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Pathogenesis of respiratory syncytial virus
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Pathogenesis of respiratory syncytial virus Sylvia van Drunen Littel-van den Hurk1,2 and Ellen R Watkiss1,3 While affecting all age groups, respiratory syncytial virus (RSV) infections can be particularly severe in infants, who develop functionally distinct immune responses, as well as in immunocompromised individuals. The extent to which environmental, viral and host factors contribute to the pathogenesis of RSV varies considerably between infected individuals. A correlation between the level of virus replication and pathogenesis has been established, and several viral proteins, in particular NS1 and NS2, modulate the immune response. Host immunity clearly contributes to RSV pathogenesis, and a number of specific cell populations may be involved. Ultimately, whether the response induced by RSV is protective or pathogenic depends on a combination of host factors, young age being one of the most important ones. Addresses 1 VIDO-Intervac, University of Saskatchewan, 120 Veterinary Rd., Saskatoon, SK, S7N 5E3, Canada 2 Microbiology and Immunology, University of Saskatchewan, 120 Veterinary Rd., Saskatoon, SK, S7N 5E3, Canada 3 Veterinary Microbiology, University of Saskatchewan, 120 Veterinary Rd., Saskatoon, SK, S7N 5E3, Canada Corresponding author: van Drunen Littel-van den Hurk, Sylvia ([email protected])
Current Opinion in Virology 2012, 2:300–305 This review comes from a themed issue on Viral pathogenesis Edited by Diane Griffin and Veronika von Messling Available online 23rd February 2012 1879-6257/$ – see front matter # 2012 Elsevier B.V. All rights reserved. DOI 10.1016/j.coviro.2012.01.008
Introduction Respiratory syncytial virus (RSV) is a negative-strand, non-segmented RNA pneumovirus of the family Paramyxoviridae, and a common human pathogen that causes cold-like symptoms in most healthy adults and children. In infants and young children predisposed to respiratory illness, however, RSV infection is more likely to move into the lower respiratory tract (LRT), leading to pneumonia and bronchiolitis. RSV can be isolated from the majority of children hospitalized for bronchiolitis during the RSV season and is the most common respiratory virus in infants [1]. An increased incidence of asthma later in life has been associated with more severe LRT infections (LRTI) [2]. In older children, RSV is thought to be an important contributor to otitis media [3]. The winter and spring months of November through April are the peak Current Opinion in Virology 2012, 2:300–305
season of RSV infections in temperate climates of the Northern hemisphere, while in tropical climates RSV outbreaks occur most frequently during the rainy season [4,5]. RSV rapidly spreads through communities, infects most of the population within their first year of life, and re-infects individuals multiple times over the course of a lifetime (reviewed in [6]). In 2005, RSV caused 34 million cases of LRTI in children <5 years of age, 10% of whom required hospitalization [5]. While during the past few years RSVassociated mortality in developed countries decreased, probably owing to improved supportive care [6], the death rates are much higher in areas where access to healthcare is limited, such as in developing nations and remote areas of North America [7]. The mortality in 2005 was 66 000– 199 000 worldwide, of which 99% occurred in developing nations [5]. These numbers are probably underestimates [6]. RSV also is an important cause of mortality in elderly patients [8]. As the morbidity and mortality of infants, immunocompromised, and elderly individuals continue to rise worldwide, there still is a need for a better understanding of the mechanism of RSV pathogenesis.
Factors playing a role in RSV pathogenesis and disease outcome There are several schools of thought on what drives the pathogenesis of RSV, in particular the extent to which environmental, viral and host factors contribute to severe disease in infants (Figure 1). The immune-mediated nature of RSV pathology was highlighted by the failed vaccine that led to severe, non-protective, Th2-biased immunopathology upon RSV infection [9]. This, in combination with the striking association between severe bouts of RSV in infancy and the development of Th2mediated conditions like asthma and allergies in childhood, led many researchers to focus on the T-helper bias of the immune response to RSV in severe and mild infections. Others are more concerned with additional T cell populations or the innate responses produced in the lungs in the form of pro-inflammatory chemokines responsible for the influx of immune cells involved in pathogenesis. Environmental and social factors
Numerous factors may contribute to RSV disease severity. The age at the onset of the RSV season is a definite risk (<3 months or even <6 months). The presence of smoking members or young siblings in the household, daycare attendance, stay in a nursing home or hospital also increase the risk of exposure and serious infection with RSV (reviewed in [10]). www.sciencedirect.com
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Figure 1
Viral factors
Environmental factors
•Viral load and isolate •Down-regulation of type I IFN response by NS1 and NS2 •Escape from neutralization (sG) •Reduction fraktalkine action (G)
•Smoking in household •Young siblings •Daycare attendance •Stay in a hospital
RSV infection of epithelial cells
Virus replication and cytopathology
Immunopathology
Genetic polymorphisms •Innate defense genes Sloughing of cells •Surfactant protein genes Reduced ciliary funcion •Host cell receptor genes •Neutrophil response genes •Th1/Th2 response genes •Gene effectors of adaptive immunity
Cell infiltration Mucus production
Host factors •Premature birth and low birth weight •Chronic lung disease of prematurity • Congenital heart disease •Incomplete development, damage or hyperreactivity of the airway •Immunodeficiency •Male gender •Multiple births •Low titer of RSV-specific maternal antibodies •Cord blood vitamin D deficiency
Bronchiolitis Airway insufficiency Current Opinion in Virology
Factors influencing the pathogenesis and clinical disease caused by RSV infection in infants and young children.
