At necropsy, performed on day 7 post infection, gross lesions typically consist of bilateral, multifocal, firm, plum-red areas of consolidation that are of variable size and depressed compared to the adjacent normal appearing lung (Figure 1). Lesions are most frequently observed in cranioventral lung lobes. On cut surface, areas of consolidation are well delineated from adjacent normal lung. In some cases, areas of consolidation surround and divide regions of pink, hyperinflated lung. Significant microscopic lesions are apparent with representative photomicrographs shown (Figure 2). Microscopically, interlobular septa are expanded by clear space interpreted to be edema (Figure 2A). Alveolar septa are thickened due to infiltrates of macrophages, lymphocytes, and lesser numbers of neutrophils (Figure 2B). Intralesional bronchioles are filled with neutrophils, sloughed epithelial cells, and necrotic cellular debris (Figure 2C). Bronchiolar epithelial cell necrosis results in attenuation of remaining epithelial cells, or complete loss of airway epithelium. In some bronchioles, epithelial cells form multinucleated syncytial cells (Figure 2D). These lesions are similar to those described by others following experimental bRSV challenge.

In the rare instances where histopathological evaluation is available from fatal hRSV cases, it was noted that airway lumina contain cellular debris from leukocytes and sloughed epithelial cells, with fibrin and a minor amount of mucin [33,34,35]. Submucosal edema and peribronchiolar infiltrates, consisting predominately of mononuclear cells with minor numbers of neutrophils, further obstructed the airways. With interstitial pneumonia, there was evidence of significant alveolar involvement including edema and cellular infiltration, as well as epithelial cell attenuation or loss.

Paramixoviruses are known to utilize different mechanisms to affect IFN signaling. hRSV and bRSV have evolved strategies to inhibit the IFN-induced cellular response that are dependent upon nonstructural (NS) proteins. As is characteristic of pneumoviruses, these viruses have two genes that encode for NS proteins. It has been shown that NS1 and NS2 proteins cooperatively mediate resistance of bRSV and hRSV to IFN-stimulated responses in a species-specific manner [40,41]. In fact, the precise mechanism whereby type I IFN responses are altered, varies between these viruses. In the case of bRSV, NS proteins block phosphorylation and activation of IRF3 [42]. By comparison, hRSV NS1 and NS2 modulation of type I IFN responsiveness involves inhibition of Stat2 expression [43].

In addition to induction of IRFs, ligation of PRRs by viral PAMPs stimulates the release of inflammatory mediators. It has been shown that RSV induces cytokine/chemokine production in airway epithelial cells via signaling through TLR3 and RLRs, which have been linked to distinct pathways controlling NF-κB activation [44]. In addition to respiratory epithelial cells, RSV infection induces inflammatory cytokines in antigen-presenting cells, the kinetics of which differs between cytokines. For example, we found peak induction of IL-1β and IL-12p40 mRNA in alveolar macrophages occurs on day 3 post-infection in the neonatal bRSV model, whereas IL-6 mRNA was higher at day 5 than day 3 of infection [29]. Recently, we have shown a significant upregulation of the innate chemokine IL-8 in lesioned lungs of bRSV-infected calves on day 7 post infection [31]. Importantly, our results fit well with data showing that IL-8 is elevated in the respiratory tract of children with hRSV bronchiolitis. Rodents lack a bona fide homologue of IL-8, although mice have what are considered to be functional homologues, CXCL1 (GRO/KC), CXCL2 (MIP-2) and CXCL5-6 (LIX), which belong to the same major cluster of chemokines. It is therefore evident that the neonatal calf model can prove useful as an in vivo platform for future exploration into pathways that specifically regulate innate immune responses during RSV infection.

Gamma delta T cells have been suggested to play a role in innate immunity to RSV based on their recognition of unprocessed and non-protein antigens independent of MHC restriction and their localization in epithelial tissues at the host-environmental interface [45]. In humans and mice, the frequency of γδ T cells in circulation or within secondary lymphoid tissues is low, generally representing less than 5-10% of the circulating peripheral lymphocyte population [46]. However, in ruminants, γδ T cells are significantly more abundant, representing up to 70% of the circulating peripheral blood lymphocytes in very young animals [47,48]. Given their low frequency and the difficulty in obtaining sufficient cell numbers, relatively few studies exist which have examined the role of γδ T cells during hRSV infection. A study in patients hospitalized for hRSV bronchiolitis revealed a significant reduction in the numbers of γδ T cells circulating in the blood compared to control patients. Interestingly, this reduction was more pronounced in patients with severe disease [49]. Aoyagi et al. recently demonstrated that γδ T cells isolated from the blood of hRSV-infected infants produce less IFNγ and more IL-4 in response to mitogen stimulation than equivalent cells isolated from rotavirus-infected infants [50], suggesting that γδ T cells may be susceptible to the Th2-skewing often associated with hRSV infection (see section on Adaptive immunity to RSV). Data from mice suggests that γδ T cells contribute to immunopathology at the site of infection, as depletion of γδ T cells prior to challenge with hRSV results in reduced lung inflammation and disease severity [51]. However, γδ T cells may play a role in limiting viral replication in the lungs as their depletion also resulted in an increase in peak viral titers [51].

