Human respiratory syncytial virus (RSV) is the leading cause of bronchiolitis and pneumonia worldwide, infecting nearly all children by 2 years of age . Severe disease occurs in premature infants and individuals with compromised respiratory, cardiac or immune system [2, 3]. Furthermore, exposure to RSV early in life may predispose susceptible individuals to asthma .
RSV belongs to the Parmyxoviridae family of enveloped RNA viruses, specifically the genus Pneumovirus. Initially isolated from a chimpanzee, RSV can be found in ruminants, such as sheep, goats and cattle; its main host, however, is humans. The virus has a negative-stranded RNA genome that codes for 10 proteins, including 4 nucleocapsid proteins (N, P, L and M2-1; necessary for RNA replication) and 3 envelope transmembrane proteins (G, F and SH; responsible for virus attachment, membrane fusion, entry and syncytium formation) . The surface fusion protein F and the glycoprotein G serve as the two main targets for antibodies. Variability in the G glycoprotein is primarily responsible for the two clinical strains of RSV, the subgroups A and B . These two subgroups differ mainly in the extracellular domains of G and the small hydrophobic protein SH. Based on sequence variability of the G protein hypervariable region, the subgroups are further divided into clades. The matrix protein (M) is important for viral RNA packaging, while the non-structural proteins have regulatory activities.
RSV infection produces serum and mucosal immune responses that includes IgM, IgA, IgG and IgE; however, the IgG response is short lived and wanes by one year . The primary immune response is not efficient against the initial infection, but results in an effective memory response against reinfection. In addition, the virus generates CD4 and CD8 T cell responses that result in classical interferon-gamma production. Both the humoral and cell-mediated immune responses play roles in virus clearance, but also contribute to the immunopathology of the respiratory tract.
The pathogenesis of RSV is related to its ability to reach the lower respiratory tract, where it can cause bronchiolitis and pneumonia . The RSV strains differ in their virulence, depending upon critical changes in their viral genomes . Overall, subgroup A causes more illness than subgroup B . In the BALB/c mouse model of RSV infection, the virus causes lower respiratory tract infection, making it a good animal model to study . Of note, wild-type inbred mice are described as semi-permissive hosts for human RSV; a very high intranasal inoculum (105 to 107 plaque-forming units per mouse) is usually administered to cause lower respiratory tract disease signs and symptoms such as weight loss, reduced activity, and ruffled fur . Nevertheless, similar to humans, constriction of the airways by mucus production represents a hallmark of disease in mice. Other findings include epithelial hypersensitivity, inflammation, and infiltration by mononuclear cells [9, 12]. Furthermore, production of IgE antibodies and skewing of the immune response towards higher Th-2 and lower Th-1 cytokines are thought to contribute to asthma symptoms observed in mice and young infants [7, 9].
The molecular pathogenesis of clinically aggressive RSV infection is poorly understood, which partially explains the inability to control the disease at the molecular level [13, 14]. For example, information concerning RSV-induced caspases (a series of cysteine, aspartate-specific proteases that mediates apoptosis) in pneumatocytes is inconsistent, and most studies are performed on cell lines. Early studies in the lung epithelial cell line A549 identified apoptotic mediators in response to RSV (e.g., interleukin-1 converting enzyme and CD95), but apoptosis was detected only in ~15% of infected cells [15, 16]. Using the same cell line, Bitko and Barik showed RSV-induced apoptosis was mediated by caspase-12 (an endoplasmic reticulum stress response caspase) . Additional in vitro data implicated RSV in inducing the anti-apoptotic factor IEX-1L, and that normal signaling through the phosphoinositide 3-kinase pathway blocked RSV-induced apoptosis [17, 18]. The study by Kotelkin et al. found transcriptional activations of pro-apoptotic and anti-apoptotic factors in response to RSV in various cell lines . In another study, RSV induced tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and its receptors and elicited apoptosis associated with activation of caspase-8 (receptor-mediated) and caspase-9 (mitochondrial-associated) .
Initiation of apoptosis requires the mitochondria to sense the injury, resulting in leakage of cytochrome c and other small molecular weight pro-apoptotic molecules from the mitochondrial intermembrane space to the cytosol . In the cytosol, cytochrome c binds to the apoptotic protease activating factor-1 (Apaf-1), triggering the caspase cascade. Caspase activation induces mitochondrial perturbations, which involve opening the permeability transition pores and collapsing the electrochemical potential. Thus, induction of apoptosis is linked to mitochondrial dysfunction.
Caspase-3 is also involved in proteolysis of proteins, including poly(ADP ribose) polymerase; it cleaves at the second aspartate in the asp-glu-val-asp sequence. Hence, the synthetic substrate N-acetyl-asp-glu-val-asp-7-amino-4-methylcoumarin (Ac-DEVD-AMC) can be used to monitor intracellular caspase-3 activity. The released fluorogenic moiety AMC can be separated on HPLC and detected with a great accuracy .
The term “cellular bioenergetics” refers to the biochemical processes involved in energy metabolism (energy conversion or transformation), and the term “cellular respiration” (mitochondrial oxygen consumption) is used to describe the delivery of metabolites and O2 to the mitochondria, oxidation of reduced metabolic fuels with the passage of electrons to O2, and synthesis of ATP.
Measuring tissue mitochondrial O2 consumption, using the principle that O2 quenches the phosphorescence of palladium II-meso-tetra-(4-sulfonatophenyl)-tetrabenzoporphyrin, has been recently reported [22–25]. This analytical tool allows in vitro monitoring of cellular respiration over several hours. Simultaneous determinations of intracellular ATP and caspase activity, however, are necessary, since uncoupling oxidative phosphorylation (accelerated respiration with collapsing cellular ATP) is common after tissue collection. Moreover, caspases are potent inhibitors of the inner mitochondrial membrane function. Therefore, the three parameters (respiration, ATP content and caspase activity) are all necessary for accurate assessment of lung tissue bioenergetics.
The status of lung tissue bioenergetics in RSV infection is currently unknown. It is also unclear whether RSV infection induces pneumatocyte apoptosis and mitochondrial perturbation. Using assays described by us [22–26], these unmet tasks are addressed in this study using a well-established RSV-mouse model system .