Exposure to smoke, whether from air pollution or cigarettes, has a detrimental effect on respiratory health in general. According to a recent study the risk for development of RSV broncheolitis is increased by trafficderived pollution [11]. A more definite link was shown between smoking in the household, and the incidence and severity of RSV infection [12]. Furthermore, in an ex vivo tissue culture system of primary airway epithelial cells cigarette smoke caused necrosis rather than virusinduced apoptosis resulting in increased inflammation and enhanced viral replication; this effect was primarily mediated by reactive aldehydes [13]. Viral factors
RSV is not a highly cytopathic virus. RSV targets both type I alveolar and nonbasilar airway epithelial cells and possibly alveolar macrophages [14]. This results in impairment of the ciliary action and sloughing of infected epithelial cells. Peribroncheal mononuclear cell infiltration, submucosal edema, mucus secretion, and sometimes syncytia are observed in the lung. When polarized, mucociliary epithelial cells were infected with RSV in vitro, replication was limited to the apical surface [15]. RSV www.sciencedirect.com
does not induce general shut-off of host cell transcription; however, changes in abundance of cell-cycle-regulatory proteins were observed in primary HBEpC cells as well as A549 cells, leading to enrichment in the G0/G1 population and increased progeny virus [16]. Despite its low cytopathicity, there is compelling evidence that the level of RSV replication correlates to the disease severity [17,18]. Furthermore, while evidence that RSV subgroup A (or an A genotype) is more virulent has been controversial, subgroup A strain 19 was recently shown to induce increased production of IL-13, mucus production and airway hypersensitivity in mice [19]. Subsequently, RSV clinical isolates were found to induce variable pathogenesis. Several RSV subgroup A isolates had similar growth properties in BEAS-2B cells, but one of them, RSV A2001/2-20, caused greater disease severity, higher lung IL-13 levels, and higher lung gob-5 levels than RSV strains A2, line 19, Long, and A2001/3-12 in BALB/cJ mice. In addition, the A2001/2-20 clinical isolate induced airway mucin expression, which was correlated to F protein characteristics. Two of the clinical isolates, RSV 2-20 and A2001/3-12, replicated to higher titers in the Current Opinion in Virology 2012, 2:300–305
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lung than laboratory RSV strains on day 1, which correlated with greater histopathologic changes. RSV 2-20 induced epithelial desquamation, bronchiolitis, airway hyperresponsiveness, and increased breathing effort [20]. Several RSV proteins play a role in modulation of the host response to infection. The NS1 and NS2 proteins block IFN regulatory factor 3 activation and inhibit the type I IFN induced signaling through the JAK/STAT pathway leading to a very effective block of IFNa/b production by the infected host. RSV further downregulates IFN production and function by inhibiting toll-like receptor (TLR) signaling through myeloid differentiation factor 88 and mitochondrial antiviral signaling protein [21], as well as induction of suppressor of cytokine signaling molecules [22] and interference with RIG-I [23]. In addition, NS1 and NS2 activate the phosphoinositidide 3-kinase pathway, which results in reduced apoptosis and thus enhanced survival of the infected cells [24]. The G protein plays a major role in immune evasion of RSV. First, it is a highly glycosylated protein, which may impede immune recognition. It also is highly variable, with only 53% amino acid identity and 1–7% antigenic relatedness between subgroups, which allows easy escape from neutralizing antibodies. Furthermore, in addition to the full-length G protein a truncated, secreted form, sG, is produced during RSV infection at 4-fold the rates present in progeny virus. This sG binds RSV-specific antibodies and thus reduces the concentrations available for RSV neutralization [25]. The G protein also has limited sequence homology to fraktalkine, and can reduce the action of host fraktalkine and the influx of CXCR1 leukocytes such as natural killer (NK) cells, and CD4 and CD8 T cells [26]. Finally, the sG protein functions as a TLR antagonist, and down-regulates the TLR2-, TLR4- and TLR9-mediated inflammatory response [27]. RSV also has the ability to infect different populations of immune cells, including macrophages and dendritic cells (DCs), affecting their antigen presenting capacity (reviewed in [28]). It is not surprising that such effects may be more prominent in neonates, which are immature at the level of DC development. Recently, a role for NS1 was found in suppression of CD103+CD8+ T cells and Th17 cells, and stimulation of Th2 cells. This was probably owing to NS1 causing reduced maturation of DCs [29]. Interestingly, ex vivo RSV-stimulated monocytederived DCs suboptimally upregulated the CCR7 receptor and thus migrated less efficiently to CCL19 than for example influenza virus. As the CCR7 receptor is critical for migration of DCs to the lymphoid tissues, this may contribute to the reduced adaptive immune responses to RSV [30]. This is in agreement with a previous observation that in children and mice, DCs persisted in the respiratory mucosa and secretions for weeks after the acute symptoms of RSV infection had resolved [31,32]. Current Opinion in Virology 2012, 2:300–305
