This study shows that pulmonary infection of mice with equivalent doses of RSV and HMPV leads to different clinical outcomes. Although RSV replicated to higher titers, HMPV caused more severe disease associated with higher levels of cytokines and a much stronger NK cell response. These findings indicate important differences in the pathogenesis of respiratory disease induced by these two related paramyxoviruses.
HMPV reached lower titers than RSV, both in the URT and LRT, with a single peak observed on day 4 in both infection models (Fig. 1A and 1B). An early report had suggested that HMPV replicates in lung tissue with biphasic kinetics reaching peak titers 7 and 14 days p.i. By contrast, more recent results in the mouse and in the cotton rat model [13, 14, 16], showed uniphasic growth kinetics, more consistent with our results and similar to what can be observed after RSV infection . Lower HMPV peak titers might reflect different susceptibility of airway epithelial cells to viral infections or viral spread; in addition, they may indicate differences in the early (innate) immune response. Indeed, we found evidence that the NK cell response is much stronger in HMPV- than RSV-infected mice.
Despite poor replication, HMPV induced considerable airway obstruction, weight loss, and histopathology, while only minimal changes occurred in RSV-infected mice.
A closer look at the inflammatory cell infiltrates revealed significant differences in two components of the innate immune response. In particular, HMPV induced a more prominent recruitment of neutrophils and NK cells to the BAL when compared to RSV (Fig. 3B and 4D). These findings support the concept that HMPV may elicit a more pronounced innate immune response that, on the one hand, is beneficial for virus control but, on the other hand, may cause more extensive immunopathology. Previous data have shown that RSV infection in the BALB/c mouse leads to recruitment of neutrophils and NK cells to the lungs, with a peak observed on day 4 p.i. . We found that, even after infection with a 5-fold higher inoculum, RSV was not able to recruit and activate NK cells to the same extent as HMPV (Fig. 4D and 4E). However, the potential role of this cell subset for HMPV pathogenesis has to be clarified by future approaches, such as in vivo depletion of NK cells.
It has been suggested that the RSV G and/or SH protein inhibit trafficking of NK cells to the lungs, since the absence of the corresponding genes markedly increases the number of NK cells in BAL . The mechanism of this inhibition is still unknown, but it might be an effect of these proteins on the profile of chemokines produced . Therefore, the structural differences between the G and the SH proteins of HMPV and RSV might be instrumental in recruiting NK cells to high levels. However, this possibility needs to be further evaluated.
Higher levels of the inflammatory cytokines IL-6, TNF-α and of the C-C chemokine MCP-1 were observed in HMPV-compared to RSV-infected mice. This is in contrast to previous findings showing that HMPV poorly activates inflammatory cytokines such as IL-1, IL-6 and TNF-α . The discrepancy can possibly be assigned to the different properties of the isolates used for the infection studies (low passage clinical isolate in our study versus extensively cell-passaged isolate ). In fact, pathogen-specific factors may be altered after extensive cell culture passages thus influencing the replication pattern of and the response to a given pathogen. For instance, it has been shown that a non-pathogenic variant of pneumonia virus of mice, another member of the subfamily Pneumovirinae, was generated during in vitro passages .
It has been reported that RSV induces significant changes in the mouse model only if given at high dose [17, 31]; therefore, the low levels of cytokines observed in our study after RSV infection could also be a consequence of the different viral dose used i.e. 2 × 105 PFU/animal in our study versus 107 PFU/animal in previous studies [16, 23]. Hence, it appears that the virus load required to trigger an inflammatory response (cytokine production as well as recruitment of inflammatory cells) is significantly lower in the HMPV than in the RSV infection process.
In contrast to the NK cell response, the T cell recruitment to the lung airways showed no major differences between the two viral infections. In the absence of defined CTL epitopes, the functional analysis of T cells was restricted to IFN-γ production following non-specific PMA/Ionomycin stimulation and was found to be slightly lower after HMPV infection than after RSV infection. However, in the absence of data on virus-specific T cell response, these findings should not be over-interpreted.
In pediatric patients, HMPV has been reported to cause a disease pattern similar to that of RSV with signs and symptoms ranging from severe cough to bronchiolitis and pneumonia [5, 32]. In some studies but not in others, HMPV infection has been associated more frequently than RSV with acute asthma exacerbations in children [8, 33, 34] and adults  and with more severe lower respiratory tract involvement leading to pneumonia [4, 8, 32]. In infants, HMPV has been reported to promote a weak inflammatory response, with low levels of cytokines and chemokines in respiratory secretions . By contrast, in a recent study, restimulation by HMPV of human PBMC from previously exposed adults resulted in markedly more robust IL-6 and significantly weaker IFN-γ response than did restimulation by RSV . Taken together, these studies indicate that, as in our mouse model, HMPV-induced pathogenesis may differ significantly from that related to RSV.