Streptococcus pneumoniae Enhances Human Respiratory Syncytial Virus Infection In Vitro and In Vivo

Abstract

Human respiratory syncytial virus (HRSV) and Streptococcus pneumoniae are important causative agents of respiratory tract infections. Both pathogens are associated with seasonal disease outbreaks in the pediatric population, and can often be detected simultaneously in infants hospitalized with bronchiolitis or pneumonia. It has been described that respiratory virus infections may predispose for bacterial superinfections, resulting in severe disease. However, studies on the influence of bacterial colonization of the upper respiratory tract on the pathogenesis of subsequent respiratory virus infections are scarce. Here, we have investigated whether pneumococcal colonization enhances subsequent HRSV infection. We used a newly generated recombinant subgroup B HRSV strain that expresses enhanced green fluorescent protein and pneumococcal isolates obtained from healthy children in disease-relevant in vitro and in vivo model systems. Three pneumococcal strains specifically enhanced in vitro HRSV infection of primary well-differentiated normal human bronchial epithelial cells grown at air-liquid interface, whereas two other strains did not. Since previous studies reported that bacterial neuraminidase enhanced HRSV infection in vitro, we measured pneumococcal neuraminidase activity in these cultures but found no correlation with the observed infection enhancement in our model. Subsequently, a selection of pneumococcal strains was used to induce nasal colonization of cotton rats, the best available small animal model for HRSV. Intranasal HRSV infection three days later resulted in strain-specific enhancement of HRSV replication in vivo. One S. pneumoniae strain enhanced HRSV both in vitro and in vivo, and was also associated with enhanced syncytium formation in vivo. However, neither pneumococci nor HRSV were found to spread from the upper to the lower respiratory tract, and neither pathogen was transmitted to naive cage mates by direct contact. These results demonstrate that pneumococcal colonization can enhance subsequent HRSV infection, and provide tools for additional mechanistic and intervention studies.

Conflict of interest statement

Competing Interests: A.D.M.E. Osterhaus founded and is chief scientific officer of ViroClinics Biosciences B.V., a company set up in collaboration with Erasmus University. R.L. de Swart has received research funding from ViroClinics Biosciences B.V. However, for clarification, no materials or support were received from the company for the studies described in this manuscript, and no agreements were in place concerning the execution or publication of this work. The other authors declare no competing interests. This does not alter the authors' adherence to PLOS ONE policies on sharing data and materials.

Figures

Fig 1. Enhancement of HRSV infection mediated by specific S. pneumoniae strains in wd-NHBE cells.

(A) wd-NHBE cells were inoculated with HRSV and one S. pneumoniae strain with mock control. Strain 8, 15A and 19F enhanced HRSV infection as evidenced by quantification of EGFP+ cells 2 d.p.i. Data are represented as mean ± SD. * p ≤ 0.05; 2-tailed Mann-Whitney U test. (B) wd-NHBE cells were treated with neuraminidase from Vibrio cholerae, followed by HRSV infection after washing. Two dpi the numbers of HRSV-infected cells were enumerated. Data are presented as means ± SD. * p ≤ 0.05; 2-tailed Mann-Whitney U test. (C) HRSV was treated with neuraminidase from Vibrio cholerae before inoculation of wd-NHBE cells. Treated virus was incubated for 1 hour, followed by washing. HRSV-infected cells were enumerated 2 dpi. Data are presented as means ± SD. * p ≤ 0.05; 2-tailed Mann-Whitney U test. (D) Apical inocula of the wd-NHBE cell co-infection experiment shown in Fig 1A were tested for neuraminidase activity using the NA-Star Influenza Neuraminidase Inhibitor Resistance Detection Kit (Applied Biosystems). Numbers correspond to Vibrio cholerae μU neuraminidase activity. Data are presented as means ± SD.

Fig 2. Bacterial and virus titers of HRSV infected S. pneumoniae colonized cotton rats.

(A) Experimental design. Pneumococcal carriage was induced by i.n. inoculation of cotton rats with 5x105 CFU S. pneumoniae in 10 μl at day 0. Three days later animals were infected with 1x104 TCID50 rHRSVB05EGFP(5) in PBS (10 μl). At day 8 animals were euthanized. (B) Bacterial titers eight days after induction of pneumococcal carriage. The geometric mean titer was about 2x105 CFU/ml for the different strains. Data symbols represent individual animals, bars represent geometric mean titers (GMT) per group. * p ≤ 0.05; 2-tailed Mann-Whitney U test. (C) Virus titers five days after HRSV infection. Significantly higher virus load were detected in groups with nasal carriage of S. pneumoniae strain 19F and 23F compared to mock-treated. * p ≤ 0.05; 2-tailed Mann-Whitney U test.

Fig 3. HRSV infection in nasal septum of a S. pneumoniae colonized cotton rat.

Composite microscopic UV images of the complete nasal septum of a cotton rat 5 d.p.i. showing a representative syncytium in the middle (scale bar represents 500 μm). The inset is a confocal laser scanning image of the same nasal septum with nuclei stained with TO-PRO3 (red; scale bar represents 100 μm).

Fig 4. In vivo transmission study HRSV infection in S. pneumoniae colonized cotton rats.

(A) Schematic representation of the time course of the experiment. Animals were initially housed in pairs. One of each pair was pneumococcus- or mock-colonized, and infected with HRSV three days later. Another two days later the matching naive contact animals were added to their cage, reuniting the original pairs. Index and contact animals were euthanized at day 8 or 11, respectively. (B) Bacterial titer of index and contact animals. The geometric mean titer (GMT) was about 2x105 CFU/ml for the index group, but no bacteria were isolated in the contact animals. Each data symbol represents one animal, bars represent GMT. (C) Virus titers of index and contact animals. Significantly higher virus loads were detected in the index group colonized with strain 19F, as compared to mock-colonized. In both contact groups no virus could be isolated out of the nose and lungs. * p ≤ 0.05; 2-tailed Mann-Whitney U test.