Another cell population, human bone marrow stromal cells (BMSCs), was recently identified as a novel target for RSV, which induced disruption of cytoskeletal actin filaments, altered chemokine/cytokine expression, decreased ability to stimulate B cell maturation, and modulated chemotaxis. RNA sequences homologous to the RSV genome were found in naive primary human BMSCs. This may have consequences for the acute RSV induced pathogenesis as well as long-term effects [33]. Host factors
Very little is known about the normal host response to RSV infection, so much of the information about the impact of host factors is based on LRTI. More severe LRT RSV infections are correlated to premature birth, chronic lung disease of prematurity, congenital heart disease, and T-cell immunodeficiency. Incomplete development or damage to the airway, and/or airway hyperreactivity of premature infants contribute to RSV-induced morbidity. Immunodeficiency, immunosuppression or old age may lead to prolonged viral replication and more severe illness. Other risk factors are male gender, low birth weight, multiple births and low titer of RSV-specific maternal antibodies (discussed in [34]). Interestingly, recent evidence suggests that cord blood vitamin D deficiency in healthy neonates is associated with increased risk of severe RSV LRTI in the first year of life [35]. Several genetic polymorphisms, including genes involved in the innate defense, surfactant protein genes, host cell receptor genes, neutrophil response genes, Th1/Th2 response genes and gene effectors of adaptive immunity, have been reported. Although the genetic factors related to RSV severity were suggested to be probably complex and polygeneic, certain loci affecting the ability of the host to control early viral replication as well as candidate loci affecting potential immunopathology, were identified as significant factors in the severity of human RSV disease (reviewed in [36]). The importance of epithelial cells and macrophages in the innate immune response to RSV was recently recognized. Several chemokines and cytokines including IL-8/ CXCL8, IP-10/CXCL10, MCP-1/CCL2, MIP-1a/CCL3, MIP-1b/CCL4, RANTES/CCL5, IL-6, TNF-a, IL-1ab, and IFN-a/b are produced by epithelial cells and macrophages in response to RSV infection, and found in enhanced amounts in respiratory secretions of children hospitalized for RSV infection [37]. Upregulation of IL8 is correlated to the severity of RSV disease [38], and leads to recruitment of neutrophils, which constitute the majority of infiltrating cells (at least 84%) [39]. While neutrophils may mediate elimination of virus-infected cells, their high numbers, ability to secrete further cytokines and chemokines, and degranulation products may contribute to RSV-induced immunopathogenesis. Indeed, instead of eosinophils as originally proposed, neutrophils www.sciencedirect.com
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constituted the predominant population in FI-RSV vaccinated children who died of subsequent RSV infection. Moreover, although increased eosinophil degranulation products were found in the respiratory secretions of children with RSV bronchiolitis [40], eosinophils represent only 1–3% of the airway leukocytes in RSV infected infants [39], and in mice can be protective [41]. Interestingly, an inverse correlation between IL-8 concentrations in the amniotic fluid and wheezing was found, suggesting that inflammatory cytokines in the amniotic fluid accelerate maturation of the fetal lung, which promotes protection from severe RSV infection [42]. While the RSV-specific T cell response plays a major role in viral clearance and the clinical outcome of infection, both Th2-biased CD4+ and CD8+ T cells have been implicated in immunopathogenesis (reviewed in [6,43]). Recently, regulatory T cells (Tregs), were shown to also play a critical role in regulating the innate and adaptive responses during the later stages of RSV lung infection; depletion of Tregs before RSV infection resulted in delayed viral clearance and increased disease severity in mice [44,45,46]. While in one study innate immune cells in lung and bronchoalveolar lavage (BAL), as well influx of CD4+ and CD8+ IFN-g producing T cells were increased [45], a delay in early CD8+ T cell recruitment into the lung [44,46], in one case followed by an increase at a later time point [44] was reported by other groups. Enhanced lung cytokine and chemokine levels, and increased NK cells and neutrophils in the BAL, were also observed [45]. As IL-17 is known to play a role in the development of asthma, its role in RSV pathogenesis was recently examined. Increased IL-6 and IL-17 levels were found in the tracheal aspirate samples from severely ill RSV-infected infants. Furthermore, IL-6, IL-17 and IL-23 were increased in RSV-infected mice, while treatment with anti-IL17 antibodies reduced inflammation, decreased viral load, and increased antigenspecific CD8+ T cells in the lung [47]. This study agrees with the recently reported detection of numerous IL-17positive cells in the lung sections of children who died of enhanced RSV disease in 1967 [42].
