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.

Effects of montelukast on patients with asthma after experimental inoculation with human rhinovirus 16.

BACKGROUND: Leukotrienes are induced by viral infections. OBJECTIVES: To determine whether treatment with montelukast would improve asthma disease control in patients with mild allergic asthma during an experimentally induced rhinovirus infection. METHODS: Patients with mild allergic asthma were randomized to receive treatment with either montelukast or placebo, and 7 days later both groups were inoculated with human rhinovirus 16. Patients were evaluated at baseline, during the acute infection phase, and during the recovery phase for asthma and cold symptoms by questionnaire. Sputum, nasal lavage fluid, and blood were analyzed for viral shedding and cellular inflammation, and peak expiratory flow was measured daily. RESULTS: A total of 19 patients (11 in the placebo group and 8 in the active group) completed the study. No significant differences were found in asthma control and cold symptom scores between the control and treatment groups. The change in peak expiratory flow from the randomization to acute illness phase was greater in the placebo group than the treatment group (mean, -22 vs 0 L/min; P = .05). During the recovery phase, the percentage of sputum eosinophils increased in the placebo group and remained at baseline levels in the montelukast group (median, 2.7% vs 0.2%; P = .05 between groups). CONCLUSIONS: In this pilot study, montelukast did not improve asthma control or cold symptom scores caused by experimental rhinovirus infection. Analysis of secondary outcomes suggests that montelukast may prevent reductions in lung function and increases in sputum eosinophils caused by common cold infections. Further studies are needed to determine whether these effects are associated with clinically significant improvements in health outcomes during natural colds. TRIAL REGISTRATION: clinicaltrials.gov Identifier: NCT00359073.

Similar colds in subjects with allergic asthma and nonatopic subjects after inoculation with rhinovirus-16.

BACKGROUND: Rhinovirus infections are frequent causes of asthma exacerbations. OBJECTIVE: This study was conducted to test whether subjects with and without allergic asthma have different responses to infection and to identify baseline patient risk factors that predict cold outcomes. METHODS: Twenty subjects with mild persistent allergic asthma and 18 healthy subjects were experimentally inoculated with rhinovirus-16. Subjects were evaluated at baseline, during the acute infection, and during recovery for asthma and cold symptoms by using a validated questionnaire. Sputum and nasal lavage fluid were evaluated for viral shedding, cytokines, and cellular inflammation. RESULTS: There were no group-specific significant differences in peak cold symptom scores (10.0 +/- 5.8 vs 11.1 +/- 6.2, asthmatic vs healthy subjects), peak nasal viral titers (log(10) 4.3 +/- 0.8 vs 3.7 +/- 1.4 50% tissue culture infective dose/mL, respectively), or changes in peak flow during the study (10% +/- 10% vs 8% +/- 6%, respectively). Rhinovirus-16 infection increased peak asthma index values in the asthmatic group (median, 6 --> 13; P = .003) but only marginally in the healthy group (median, 4 --> 7; P = .09). More asthmatic subjects had detectable eosinophils in nasal lavage and sputum samples at baseline and during infection, but otherwise, cellular and cytokine responses were similar. Baseline sputum eosinophilia and CXCL8 (IL-8) levels were positively associated with cold symptoms, whereas CCL2 (monocyte chemotactic protein 1) levels were inversely associated with nasal viral shedding. CONCLUSIONS: These findings suggest that subjects with mild allergic asthma and healthy subjects have similar cold symptoms and inflammatory and antiviral responses. In addition, eosinophilia and other selective baseline measures of airway inflammation in subjects with or without asthma might predict respiratory outcomes with rhinovirus infection.

Distribution and seasonality of rhinovirus and other respiratory viruses in a cross-section of asthmatic children in Trinidad, West Indies.