While important in anti-viral immunity, the RSV-specific CD4 T cell response is also thought to contribute to disease pathogenesis and damaging immunopathology [58,59,60]. CD4 T cells, and the cytokines they produce, are key in shaping the nature of the adaptive virus-specific immune response. HRSV infection induces a mixed Th1 and Th2-type cytokine response [61]. Production of IL-12 by dendritic cells, and early IFNγ is required for the priming of an effective Th1 type cytokine response; however, hRSV has been shown to interfere with dendritic cell cytokine production and their ability to initiate the development of a Th1 response [56,62,63]. The ensuing Th2 polarized response leads to increased disease severity and lung injury, and is thought to block the development of an effective CD8 T cell response during both primary and secondary challenge [56,64,65,66]. Much of our understanding about the adaptive immune response to hRSV is derived from small rodent models; however, evidence from humans, while limited, suggests a similar mechanism of disease pathogenesis. Infants infected with hRSV exhibit Th2 polarization with increased IL-4 production in the lungs and the establishment of eosinophilia [67,68]. Interestingly, eosinophilia was also apparent in the lungs of infants that exhibited vaccine enhanced disease after receiving a formalin-inactivated hRSV vaccine (see section on RSV Vaccines). However, cytokine balance and the resulting disease outcome likely depends upon genetic background, as some infants have also been shown to contain significant numbers IFNγ-producing cells in the lungs, and these increased levels did not correlate to disease severity or outcome [69].

Like humans, calves infected with bRSV develop a mixed cytokine response, but favor the development of a Th2-type immune response following infection. Studies of the cells and lymph fluid from bRSV infected calves reveal enhanced IL-4 and IL-13 production in the serum and tissues as early as day 4 post infection, and increased serum levels of virus-specific IgE, indicating the establishment of a Th2-type response [70,71,72]. Calves also develop IFNγ producing cells and levels of the cytokine increase in the serum, but, as with humans, neither cell numbers nor IFNγ levels correlate with positive disease outcome [73]. Evidence from humans has suggested that, due to the Th2 nature of the anti-viral immune response, hRSV infection may predispose children to the development of allergies and asthma later in life [74,75]. Interestingly, bRSV infection in calves has also been shown to predispose to allergic sensitization [76,77]. Gershwin et al. demonstrated that exposure to the model allergen ovalbumin during bRSV infection resulted in significantly increased levels of IL-4, IL-13 and ovalbumin-specific IgE compared to uninfected control calves [77].

Cytotoxic CD8 T cells play a critical role in the control and clearance of RSV infection. Infection of human infants results in a significant influx of activated CD8 T cells into the airways [78], and calves infected with bRSV exhibit increased CD8 T cell infiltration in the lungs, trachea and nasopharynx [79]. Depletion of CD8 T cells from mice [59] or bRSV infected calves [53] results in more severe disease and increased and sustained viral shedding compared to non-depleted control animals. In humans, the hRSV-specific HLA class I response is primarily targeted against the M2, F and N proteins [80,81,82]. Bovine CD8 T cells target the M2, F, N and G proteins of bRSV [83,84]. Interestingly, while G-specific CD8 T cells are readily detectable in cattle, they have not been demonstrated in humans [85]. It has been reported that the strong Th2 skewing that occurs during RSV infection acts to inhibit the development of an efficient CD8 T cell response and prevent the establishment of long-lived memory [56]. Anecdotally, this is evidenced by the recurring infections that occur commonly in both humans and calves [16,18]. In the mouse model, hRSV-specific CTLs appear impaired in both cytokine secretion and cytotoxicity [56,86,87], while calves infected with bRSV exhibit a similar phenotype, displaying limited bRSV-specific cytotoxicity during primary infections and impaired memory responses following challenge or vaccination [88,89].