While the mouse model has advantages, in particular the availability of reagents and knockout strains, the host specificity of RSV makes studies in mice, as well as cotton rats, difficult to interpret in the context of human infections. Another limiting factor in most RSV studies is the use of adult mice. RSV-infected neonatal mice (7 days of age) were shown to develop long-term asthma-like disease including increased airway hypersensitivity, mucus hyperproduction, airway remodeling, and Th2 cytokines and cellular responses, which makes this a potentially more attractive model (reviewed in [48]). Alternative animal models include pneumonia virus of mice. As a natural pathogen in rodents, a low-dose inoculum replicates to high levels and causes immunemediated respiratory illness that is quite similar to that described for RSV in children (reviewed in [49]). Bovine RSV (BRSV) infection of calves constitutes another appropriate model, as the pathologic lesions caused by human and bovine RSV are very similar (reviewed in [50]). Furthermore, as for RSV, age-dependent differences in BRSV-induced pathogenesis were recently demonstrated [51]. However, the expense of using calves as a model is a limiting factor. This applies even more so to non-human primates, even though they are a natural target for RSV. Recently, a newborn lamb model was established, both for human and bovine RSV [52]. The pathology of RSV in lambs is similar to that in human infants, that is, mild peribronchiolar infiltrates of lymphocytes and plasma cells, and bronchiolitis characterized by degeneration and sloughing of epithelial cells, and intraluminal infiltrates of neutrophils [52]. The airway structure and function of newborn lambs are more similar to those in infants compared to mice [53]. Furthermore, alveolar development occurs preterm in both humans and lambs [54], and pre-term lambs can be examined for their ability to respond to RSV infection [55], making this an attractive model to study RSV pathogenesis.
Acknowledgements Animal models of pathogenesis Studying the pathogenesis of RSV in humans is difficult for a number of reasons. Many RSV infections in healthy individuals are mild enough to remain untreated and unreported, so often only the most severe infections requiring medical intervention can be studied. Also, the site of infection in the LRT is not readily accessible, limiting studies to non-invasive procedures such as the analysis of peripheral blood cells, nasal washes, lung aspirates from intubated infants, and post-mortem lung tissue. The availability of appropriate control samples and the ethical implications of sampling the LRT in severely ill infants pose further limits. Animal models of RSV infection overcome these limitations by providing access to the site of inflammation. www.sciencedirect.com
The contributions of all current and previous members of the laboratory, colleagues and the animal care group at VIDO-Intervac are gratefully acknowledged. Work in the author’s laboratory is currently supported by the Natural Sciences and Engineering Research Council of Canada, Canadian Institutes of Health Research, Krembil Foundation, Saskatchewan Health Research Foundation, Agricultural Development Fund of Saskatchewan, and Alberta Livestock and Meat Agency.
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40. Harrison AM, Bonville CA, Rosenberg HF, Domachowske JB: Respiratory syncytical virus-induced chemokine expression in the lower airways: eosinophil recruitment and degranulation. Am J Respir Crit Care Med 1999, 159:1918-1924. 41. Phipps S, Lam CE, Mahalingam S, Newhouse M, Ramirez R, Rosenberg HF, Foster PS, Matthaei KI: Eosinophils contribute to innate antiviral immunity and promote clearance of respiratory syncytial virus. Blood 2007, 110:1578-1586. 42. van Bleek GM, Osterhaus AD, de Swart RL: RSV 2010: recent advances in research on respiratory syncytial virus and other pneumoviruses. Vaccine 2011, 29:7285-7291. 43. van Drunen Littel-van den Hurk S, Mapletoft JW, Arsic N, KovacsNolan J: Immunopathology of RSV infection: prospects for developing vaccines without this complication. Rev Med Virol 2007, 17:5-34. 44. Fulton RB, Meyerholz DK, Varga SM: Foxp3+ CD4 regulatory T cells limit pulmonary immunopathology by modulating the CD8 T cell response during respiratory syncytial virus infection. J Immunol 2010, 185:2382-2392. A rapid accumulation of regulatory T-cells (Tregs) in the mediastinal lymph nodes and lungs of RSV infected mice was shown. In vivo depletion of Tregs before RSV infection led to delayed virus clearance and enhanced disease. This indicates that pulmonary Tregs play a critical role in limiting immunopathology during acute RSV infection.