BACKGROUND: Childhood asthma in the Caribbean is advancing in prevalence and morbidity. Though viral respiratory tract infections are reported triggers for exacerbations, information on these infections with asthma is sparse in Caribbean territories. We examined the distribution of respiratory viruses and their association with seasons in acute and stable asthmatic children in Trinidad. METHODS: In a cross-sectional study of 70 wheezing children attending the emergency department for nebulisation and 80 stable control subjects (2 to 16 yr of age) in the asthma clinic, nasal specimens were collected during the dry (n = 38, January to May) and rainy (n = 112, June to December) seasons. A multitarget, sensitive, specific high-throughput Respiratory MultiCode assay tested for respiratory-virus sequences for eight distinct groups: human rhinovirus, respiratory syncytial virus, parainfluenza virus, influenza virus, metapneumovirus, adenovirus, coronavirus, and enterovirus. RESULTS: Wheezing children had a higher [chi(2 )= 5.561, p = 0.018] prevalence of respiratory viruses compared with stabilized asthmatics (34.3% (24) versus (vs.) 17.5% (14)). Acute asthmatics were thrice as likely to be infected with a respiratory virus (OR = 2.5, 95% CI = 1.2 - 5.3). The predominant pathogens detected in acute versus stable asthmatics were the rhinovirus (RV) (n = 18, 25.7% vs. n = 7, 8.8%; p = 0.005), respiratory syncytial virus B (RSV B) (n = 2, 2.9% vs. n = 4, 5.0%), and enterovirus (n = 1, 1.4% vs. n = 2, 2.5%). Strong odds for rhinoviral infection were observed among nebulised children compared with stable asthmatics (p = 0.005, OR = 3.6, 95% CI = 1.4 - 9.3,). RV was prevalent throughout the year (Dry, n = 6, 15.8%; Rainy, n = 19, 17.0%) and without seasonal association [chi(2 )= 0.028, p = 0.867]. However it was the most frequently detected virus [Dry = 6/10, (60.0%); Rainy = 19/28, (67.9%)] in both seasons. CONCLUSION: Emergent wheezing illnesses during childhood can be linked to infection with rhinovirus in Trinidad's tropical environment. Viral-induced exacerbations of asthma are independent of seasons in this tropical climate. Further clinical and virology investigations are recommended on the role of infections with the rhinovirus in Caribbean childhood wheeze.

Distribution and seasonality of rhinovirus and other respiratory viruses in a cross-section of asthmatic children in Trinidad, WestIndies

BACKGROUND: Childhood asthma in the Caribbean is advancing in prevalence and morbidity. Though viral respiratory tract infections are reported triggers for exacerbations, information on these infections with asthma is sparse in Caribbean territories. We examined the distribution of respiratory viruses and their association with seasons in acute and stable asthmatic children in Trinidad. METHODS: In a cross-sectional study of 70 wheezing children attending the emergency department for nebulisation and 80 stable control subjects (2 to 16 yr of age) in the asthma clinic, nasal specimens were collected during the dry (n = 38, January to May) and rainy (n = 112, June to December) seasons. Amultitarget, sensitive, specific high-throughput Respiratory MultiCode assay tested for respiratory-virus sequences for eight distinct groups: human rhinovirus, respiratory syncytial virus, parainfluenza virus,influenza virus, metapneumovirus, adenovirus, coronavirus, and enterovirus. RESULTS: Wheezing children had a higher [c2 = 5.561, p = 0.018] prevalence of respiratory viruses compared with stabilized asthmatics (34.3% (24) versus (vs.) 17.5% (14)).... CONCLUSION: Emergent wheezing illnesses during childhood can be linked to infection with rhinovirus in Trinidad's tropical environment. Viral-induced exacerbations of asthma are independent of seasons in this tropical climate. Further clinical and virology investigations are recommended on the role of infections with the rhinovirus in Caribbean childhood wheeze.

Distribution and seasonality of rhinovirus and other respiratory viruses in a cross-section of asthmatic children in Trinidad, West Indies.