Humoral immunity plays an important role in defending the host from RSV infection. While not fully effective, maternal antibodies may provide some level of protection from severe RSV infection in both humans [90,91,92] and calves [93]; however, their presence has also been described to suppress the development of antibody and T cell responses during acute infection [94,95]. Although humans initiate responses to several proteins of hRSV, only antibodies that are reactive to the major surface glycoproteins F and G appear to be important for protection [96,97]. The F protein is highly conserved and the majority of the F specific response is cross-reactive, making it more important for protection across hRSV strains [98]. The G protein is more divergent, thus few antibodies cross-react between virus strains [98,99]. Further, the G-specific response is particularly inefficient and it has been proposed that this may be due to the secreted form of the G protein acting as a decoy for the host’s humoral response [100,101].

Like humans, calves mount antibody responses to several bRSV antigens, but the primary targets for protective humoral responses are the F, G and NP proteins [84]. bRSV-specific IgM and IgA can be detected in the nasal secretions and serum of bRSV infected calves as early as 8 days post infection [95]. BRSV-specific IgG2, the antibody isotype associated with a Th1 response, is not detected in the serum until 1-3 months post infection. As evidence of the cytokine skewing that occurs during RSV infection of calves, virus-specific IgG1, the isotype associated with a Th2 phenotype, is detectable in the serum starting at 13 days post infection [95]. In a separate study, virus-specific IgE, another antibody associated with Th2 skewing and airway hyperresponsiveness, was detectable in the serum concurrent with the development of clinical signs [71,72].

There are multiple vaccines currently marketed for bRSV (killed and modified-live) that are generally provided as part of multivalent products. As with humans, similar vaccine-enhanced disease has been reported from two cases of natural bRSV infections in calves [112,113]. In the former case, an outbreak of respiratory tract disease among 5- to 7-mo old calves on a beef-fattening farm in the Netherlands started two days after administration of a modified-live bRSV vaccine [112]. The disease was severe among vaccinates, but absent in non-vaccinated calves 8 mo of age or older. In the latter case, 30% of 8-mo old Belgian Blue calves vaccinated with a beta-propiolactone inactivated bRSV vaccine died during a naturally occurring bRSV outbreak. Interestingly, no deaths were recorded among younger calves not vaccinated [113]. Experimentally, vaccine-enhanced disease has been reproduced in some studies of calves vaccinated with formalin-inactivated bRSV preparations [114,115], but not in others [116,117]. Where bRSV vaccine-enhanced disease has been observed, there is a bias toward a Th2-like response characterized by increases in serum IgE [114,118] and eosinophils in lavage fluid [114].

Given the significant disease burden associated with hRSV and bRSV infections, there is a profound interest in developing new and more efficacious vaccines for both species. RSV vaccine development in both cattle and humans has been the topic of several recent and excellent reviews [55,101,119]. As such, we will only briefly describe some of the approaches currently being pursued in the field.

RSV and its target populations pose several obstacles with respect to vaccine development, particularly the need to vaccinate populations with immature immune systems, to induce a response in the face of maternal antibodies, and to induce an appropriate, robust and long-lasting immune response. To address these challenges, a variety of new hRSV vaccines have recently been tested in animal models and, in some cases, clinical trials. The primary focus of these new platforms is to induce a more robust response using actual replicating agents, as opposed to the use of inactivated virus, and to induce Th1-type CD4 T cell response through the use of known CD4 T cell targeted proteins or Th1 activating adjuvants. Amongst these new candidates are synthetic and subunit vaccines that target whole or fragments of the F, N, G and M proteins, singly or in combination. Other examples include the use of live attenuated hRSV, such as strains lacking nonessential genes such as the NS1, NS2, SH, G or M2-2 genes and/or cold-passaged temperature-sensitive mutants [55,119]. One such temperature-sensitive mutant, which lacks the SH gene and contains a mutation in the L gene, proved promising in early phase I trials but lacked efficacy during phase II clinical trials in seronegative infants [120,121]. The stability of the mutant has recently been improved and shown success in trials using seronegative chimpanzees [121]. The use of nanoparticles, virosomes and virus-like particles, as well as vector-based approaches using DNA, virus or bacterial-based vectors that express hRSV proteins have all proven promising [55,119]. One of the vectors that has shown potential is a chimeric bovine/human parainfluenza virus type 3-based vaccine that has been engineered to express the hRSV F protein [122]. This vaccine has proven safe in phase I clinical trials and is currently being tested in a large international clinical trial in infants [123,124].

The authors declare no conflict of interest.