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52. Sow FB, Gallup JM, Olivier A, Krishnan S, Patera AC, Suzich J, Ackermann MR: Respiratory syncytial virus is associated with an inflammatory response in lungs and architectural remodeling of lung-draining lymph nodes of newborn lambs. Am J Physiol Lung Cell Mol Physiol 2011, 300:L12-L24. A detailed characterization of the pathology induced by RSV A2 in newborn lambs demonstrated moderate clinical disease with increased virus titers, accumulation of macrophages, CD4+ and CD8+ T cells, and a transcriptional response of genes involved in inflammation, chemotaxis, and interferon response. 53. Scheerlinck JP, Snibson KJ, Bowles VM, Sutton P: Biomedical applications of sheep models: from asthma to vaccines. Trends Biotechnol 2008, 26:259-266. 54. Flecknoe SJ, Wallace MJ, Cock ML, Harding R, Hooper SB: Changes in alveolar epithelial cell proportions during fetal and postnatal development in sheep. Am J Physiol Lung Cell Mol Physiol 2003, 285:L664-L670. 55. Meyerholz DK, Grubor B, Fach SJ, Sacco RE, Lehmkuhl HD, Gallup JM, Ackermann MR: Reduced clearance of respiratory syncytial virus infection in a preterm lamb model. Microbes Infect 2004, 6:1312-1319.
Current Opinion in Virology 2012, 2:300–305
Pathogenesis of respiratory syncytial virus Sylvia van Drunen Littel-van den Hurk1,2 and Ellen R Watkiss1,3 While affecting all age groups, respiratory syncytial virus (RSV) infections can be particularly severe in infants, who develop functionally distinct immune responses, as well as in immunocompromised individuals. The extent to which environmental, viral and host factors contribute to the pathogenesis of RSV varies considerably between infected individuals. A correlation between the level of virus replication and pathogenesis has been established, and several viral proteins, in particular NS1 and NS2, modulate the immune response. Host immunity clearly contributes to RSV pathogenesis, and a number of specific cell populations may be involved. Ultimately, whether the response induced by RSV is protective or pathogenic depends on a combination of host factors, young age being one of the most important ones. Addresses 1 VIDO-Intervac, University of Saskatchewan, 120 Veterinary Rd., Saskatoon, SK, S7N 5E3, Canada 2 Microbiology and Immunology, University of Saskatchewan, 120 Veterinary Rd., Saskatoon, SK, S7N 5E3, Canada 3 Veterinary Microbiology, University of Saskatchewan, 120 Veterinary Rd., Saskatoon, SK, S7N 5E3, Canada Corresponding author: van Drunen Littel-van den Hurk, Sylvia ([email protected])
Current Opinion in Virology 2012, 2:300–305 This review comes from a themed issue on Viral pathogenesis Edited by Diane Griffin and Veronika von Messling Available online 23rd February 2012 1879-6257/$ – see front matter # 2012 Elsevier B.V. All rights reserved. DOI 10.1016/j.coviro.2012.01.008
Introduction Respiratory syncytial virus (RSV) is a negative-strand, non-segmented RNA pneumovirus of the family Paramyxoviridae, and a common human pathogen that causes cold-like symptoms in most healthy adults and children. In infants and young children predisposed to respiratory illness, however, RSV infection is more likely to move into the lower respiratory tract (LRT), leading to pneumonia and bronchiolitis. RSV can be isolated from the majority of children hospitalized for bronchiolitis during the RSV season and is the most common respiratory virus in infants [1]. An increased incidence of asthma later in life has been associated with more severe LRT infections (LRTI) [2]. In older children, RSV is thought to be an important contributor to otitis media [3]. The winter and spring months of November through April are the peak Current Opinion in Virology 2012, 2:300–305
season of RSV infections in temperate climates of the Northern hemisphere, while in tropical climates RSV outbreaks occur most frequently during the rainy season [4,5]. RSV rapidly spreads through communities, infects most of the population within their first year of life, and re-infects individuals multiple times over the course of a lifetime (reviewed in [6]). In 2005, RSV caused 34 million cases of LRTI in children <5 years of age, 10% of whom required hospitalization [5]. While during the past few years RSVassociated mortality in developed countries decreased, probably owing to improved supportive care [6], the death rates are much higher in areas where access to healthcare is limited, such as in developing nations and remote areas of North America [7]. The mortality in 2005 was 66 000– 199 000 worldwide, of which 99% occurred in developing nations [5]. These numbers are probably underestimates [6]. RSV also is an important cause of mortality in elderly patients [8]. As the morbidity and mortality of infants, immunocompromised, and elderly individuals continue to rise worldwide, there still is a need for a better understanding of the mechanism of RSV pathogenesis.