BACKGROUND: Childhood asthma in the Caribbean is advancing in prevalence and morbidity. Though viral respiratory tract infections are reported triggers for exacerbations, information on these infections with asthma is sparse in Caribbean territories. We examined the distribution of respiratory viruses and their association with seasons in acute and stable asthmatic children in Trinidad. METHODS: In a cross-sectional study of 70 wheezing children attending the emergency department for nebulisation and 80 stable control subjects (2 to 16 yr of age) in the asthma clinic, nasal specimens were collected during the dry (n=38, January to May) and rainy (n=112, June to December) seasons. A multitarget, sensitive, specific high-throughput Respiratory MultiCode assay tested for respiratory-virus sequences for eight distinct groups: human rhinovirus, respiratory syncytial virus, parainfluenza virus, influenza virus, metapneumovirus, adenovirus, coronavirus, and enterovirus. RESULTS: Wheezing children had a higher [chi square =5.561, p=0.018] prevalence of respiratory viruses compared with stabilized asthmatics (34.3 per cent (24) versus (vs.) 17.5 per cent (14)). Acute asthmatics were thrice as likely to be infected with a respiratory virus (OR = 2.5, 95 per cent CI = 1.2 - 5.3). The predominant pathogens detected in acute versus stable asthmatics were the rhinovirus (RV) (n=18, 25.7 per cent vs. n=7, 8.8 per cent; p=0.005), respiratory syncytial virus B (RSV B) (n=2, 2.9 per cent vs. n=4, 5.0 per cent), and enterovirus (n=1, 1.4 per cent vs. n=2, 2.5 per cent). Strong odds for rhinovirus infection were observed among nebulised children compared with stable asthmatics (p=0.005, OR = 3.6, 95 per cent CI = 1.4 - 9.3,). RV was prevalent throughout the year (Dry, n=6, 15.8 per cent; Rainy, n=19, 17.0 per cent) and without seasonal association [chi square =0.028, p=0.867]...

Inhibition of rhinovirus replication in vitro and in vivo by acid-buffered saline.

Human rhinoviruses (HRVs) are quite sensitive to low pH. To determine whether this characteristic might be a therapeutic target, we evaluated the sensitivity of HRV to low-pH buffers in vitro and in vivo. Our findings confirm that low pH inhibited replication of most HRVs and reduced the replication of influenza virus. Preliminary experiments verified that the surface pH of the human nasopharynx could be transiently lowered to pH approximately 4.0 by topical administration of citrate/phosphate (CP) buffers, which was well tolerated. In a pilot experimental colds study, intranasal administration of CP buffer, compared with normal saline, reduced viral shedding by 1 log unit (10(3) vs. 10(4) 50% tissue culture infective dose/mL; P<.01), although respiratory symptoms were not significantly reduced. These findings demonstrate that low-pH buffers have antiviral activity in vivo and suggest that a larger clinical trial is warranted to determine whether this approach could reduce rates of viral transmission.

Neural circuitry underlying the interaction between emotion and asthma symptom exacerbation.

Asthma, like many inflammatory disorders, is affected by psychological stress, suggesting that reciprocal modulation may occur between peripheral factors regulating inflammation and central neural circuitry underlying emotion and stress reactivity. Despite suggestions that emotional factors may modulate processes of inflammation in asthma and, conversely, that peripheral inflammatory signals influence the brain, the neural circuitry involved remains elusive. Here we show, using functional magnetic resonance imaging, that activity in the anterior cingulate cortex and insula to asthma-relevant emotional, compared with valence-neutral stimuli, is associated with markers of inflammation and airway obstruction in asthmatic subjects exposed to antigen. This activation accounts for > or =40% of the variance in the peripheral markers and suggests a neural basis for emotion-induced modulation of airway disease in asthma. The anterior cingulate cortex and insula have been implicated in the affective evaluation of sensory stimulation, regulation of homeostatic responses, and visceral perception. In individuals with asthma and other stress-related conditions, these brain regions may be hyperresponsive to disease-specific emotional and afferent physiological signals, which may contribute to the dysregulation of peripheral processes, such as inflammation.

Quantitative and qualitative analysis of rhinovirus infection in bronchial tissues.

Although rhinovirus (RV) infections can cause asthma exacerbations and alter lower airway inflammation and physiology, it is unclear how important bronchial infection is to these processes. To study the kinetics, location, and frequency of RV appearance in lower airway tissues during an acute infection, immunohistochemistry and quantitative polymerase chain reaction analysis were used to analyze the presence of virus in cells from nasal lavage, sputum, bronchoalveolar lavage, bronchial brushings, and biopsy specimens from 19 subjects with an experimental RV serotype 16 (RV16) cold. RV was detected by polymerase chain reaction analysis on cells from nasal lavage and induced sputum samples from all subjects after RV16 inoculation, as well as in 5 of 19 bronchoalveolar lavage cell samples and in 5 of 18 bronchial biopsy specimens taken 4 days after virus inoculation. Immunohistochemistry detected RV16 in 39 and 36% of all biopsy and brushing samples taken 4 and 15 days, respectively, after inoculation. Infected cells were primarily distributed in discrete patches on the epithelium. These results confirm that infection of lower airway tissues is a frequent finding during a cold and further demonstrate a patchy distribution of infected cells, a pattern similar to that reported in upper airway tissues.

