Eight to 10-week-old, specific-pathogen-free female BALB/c mice were obtained from Charles River (Sulzfeld, Germany). The mice were kept in a venti-rack at the Institute for Medical Microbiology and Hygiene, Freiburg and fed sterilized water and food ad libitum. All experiments were performed in accordance with the local animal care commission.

Rhesus monkey kidney cells (LLC-MK2 and Vero) were maintained in D-MEM supplemented with 10% FCS, 1% L-glutamine, and penicillin/streptomycin (complete medium) and Eagles's MEM complete medium, respectively. HMPV was propagated in LLC-MK2 cells cultured in D-MEM w/o serum supplemented with 1% L-glutamine, penicillin/streptomycin antibiotic mix and 5 μg/ml trypsin (Sigma-Aldrich, Munich, Germany) (trypsin medium) whereas RSV was grown on HEp-2 cells cultured in Eagles's MEM complete medium.

For HMPV and RSV titration on Vero cells, Eagle's MEM trypsin medium and Eagle's MEM supplemeted with 5% FCS were used, respectively.

The HMPV strain D03-574 (subgroup A2a) was isolated from an infant with bronchiolitis in our laboratory, propagated 4 times on LLC-MK2 cells and used to prepare a virus stock. Virus was harvested 3 days p.i., snap-frozen, and kept in liquid nitrogen. The infectious virus titer of the stock was 2,5 × 10⁶ plaque froming units/ml (PFU/ml). The RSV A2 strain is a common laboratory strain and was originally obtained from Peter Openshaw (Imperial College, London, GB), grown on HEp-2 cells over 4 passages, and kept in liquid nitrogen. The infectious virus titer of the stock on Vero cells was 3,3 × 10⁷ PFU/ml.

BALB/c mice were lightly anesthetized by intraperitoneal injection of ketamine (2 mg/mouse) and xylazine (0.15 mg/mouse), and infected intranasally (i.n.) with 2 × 10⁵ PFU HMPV strain D03-574 or 2 × 10⁵(when indicated 10⁶) PFU RSV strain A2 in 80 μl serum-free (SF) Eagle's MEM.

At the indicated time points, anesthetized mice were exsanguinated, and lungs and nasal turbinates were harvested separately for virus quantification by plaque assay. Lungs were homogenized using a Teflon pestle in a volume of 1.5 ml of SF culture medium, whereas nasal turbinates were homogenized in 3 ml by grinding with sterile sand. Total homogenates were quickly spun down, and supernatants were frozen in liquid nitrogen until use. To determine HMPV titers, 150 μl of 10-fold serial dilutions of the clarified homogenates were added in duplicate to confluent Vero cell monolayers in a 24-well plate and cultured for 5 days under 0.8% methylcellulose, followed by fixation with 80% methanol. HMPV plaques were visualized by incubation with anti-HMPV rabbit serum (kindly provided by U. Buchholz, NIH, Bethesda, MD, USA) followed by incubation with anti-rabbit IgG coupled to biotin (Perbio Science Deutschland, Bonn, Germany) and with streptavidin coupled to horseradish peroxidase (SA-HRP; BD PharMingen, San Diego, CA). Finally, the plaques were enumerated after addition of 3',3'-diaminobenzidine substrate (DAB; Merck, Darmstadt, Germany). Similarly, RSV titers were determined on Vero cells and detected using a biotin-labelled anti-RSV antibody (Biogenesis, Berlin, Germany) followed by incubation with SA-HRP and DAB substrate as previously described 20.

Whole-body plethysmography (Buxco Electronics Inc. Troy, NY) was used, to monitor the respiratory dynamics of mice in a quantitative manner. Penh is a dimensionless value that represents a function of the ratio of peak expiratory flow to peak inspiratory flow and a function of the timing of expiration, and it correlates with pulmonary airflow resistance. Penh has previously been validated in animal models of airway hyperresponsiveness (AHR) 2122 and infection-associated airway obstruction (AO) 23. Baseline airway resistance (with or without infection) is described as AO and the transient airway resistance in response to methacholine as AHR. Before exposure to methacholine, mice were allowed to acclimate to the plethysmograph chamber, and then baseline readings were recorded to determine AO. Mice were exposed to increasing doses of aerosolized methacholine/mL (Sigma-Aldrich). Plethysmograph readings were recorded again to determine AHR. Groups of infected and control mice were always evaluated in parallel.

Pulmonary inflammatory cells were obtained by bronchoalveolar lavage (BAL) as previously described 20 and used for NK assay or antibody staining without further manipulations. For microscopic differentiation of BAL macrophages, neutrophils, and lymphocytes, 10⁴ BAL cells were used for cytospin preparation on glass slides utilizing a Shandon Cytospin centrifuge (Thermo Electron, Waltham, MA). After air-drying, 2 ml Wright's staining solution (FLUKA, Buchs, Austria) was added for 6 minutes, followed by water for 6 minutes. After washing and air-drying, a differential cell count was performed with 200 cells.