Factors playing a role in RSV pathogenesis and disease outcome There are several schools of thought on what drives the pathogenesis of RSV, in particular the extent to which environmental, viral and host factors contribute to severe disease in infants (Figure 1). The immune-mediated nature of RSV pathology was highlighted by the failed vaccine that led to severe, non-protective, Th2-biased immunopathology upon RSV infection [9]. This, in combination with the striking association between severe bouts of RSV in infancy and the development of Th2mediated conditions like asthma and allergies in childhood, led many researchers to focus on the T-helper bias of the immune response to RSV in severe and mild infections. Others are more concerned with additional T cell populations or the innate responses produced in the lungs in the form of pro-inflammatory chemokines responsible for the influx of immune cells involved in pathogenesis. Environmental and social factors
Numerous factors may contribute to RSV disease severity. The age at the onset of the RSV season is a definite risk (<3 months or even <6 months). The presence of smoking members or young siblings in the household, daycare attendance, stay in a nursing home or hospital also increase the risk of exposure and serious infection with RSV (reviewed in [10]). www.sciencedirect.com
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Figure 1
Viral factors
Environmental factors
•Viral load and isolate •Down-regulation of type I IFN response by NS1 and NS2 •Escape from neutralization (sG) •Reduction fraktalkine action (G)
•Smoking in household •Young siblings •Daycare attendance •Stay in a hospital
RSV infection of epithelial cells
Virus replication and cytopathology
Immunopathology
Genetic polymorphisms •Innate defense genes Sloughing of cells •Surfactant protein genes Reduced ciliary funcion •Host cell receptor genes •Neutrophil response genes •Th1/Th2 response genes •Gene effectors of adaptive immunity
Cell infiltration Mucus production
Host factors •Premature birth and low birth weight •Chronic lung disease of prematurity • Congenital heart disease •Incomplete development, damage or hyperreactivity of the airway •Immunodeficiency •Male gender •Multiple births •Low titer of RSV-specific maternal antibodies •Cord blood vitamin D deficiency
Bronchiolitis Airway insufficiency Current Opinion in Virology
Factors influencing the pathogenesis and clinical disease caused by RSV infection in infants and young children.
Exposure to smoke, whether from air pollution or cigarettes, has a detrimental effect on respiratory health in general. According to a recent study the risk for development of RSV broncheolitis is increased by trafficderived pollution [11]. A more definite link was shown between smoking in the household, and the incidence and severity of RSV infection [12]. Furthermore, in an ex vivo tissue culture system of primary airway epithelial cells cigarette smoke caused necrosis rather than virusinduced apoptosis resulting in increased inflammation and enhanced viral replication; this effect was primarily mediated by reactive aldehydes [13]. Viral factors
RSV is not a highly cytopathic virus. RSV targets both type I alveolar and nonbasilar airway epithelial cells and possibly alveolar macrophages [14]. This results in impairment of the ciliary action and sloughing of infected epithelial cells. Peribroncheal mononuclear cell infiltration, submucosal edema, mucus secretion, and sometimes syncytia are observed in the lung. When polarized, mucociliary epithelial cells were infected with RSV in vitro, replication was limited to the apical surface [15]. RSV www.sciencedirect.com
does not induce general shut-off of host cell transcription; however, changes in abundance of cell-cycle-regulatory proteins were observed in primary HBEpC cells as well as A549 cells, leading to enrichment in the G0/G1 population and increased progeny virus [16]. Despite its low cytopathicity, there is compelling evidence that the level of RSV replication correlates to the disease severity [17,18]. Furthermore, while evidence that RSV subgroup A (or an A genotype) is more virulent has been controversial, subgroup A strain 19 was recently shown to induce increased production of IL-13, mucus production and airway hypersensitivity in mice [19]. Subsequently, RSV clinical isolates were found to induce variable pathogenesis. Several RSV subgroup A isolates had similar growth properties in BEAS-2B cells, but one of them, RSV A2001/2-20, caused greater disease severity, higher lung IL-13 levels, and higher lung gob-5 levels than RSV strains A2, line 19, Long, and A2001/3-12 in BALB/cJ mice. In addition, the A2001/2-20 clinical isolate induced airway mucin expression, which was correlated to F protein characteristics. Two of the clinical isolates, RSV 2-20 and A2001/3-12, replicated to higher titers in the Current Opinion in Virology 2012, 2:300–305