Rhinovirus-induced interferon-gamma and airway responsiveness in asthma.

The majority of asthma exacerbations are caused by respiratory infections, with rhinovirus (RV) being the most common virus. Recent evidence has suggested that decreased generation of IFN-gamma is associated with more severe colds and delayed elimination of virus. Whether the generation of IFN-gamma also has any relationship to general features of asthma severity has yet to be determined. To evaluate this hypothesis, peripheral blood mononuclear cells from 19 subjects with atopy and asthma were incubated with RV16 for 6 days to determine IFN-gamma and interleukin (IL)-5 production; these responses were then compared with measurements of airflow obstruction and airway responsiveness. RV16-induced IFN-gamma production correlated significantly with the methacholine PD (r = 0.50, p = 0.03), and the ratio of RV16-induced IFN-gamma:IL-5 correlated with % predicted FEV1 (r = 0.53, p = 0.02). In contrast, there were no significant associations between measures of asthma severity and RV-induced IL-5. These findings suggest that a cytokine imbalance with a deficient Th1 response to RV, but not a Th2 response, is associated with measures of asthma severity and support the concept that impaired antiviral responses may be associated with asthma severity.

Comparison of the effects of repetitive low-dose and single-dose antigen challenge on airway inflammation.

BACKGROUND: Airway allergen provocation provides a model to study allergic inflammation in relationship to pulmonary physiology. Allergen provocation is usually administered as a relatively large single-dose challenge that might not reflect a chronic, natural, low-dose airborne allergen exposure. OBJECTIVE: We sought to compare the magnitude, characteristic features, and kinetics of airway inflammation induced by means of repetitive low-dose antigen challenges with those factors induced by means of an equivalent single-dose allergen challenge in allergic asthma. METHODS: This was a 2-period crossover study. During separate phases, each subject was administered either a predetermined single-dose antigen challenge or 25% of that dose on each of 4 consecutive days. The airway response to allergen challenge was determined by means of measurement of pulmonary function and sputum features of inflammation, including eosinophil, eosinophil-derived neurotoxin, and fibronectin levels. RESULTS: Both models of antigen challenge caused significant and equivalent sputum eosinophilia. The immediate decrease in FEV(1) and the FEV(1)/forced vital capacity ratio and the increase in sputum eosinophilia, eosinophil-derived neurotoxin, and fibronectin levels occurred gradually over the first 3 low doses and then reached a plateau or tended to decrease with the fourth antigen exposure. CONCLUSION: Our data suggest that although the 2 challenge models had similar quantitative effects on lung function and sputum eosinophilia, the qualitative responses and kinetics of these changes were distinct. Repetitive low doses of antigen, as might mimic natural allergy exposure, produced an equivalent inflammatory response to the large single-dose challenge but with a smaller amount of antigen, suggesting that priming and accumulative effects might have occurred. Moreover, our limited data also suggest that immunologic tolerance might be induced by frequent challenges.

School examinations enhance airway inflammation to antigen challenge.

Psychological stress can lead to asthma exacerbations in some patients. It is our hypothesis that the stress effect can occur through an enhancement of allergic inflammatory response. To investigate this possibility, airway antigen challenge was evaluated in 20 college students with mild asthma during both a low-stress phase (midsemester or two weeks postfinal examination) and a stress phase (final examination week). Subjects completed questionnaires to assess psychological state and underwent inhaled antigen challenge. Sputum samples were collected before challenge, and six and 24 hours and seven days postchallenge. Leukocytes were counted and eosinophil-derived neurotoxin (EDN) was measured in sputum supernates. Sputum cells were cultured and stimulated ex vivo with phytohemagglutinin (10 microg/ml), and culture supernates were assayed for interleukin-5 (IL-5) and interferon-gamma by enzyme-linked immunosorbent assay. Sputum eosinophils and EDN levels significantly increased at six and 24 hours postchallenge and were enhanced during the stress phase (p < 0.01). IL-5 generation by sputum cells was also increased at 24 hours during stress and correlated with airway eosinophils (r(s) = 0.65, p < 0.05). Students' anxiety and depression scores were significantly higher during the examination period. Our findings suggest that stress associated with final examinations can act as a cofactor to increase eosinophilic airway inflammation to antigen challenge and thus may enhance asthma severity.