The NK cell assay was performed under "mini-killer" conditions as previously described 24. Briefly, effector BAL cells were plated in two-fold dilutions starting with 5 × 10⁴ cells per well in a volume of 50 μl in a 96-well V-bottom plate (Greiner Labortechnik, Solingen, Germany). YAC-1, labelled with (⁵¹Cr), were used as target cells, and 2 × 10³ cells were added in a volume of 50 μl per well for an initial effector:target ratio of 25.

Single-cell suspensions of BAL cells (10⁵) were surface-stained for 30 min at 4°C with the following antibody combinations: (i) anti-CD8-FITC (Ly-2; clone 53-6.7), anti-CD4-PE (L3T4; clone RM 4–5) and anti-CD3-APC (CD3 ε chain; clone 145-2C11); (ii) anti-CD3-APC and anti-DX5-bio (CD49b/Pan-NK), followed by incubation with SA-Cy (all from BD PharMingen, San Diego, CA).

To detect intracellular cytokines, cells (1–2 × 10⁵) were incubated with 50 ng/ml phorbol myristate acetate (PMA) (Sigma-Aldrich), 500 ng of ionomycin (Calbiochem, San Diego, CA) and 1 μl/ml monensin (Golgistop, BD PharMingen) for 5 h at 37°C. Cells were harvested, washed and surface-stained with anti-CD3-APC and anti-CD8-FITC and then subjected to intracellular cytokine staining using the cytofix/cytoperm kit according to the manufacturer's instruction (BD PharMingen). Cells were stained with anti-IFN-γ-PE or with isotype-control antibody (BD PharMingen). After 30 min, cells were washed and analyzed on a fluorescence-activated cell sorter (FACScan and Cellquest Software, Becton Dickinson, Heidelberg, Germany), collecting data on at least 10,000 lymphocytes. Calculations of percentages were based on live cells as determined by FSC/SSC analysis.

BAL supernatants obtained by centrifugation of BAL cells for 10 min at 1600 rpm were harvested and stored at -70°C until cytokine testing was performed. IL-6, IL-10, IL-12, MCP-1, TNF-α, and IFN-γ were detected simultaneously using the Cytometric Bead Array (CBA) Mouse Inflammation Kit (BD PharMingen). Briefly, 50 μl of each sample was mixed with 50 μl of mixed capture beads and 50 μl of the mouse Th1/Th2 PE detection reagent consisting of PE-conjugated anti-mouse IL-6, IL-10, IL-12, MCP-1, TNF-α, and IFN-γ. The samples were incubated at room temperature for 3 h in the dark. After incubation with the PE detection reagent, the samples were washed once and resuspended in 300 μl of wash buffer before acquisition on a FACScan cytometer. Data were analyzed using CBA software (BD PharMingen). Standard curves were generated for each cytokine using the mixed cytokine standard provided by the kit. The concentration for each cytokine in cell supernatants was determined by interpolation from the corresponding standard curve. The range of detection was 20–5000 pg/ml for each cytokine measured by CBA.

For histological examination, lung specimens from RSV- and HMPV-infected mice and normal control mice were collected and fixed in 4% buffered formalin. Paraffin-embedded tissue blocks were cut at 4 μm. Deparaffinized sections were evaluated following hematoxylin & eosin (H&E) staining or by immunolabelling using anti-HMPV rabbit serum. Briefly, immunohistochemistry was performed after antigen retrieval and incubation for 20 min with 10% blocking serum (Biotrend, Cologne, Germany). Sections were stained semiautomatically (Autostainer instrument, Dako, Hamburg, Germany) with the primary antibody and a biotinylated detection antibody. Antibody binding was detected by the labelled streptavidin-biotin (LSAB) method 25 (ChemMate K5005 Alkaline Phosphatase/Red detection kit, Dako). Nuclei were counterstained with Mayer's hemalaun solution.

All data are expressed as mean +/- standard deviation. Student's unpaired t-test was used to compare HMPV-infected and RSV-infected animals at the same time point (significance level set at P < 0.05).

To assess whether our HMPV isolate is able to replicate in the respiratory tract of BALB/c mice, animals were infected with 2 × 10⁵ PFU of HMPV and the kinetics of viral load was determined in the nasal turbinates (upper respiratory tract; URT) and in the lungs (lower respiratory tract; LRT). A virus peak was observed between day 4 and day 6. The virus was eliminated by day 7 from the lungs and between day 10 and day 15 from the URT (Fig 1A). There was no evidence for long-term viral persistence as shown by sensitive real time PCR at day 120 p.i. (data not shown).

To compare HMPV and RSV replication in both compartments, BALB/c mice were infected i.n. with 2 × 10⁵ PFU of either virus preparation, and virus titers were determined in the URT and in the LRT. On day 4 p.i., titers were higher for RSV than for HMPV (Fig 1B). Consistent with this, HMPV was eliminated from the lungs by day 7 p.i., while low titers of RSV were still present. By contrast, virus elimination from the nasal turbinates was more efficient for RSV whereas significant titers of HMPV were still measured on day 7 p.i.