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lung than laboratory RSV strains on day 1, which correlated with greater histopathologic changes. RSV 2-20 induced epithelial desquamation, bronchiolitis, airway hyperresponsiveness, and increased breathing effort [20]. Several RSV proteins play a role in modulation of the host response to infection. The NS1 and NS2 proteins block IFN regulatory factor 3 activation and inhibit the type I IFN induced signaling through the JAK/STAT pathway leading to a very effective block of IFNa/b production by the infected host. RSV further downregulates IFN production and function by inhibiting toll-like receptor (TLR) signaling through myeloid differentiation factor 88 and mitochondrial antiviral signaling protein [21], as well as induction of suppressor of cytokine signaling molecules [22] and interference with RIG-I [23]. In addition, NS1 and NS2 activate the phosphoinositidide 3-kinase pathway, which results in reduced apoptosis and thus enhanced survival of the infected cells [24]. The G protein plays a major role in immune evasion of RSV. First, it is a highly glycosylated protein, which may impede immune recognition. It also is highly variable, with only 53% amino acid identity and 1–7% antigenic relatedness between subgroups, which allows easy escape from neutralizing antibodies. Furthermore, in addition to the full-length G protein a truncated, secreted form, sG, is produced during RSV infection at 4-fold the rates present in progeny virus. This sG binds RSV-specific antibodies and thus reduces the concentrations available for RSV neutralization [25]. The G protein also has limited sequence homology to fraktalkine, and can reduce the action of host fraktalkine and the influx of CXCR1 leukocytes such as natural killer (NK) cells, and CD4 and CD8 T cells [26]. Finally, the sG protein functions as a TLR antagonist, and down-regulates the TLR2-, TLR4- and TLR9-mediated inflammatory response [27]. RSV also has the ability to infect different populations of immune cells, including macrophages and dendritic cells (DCs), affecting their antigen presenting capacity (reviewed in [28]). It is not surprising that such effects may be more prominent in neonates, which are immature at the level of DC development. Recently, a role for NS1 was found in suppression of CD103+CD8+ T cells and Th17 cells, and stimulation of Th2 cells. This was probably owing to NS1 causing reduced maturation of DCs [29]. Interestingly, ex vivo RSV-stimulated monocytederived DCs suboptimally upregulated the CCR7 receptor and thus migrated less efficiently to CCL19 than for example influenza virus. As the CCR7 receptor is critical for migration of DCs to the lymphoid tissues, this may contribute to the reduced adaptive immune responses to RSV [30]. This is in agreement with a previous observation that in children and mice, DCs persisted in the respiratory mucosa and secretions for weeks after the acute symptoms of RSV infection had resolved [31,32]. Current Opinion in Virology 2012, 2:300–305
Another cell population, human bone marrow stromal cells (BMSCs), was recently identified as a novel target for RSV, which induced disruption of cytoskeletal actin filaments, altered chemokine/cytokine expression, decreased ability to stimulate B cell maturation, and modulated chemotaxis. RNA sequences homologous to the RSV genome were found in naive primary human BMSCs. This may have consequences for the acute RSV induced pathogenesis as well as long-term effects [33]. Host factors
Very little is known about the normal host response to RSV infection, so much of the information about the impact of host factors is based on LRTI. More severe LRT RSV infections are correlated to premature birth, chronic lung disease of prematurity, congenital heart disease, and T-cell immunodeficiency. Incomplete development or damage to the airway, and/or airway hyperreactivity of premature infants contribute to RSV-induced morbidity. Immunodeficiency, immunosuppression or old age may lead to prolonged viral replication and more severe illness. Other risk factors are male gender, low birth weight, multiple births and low titer of RSV-specific maternal antibodies (discussed in [34]). Interestingly, recent evidence suggests that cord blood vitamin D deficiency in healthy neonates is associated with increased risk of severe RSV LRTI in the first year of life [35]. Several genetic polymorphisms, including genes involved in the innate defense, surfactant protein genes, host cell receptor genes, neutrophil response genes, Th1/Th2 response genes and gene effectors of adaptive immunity, have been reported. Although the genetic factors related to RSV severity were suggested to be probably complex and polygeneic, certain loci affecting the ability of the host to control early viral replication as well as candidate loci affecting potential immunopathology, were identified as significant factors in the severity of human RSV disease (reviewed in [36]). The importance of epithelial cells and macrophages in the innate immune response to RSV was recently recognized. Several chemokines and cytokines including IL-8/ CXCL8, IP-10/CXCL10, MCP-1/CCL2, MIP-1a/CCL3, MIP-1b/CCL4, RANTES/CCL5, IL-6, TNF-a, IL-1ab, and IFN-a/b are produced by epithelial cells and macrophages in response to RSV infection, and found in enhanced amounts in respiratory secretions of children hospitalized for RSV infection [37]. Upregulation of IL8 is correlated to the severity of RSV disease [38], and leads to recruitment of neutrophils, which constitute the majority of infiltrating cells (at least 84%) [39]. While neutrophils may mediate elimination of virus-infected cells, their high numbers, ability to secrete further cytokines and chemokines, and degranulation products may contribute to RSV-induced immunopathogenesis. Indeed, instead of eosinophils as originally proposed, neutrophils www.sciencedirect.com
