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.

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.

Attenuated innate mechanisms of interferon-gamma production in rats susceptible to postviral airway dysfunction.

After Sendai virus (SeV)-induced bronchiolitis as weanlings, BN, but not F344, rats develop a postbronchiolitis asthma-like phenotype, which can be prevented by supplemental interferon (IFN)-gamma treatment. We have shown that splenocytes from BN weanlings, compared with those from F344 weanlings, have a markedly reduced capacity for IFN-gamma production. We hypothesized that SeV-induced IFN-gamma production occurs via innate mechanisms that are attenuated in BN weanlings. Therefore, we investigated potential mechanisms of SeV-induced IFN-gamma production in BN and F344 weanlings. SeV-stimulated splenocytes secreted the IFN-gamma-inducing cytokines, interleukin (IL)-12 and IL-18. BN splenocytes produced significantly less IL-12 (P = 0.001) and IL-18 (P < 0.001) than did F344 splenocytes. Depletion studies demonstrated that natural killer cells were the primary source of SeV-induced IFN-gamma production. Anti-IL-12 antibody, IL-12 p40 homodimer, and IL-18 binding protein each inhibited SeV-induced IFN-gamma production by 82-94%, and the combination of IL-12 p40 homodimer and IL-18 binding protein abolished SeV-induced IFN-gamma production, demonstrating synergism between IL-12 and IL-18. Therefore, SeV-induced IFN-gamma production occurred via innate IL-12-, IL-18-, and natural killer cell-dependent mechanisms, which were attenuated in BN weanlings. Attenuation of innate IFN-gamma-producing responses to SeV in BN weanlings may be a critical factor in their susceptibility to postbronchiolitis chronic airway dysfunction.

Persistence of viral RNA in 2 rat strains differing in susceptibility to postbronchiolitis airway dysfunction.

After viral bronchiolitis at an early age, a chronic asthma-like syndrome develops in BN, but not F344, rats. We hypothesized that the BN strain is less effective at clearing virus from the involved tissues. Weanling BN and F344 rats were inoculated with Sendai virus, and lung and peribronchial lymph nodes were harvested from each strain at 5 to 84 days after infection; control tissues were obtained from noninfected rats. Lung viral titers were similar for the 2 strains, with no infectious virus detectable by day 10. However, viral RNA was detected consistently by means of RT-PCR analyses in lungs and lymph nodes of both strains from days 10 to 27 and was still present at day 84 in some of the tissues from each strain. In contrast, there were strain-related differences in immune responses because IL-13 levels remained increased in the lung secretions of BN rats at 4 weeks after inoculation. Thus although Sendai virus could persist for at least 3 months after an acute infection in rats, this did not differ with strain. The persistent increase in IL-13 suggests instead that the strain-related variability in virus-associated airway pathology might be determined by the host response to infection rather than by the intensity or duration of infection.

Chronic postbronchiolitis airway instability induced with anti-IFN-gamma antibody in F344 rats.

Analogous to childhood-onset asthma in humans, rats may develop a chronic asthmalike phenotype, depending on their genetic background and the age at which they experience a viral airway injury. Brown Norway rats develop a postbronchiolitis asthmalike phenotype that may be prevented with supplements of interferon-gamma (IFN-gamma); we hypothesized that the normally resistant F344 rat strain would develop the asthmalike phenotype if the IFN-gamma response were suppressed during viral illness. Weanling F344 rats were pretreated with anti-IFN-gamma or control antibody, and inoculated with Sendai virus or vehicle. Anti-IFN-gamma treatment reduced lung IFN-gamma and increased IL-4 mRNA during the infection. Physiologic studies performed 8 wk later revealed premature airway closure (p = 0.03) and elevated specific pulmonary resistance (p < 0.01) in the postbronchiolitis anti-IFN-gamma group compared with noninfected controls and untreated postbronchiolitis rats. However, unlike the postbronchiolitis asthmalike phenotype in Brown Norway rats, bronchiolar inflammation and fibrosis were absent in the F344 rats. Lung elastic recoil and alveolar surface density also were unchanged compared with noninfected control rats. We conclude that there is an interactive effect of a weak IFN-gamma response and viral bronchiolitis at an early age that may result in persistent postbronchiolitis airway dysfunction. The presence of premature airway closure that is independent of airway wall inflammation or changes in lung elastic recoil suggests peripheral airway instability as a mechanism for the airway obstruction.

Interferon-gamma enhances rhinovirus-induced RANTES secretion by airway epithelial cells.

Respiratory viruses, including rhinoviruses, infect respiratory epithelium and induce a variety of cytokines and chemokines that can initiate an inflammatory response. Cytokines, such as interferon (IFN)-gamma and tumor necrosis factor (TNF)-alpha, could enhance epithelial cell activation by inducing virus receptors. To test this hypothesis, effects of IFN-gamma or TNF-alpha on expression of intercellular adhesion molecule (ICAM)-1, rhinovirus binding, and virus-induced chemokine secretion on A549 and human bronchial epithelial cells (HBEC) were determined. The results varied with the type of cell. IFN-gamma was a stronger inducer of ICAM-1 and viral binding on HBEC, whereas TNF-alpha had greater effects on A549 cells. In addition, IFN-gamma, but not TNF-alpha, synergistically enhanced regulated on activation, normal T cells expressed and secreted (RANTES) mRNA expression and protein secretion induced by RV16 or RV49. To determine whether IFN-gamma could enhance RANTES secretion independent of effects on ICAM-1 and RV binding, HBEC were transfected with RV16 RNA in the presence or absence of IFN-gamma. RV16 RNA alone stimulated RANTES secretion, and this effect was enhanced by IFN-gamma. These results demonstrate that IFN-gamma can enhance rhinovirus-induced RANTES secretion by increasing viral binding, and through a second receptor-independent pathway. These findings suggest that IFN-gamma, by upregulating RANTES secretion, could be an important regulator of the initial immune response to rhinovirus infections.

Similar frequency of rhinovirus-infectible cells in upper and lower airway epithelium.

Rhinovirus (RV) infections can alter lower airway physiology and inflammation, yet the characteristics of RV replication in lower airway cells are incompletely understood. An RV serotype 16 (RV16)-specific monoclonal antibody was identified. Immunohistochemistry and an infectious center assay were used to quantitate the infectivity of RV16 in primary bronchial and adenoidal epithelial cells. The proportion of infectible epithelial cells increased with the inoculum but did not exceed 10%. Analysis of bronchial tissue samples infected ex vivo demonstrated a small subset of RV-infected cells in the epithelial layer. These data confirm previous reports that RV infects only a small subset of epithelial cells in upper airway tissues and indicate that lower airway epithelial cells have a similar susceptibility to RV infection. In confirming that RV can infect cells in the lower airway, these results suggest that lower airway dysfunction occurs through this mechanism in susceptible persons.