression, caused by the prior double-stranded RNA treatment, led to a lack of IFN production upon additional treatment, suggesting receptor desensitization. In follow-up studies, TICAM1 down-regulation was found to be dependent on TLR3 but not RIG1, MDA5, or PKR and appeared to be regulated post-translationally. Neither proteasome nor caspase inhibitors could prevent TICAM1 down-regulation. Instead, a lysosome-mediated process appeared to be involved, suggesting a novel mechanism that is different from previous reports. In conclusion, TICAM1 down-regulation is an essential step in TLR3 activation, and its function is to stop TLR3-mediated IFN production.

Evidence for a causal relationship between allergic sensitization and rhinovirus wheezing in early life.

RATIONALE: Aeroallergen sensitization and virus-induced wheezing are risk factors for asthma development during early childhood, but the temporal developmental sequence between them is incompletely understood. OBJECTIVE: To define the developmental relationship between aeroallergen sensitization and virus-induced wheezing. METHODS: A total of 285 children at high risk for allergic disease and asthma were followed prospectively from birth. The timing and etiology of viral respiratory wheezing illnesses were determined, and aeroallergen sensitization was assessed annually for the first 6 years of life. The relationships between these events were assessed using a longitudinal multistate Markov model. MEASUREMENTS AND MAIN RESULTS: Children who were sensitized to aeroallergens had greater risk of developing viral wheeze than nonsensitized children (hazard ratio [HR], 1.9; 95% confidence interval [CI], 1.2-3.1). Allergic sensitization led to an increased risk of wheezing illnesses caused by human rhinovirus (HRV) but not respiratory syncytial virus. The absolute risk of sensitized children developing viral wheeze was greatest at 1 year of age; however, the relative risk was consistently increased at every age assessed. In contrast, viral wheeze did not lead to increased risk of subsequent allergic sensitization (HR, 0.76; 95% CI, 0.50-1.1). CONCLUSIONS: Prospective, repeated characterization of a birth cohort demonstrated that allergic sensitization precedes HRV wheezing and that the converse is not true. This sequential relationship and the plausible mechanisms by which allergic sensitization can lead to more severe HRV-induced lower respiratory illnesses support a causal role for allergic sensitization in this developmental pathway. Therefore, therapeutics aimed at preventing allergic sensitization may modify virus-induced wheezing and the development of asthma.