Mice infected with 2 × 10⁵ PFU of HMPV or RSV were observed daily for development of clinical symptoms. Unexpectedly and in contrast to the viral replication kinetics, HMPV but not RSV infection was associated with severe weight loss (Fig. 1C). At the peak of weight loss, mice had ruffled fur and showed heavy breathing as well as reduced activity, but all mice finally recovered.

Penh values, representing airway obstruction (AO), were measured on day 7 p.i., in both groups of mice. In accordance with the weight loss and other clinical signs of disease, AO was significantly higher in HMPV- than in RSV-infected mice or uninfected controls. This confirmed the inverse relationship between viral replication and the pathological changes (Fig. 1D).

Due to high baseline Penh values in HMPV-infected mice, AHR measurement after methacoline treatment did not yield significant results (data not shown).

Hematoxylin-eosin-stained lung sections obtained from RSV-infected mice on day 7 p.i. revealed rare foci of mild inflammatory infiltration of bronchioles and adjacent alveoli, while no histopathology was found in uninfected animals (Fig. 2A and 2B). By contrast, bronchioli and pulmonary parenchyma of HMPV inoculated mice showed severe bronchopneumonia. Bronchioli and alveolar spaces were densely packed with neutrophils, lymphocytes, macrophages, desquamated pneumocytes, and fibrin (Fig. 2C). Immunolabelling revealed groups of intraalveolar macrophages and pneumocytes expressing HMPV antigens (Fig. 2D).

To further understand the inverse correlation between viral replication and clinical disease during infection with HMPV versus RSV, we analyzed the inflammatory cells recruited to the lungs on day 4 and 7 p.i.. Day 4 represents the time of maximal virus replication and accumulation of innate immune cells including NK cells, while day 7 is the time point of maximal T cell recruitment and activity in RSV-infected mice. The number of inflammatory cells eluted from the lung airways by BAL was similar on day 4 p.i. in the HMPV- and in the RSV-infected mice. On day 7 p.i., an increase of total BAL cells was observed in both groups, but HMPV-infected mice had significantly higher numbers of BAL cells (Fig. 3A). Microscopic differentiation of BAL cells revealed that lymphocytes represent the main population at both time points, with little differences among the two groups (Fig. 3B). By contrast, the percentage and absolute numbers of neutrophils was significantly higher in the HMPV- than in the RSV-infected mice at both time points, while macrophages were more prominent at day 7 in RSV-infected mice. Analysis by flow cytometry showed that 7 days after infection CD8+ T cells were the predominant population of lymphocytes in both infection models (Fig. 4A). The percentage of CD4+ T cells was slightly higher after RSV infection on day 4, but no significant differences were observed on day 7 (Fig. 4B). Functional activity of CD8+ T cells at day 7 was evaluated by intracellular IFN-γ staining after in vitro stimulation with PMA-ionomycin (Fig. 4C). A high proportion of CD8+ T cells produced IFN-γ after both infections, but there was a trend towards higher levels of IFN-γ-producing cells after RSV infection. Overall, there were little differences in T cell recruitment to the respiratory tract during the two infections arguing against a role for T cells in the differences between HMPV and RSV-induced pathology.

NK cell accumulation in the BAL of HMPV- and RSV-infected animals was determined by flow cytometry. The percentage of NK cells (DX5+/CD3- cells) recruited to the lungs of HMPV-infected mice at day 4 p.i. was significantly higher than in RSV-infected mice. The DX5+/CD3- population declined thereafter and at day 7 comparable numbers of NK cells were present following infection with both viruses (Fig. 4D). At day 4 p.i., NK cell cytotoxicity was assessed by direct ex vivo cytotoxicity of BAL cells against YAC-1 target cells. NK cell activity was significantly higher after infection with HMPV than after infection with the same dose of RSV (Fig. 4E). NK cell recruitment and activity was increased in mice infected with a 5-fold higher RSV inoculum (10⁶ PFU/mouse) but was still less than that observed in mice infected with the lower HMPV dose (Fig. 4E and data not shown). Thus, HMPV appears to be a more potent inducer of NK cell activity than RSV.

To further characterize the factors that regulate HMPV pathogenesis in the mouse model, we analyzed the production of cytokines and chemokines by BAL cells. For this purpose, cell-free BAL fluids obtained at day 4 and at day 7 p.i. were analyzed by cytometric bead array for the presence of TNF-α, IFN-γ, IL-6, IL-10, and MCP-1. Consistent with the levels of cellular infiltration observed, HMPV-infected mice produced significantly higher levels of TNF-α, IL-6, and MCP-1 than RSV-infected mice at both, day 4 and day 7 p.i.(Fig. 4F), but similar levels of IFN-γ were measured at day 7 p.i.. Interestingly, infection with RSV, but not with HMPV, seemed to downregulate IL-10, which is produced to discrete levels after mock infection. Overall significant differences were observed in the amount of inflammatory mediators after the two infections.