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constituted the predominant population in FI-RSV vaccinated children who died of subsequent RSV infection. Moreover, although increased eosinophil degranulation products were found in the respiratory secretions of children with RSV bronchiolitis [40], eosinophils represent only 1–3% of the airway leukocytes in RSV infected infants [39], and in mice can be protective [41]. Interestingly, an inverse correlation between IL-8 concentrations in the amniotic fluid and wheezing was found, suggesting that inflammatory cytokines in the amniotic fluid accelerate maturation of the fetal lung, which promotes protection from severe RSV infection [42]. While the RSV-specific T cell response plays a major role in viral clearance and the clinical outcome of infection, both Th2-biased CD4+ and CD8+ T cells have been implicated in immunopathogenesis (reviewed in [6,43]). Recently, regulatory T cells (Tregs), were shown to also play a critical role in regulating the innate and adaptive responses during the later stages of RSV lung infection; depletion of Tregs before RSV infection resulted in delayed viral clearance and increased disease severity in mice [44,45,46]. While in one study innate immune cells in lung and bronchoalveolar lavage (BAL), as well influx of CD4+ and CD8+ IFN-g producing T cells were increased [45], a delay in early CD8+ T cell recruitment into the lung [44,46], in one case followed by an increase at a later time point [44] was reported by other groups. Enhanced lung cytokine and chemokine levels, and increased NK cells and neutrophils in the BAL, were also observed [45]. As IL-17 is known to play a role in the development of asthma, its role in RSV pathogenesis was recently examined. Increased IL-6 and IL-17 levels were found in the tracheal aspirate samples from severely ill RSV-infected infants. Furthermore, IL-6, IL-17 and IL-23 were increased in RSV-infected mice, while treatment with anti-IL17 antibodies reduced inflammation, decreased viral load, and increased antigenspecific CD8+ T cells in the lung [47]. This study agrees with the recently reported detection of numerous IL-17positive cells in the lung sections of children who died of enhanced RSV disease in 1967 [42].
While the mouse model has advantages, in particular the availability of reagents and knockout strains, the host specificity of RSV makes studies in mice, as well as cotton rats, difficult to interpret in the context of human infections. Another limiting factor in most RSV studies is the use of adult mice. RSV-infected neonatal mice (7 days of age) were shown to develop long-term asthma-like disease including increased airway hypersensitivity, mucus hyperproduction, airway remodeling, and Th2 cytokines and cellular responses, which makes this a potentially more attractive model (reviewed in [48]). Alternative animal models include pneumonia virus of mice. As a natural pathogen in rodents, a low-dose inoculum replicates to high levels and causes immunemediated respiratory illness that is quite similar to that described for RSV in children (reviewed in [49]). Bovine RSV (BRSV) infection of calves constitutes another appropriate model, as the pathologic lesions caused by human and bovine RSV are very similar (reviewed in [50]). Furthermore, as for RSV, age-dependent differences in BRSV-induced pathogenesis were recently demonstrated [51]. However, the expense of using calves as a model is a limiting factor. This applies even more so to non-human primates, even though they are a natural target for RSV. Recently, a newborn lamb model was established, both for human and bovine RSV [52]. The pathology of RSV in lambs is similar to that in human infants, that is, mild peribronchiolar infiltrates of lymphocytes and plasma cells, and bronchiolitis characterized by degeneration and sloughing of epithelial cells, and intraluminal infiltrates of neutrophils [52]. The airway structure and function of newborn lambs are more similar to those in infants compared to mice [53]. Furthermore, alveolar development occurs preterm in both humans and lambs [54], and pre-term lambs can be examined for their ability to respond to RSV infection [55], making this an attractive model to study RSV pathogenesis.
Acknowledgements Animal models of pathogenesis Studying the pathogenesis of RSV in humans is difficult for a number of reasons. Many RSV infections in healthy individuals are mild enough to remain untreated and unreported, so often only the most severe infections requiring medical intervention can be studied. Also, the site of infection in the LRT is not readily accessible, limiting studies to non-invasive procedures such as the analysis of peripheral blood cells, nasal washes, lung aspirates from intubated infants, and post-mortem lung tissue. The availability of appropriate control samples and the ethical implications of sampling the LRT in severely ill infants pose further limits. Animal models of RSV infection overcome these limitations by providing access to the site of inflammation. www.sciencedirect.com
The contributions of all current and previous members of the laboratory, colleagues and the animal care group at VIDO-Intervac are gratefully acknowledged. Work in the author’s laboratory is currently supported by the Natural Sciences and Engineering Research Council of Canada, Canadian Institutes of Health Research, Krembil Foundation, Saskatchewan Health Research Foundation, Agricultural Development Fund of Saskatchewan, and Alberta Livestock and Meat Agency.
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