Several studies have described a clear association between respiratory syncytial virus (RSV) lower respiratory tract infection in infancy and the subsequent development of persistent wheezing in children. Using the mouse model we demonstrated that RSV induces long-term airway disease characterized by chronic airway inflammation and airway hyperreactivity (AHR). The RSV murine model offers great advantages to study the immunopathogenesis of RSV-induced long-term airway disease. Mice can be challenged with aerosolized methylcholine to determine the presence of AHR. We can apply the reverse transcription-polymerase chain reaction assay (RT-PCR) to detect RSV RNA in the respiratory tract and we can perform lung gene expression analysis to further characterize the chronic changes induced by RSV infection. Compared with sham-inoculated controls, RSV-infected mice developed chronic airway disease characterized by AHR and persistent airway inflammation. Forty-two days after RSV infection, a time point when RSV could no longer be isolated, RT-PCR demonstrated, quite unexpectedly, the presence of RSV RNA in the lower respiratory tract of mice. The presence of genomic RNA persisted for months after inoculation. Furthermore, preliminary studies also demonstrated that on day 42 there were a number of genes differentially expressed in RSV-infected mice compared with controls. RSV-infected mice with persistent AHR exhibited presence of abnormal chronic inflammatory changes, altered gene expression profiles, and persistence of RSV RNA, which may contribute to long-term airway disease induced by RSV. Future studies are needed to define the significance of persistent RSV RNA in the mouse model, and its potential role in the pathogenesis of RSV-induced persistent wheezing in children.
From the *Department of Pediatrics, Division of Pediatric Infectious Diseases; and †Department of Pathology, The University of Texas Southwestern Medical Center and Children's Medical Center, Dallas, TX.
Disclosure: A.M. has received the Pediatric Fellowship Award in Viral Respiratory Infectious Diseases supported by MedImmune, Inc., at the PAS-SPR Annual Meeting and was supported in part by the RGK Foundation Award in Infectious Diseases at Children's Medical Center Dallas. O.R. was supported in part by research grants from the NIH 1U19AI057234-010003 and 1R21 AI054990-01A2 and DANA Foundation, has received research grants from MedImmune, Inc. and Abbott, and has served as member of the MedImmune, Inc., Abbott and Roche Advisory Boards. S.C.B., A.M.G., C.S., D.E., J.P.T., H.S.J. all report no conflicts of interest.
Address for correspondence: Octavio Ramilo, MD, Department of Pediatrics, Room F3.202, 5323 Harry Hines Blvd., Dallas, TX 75390-9063. E-mail: Octavio.Ramilo@UTSouthwestern.edu.
Respiratory syncytial virus (RSV) is the leading cause of hospitalization in infants and young children worldwide. In addition to the acute morbidity, the association between RSV lower respiratory infection (LRTI) and the development of recurrent wheezing has been established in several well-controlled prospective studies conducted in different parts of the world.1–3 A recent case-cohort multicenter study conducted in Europe and Canada demonstrated that preterm infants who received anti-RSV prophylaxis with palivizumab had a significant lower risk of subsequent recurrent wheezing at 24-month follow-up compared with untreated matched-controls.4 Among children admitted to the hospital for severe RSV bronchiolitis, more than 30% will develop persistent wheezing up to 13 years of age which may extend into early adulthood.5
Three theories have been proposed to explain the link between early RSV infection and subsequent wheezing: (1) a causal relationship in which changes induced by the virus early in life will alter the normal development of the infant's lung; (2) an “effect” relationship in which RSV infection early in life in infants with pre-existing abnormal immune response and/or airway function serves as a triggering event that leads to subsequent episodes of wheezing; or (3) a multifactorial relationship: the response to RSV will depend on both the host predisposition (genetic makeup,6,7 airway, and immune system maturation at the time of the infection) coupled with external or environmental factors.
Recent evidence derived from in vitro experiments,8 animal models,9–13 and even from studies in humans14–16 suggest that RSV may persist “latently” and/or at low level replication in immunologically privileged sites within the lung. On the basis of these observations, investigators began to examine whether the persistence of the virus could contribute to the long-term airway disease observed in children after RSV LRTI.
This article briefly summarizes some recent studies focusing on novel perspectives and strategies to understand the pathogenesis and consequences of long-term RSV infection. The following topics will be reviewed: chronic manifestations of RSV infection, evidence and significance of RSV RNA persistence, and the differences on gene expression profiles elicited by RSV in the mouse model.
RSV Persistence: Evidence in the Mouse Model
Our laboratory has established a mouse model of RSV lower respiratory tract infection that allows assessment of viral replication, disease severity, and characterization of the immune response during both the acute and chronic phases of RSV disease.9,10,17,18
RSV Induces Chronic Pulmonary Morbidity.
After clearing the virus and recovering from the acute disease, RSV-infected mice progressed into a chronic phase characterized by airway hyperreactivity (AHR) and persistent airway inflammation for up to 154 days after the infection. Methacholine challenge elicited a modest increase in AHR in sham-inoculated controls. However, the magnitude of the response was significantly elevated in RSV-infected animals for up to 154 days after infection.9,17 The persistent AHR observed was associated with the presence of a chronic inflammatory mononuclear infiltrate in the lungs of RSV-infected mice, which was quite different from the infiltrate observed during the acute phase of the disease. On day 154, the inflammatory infiltrates were localized mainly around the larger central vessels and airways and consisted of lymphocytes, macrophages, and scattered plasma cells. Sham-inoculated mice showed rare, small lymphocytic infiltrates unchanged over time.17 These findings provide a histologic correlation to the abnormal pulmonary responses documented by plethysmography.9,17
In addition, the administration of an anti-RSV neutralizing monoclonal antibody significantly reduced RSV replication, which was associated with parallel reductions in lung inflammation and concentrations of proinflammatory cytokines, as well as significant modulation of pulmonary function abnormalities, including long-term AHR, compared with untreated controls.10,18
RSV RNA Persistence: An Unexpected Observation.
To determine whether the virus could be detected when it is no longer identified by plaque assay cultures, bronchoalveolar lavage (BAL) and lung specimens from mice inoculated with live and inactivated RSV were obtained during the acute and chronic phases of the disease and assayed for the presence of RSV RNA using a real-time reverse transcription polymerase chain reaction assay (RT-PCR) targeting the N gene.9–11 In contrast to viral cultures, which were consistently negative on day 7 postinoculation, RT-PCR detected RSV RNA in infected mice up to day 77 postinoculation. RSV RNA persistence was independent of the mouse strain evaluated (BALB/c versus C57Bl/6). In addition, RSV RNA copy number and AHR significantly correlated on day 14 postinoculation.9
We have recently confirmed these findings in a new set of experiments in which mice inoculated with live RSV or RSV inactivated by heat or ultraviolet light were followed over time to measure RSV load by culture and RT-PCR, as well as pulmonary function. Live RSV induced acute disease and long-term AHR, and was associated with persistence of RSV RNA. However, there were no RSV RNA detected nor development of AHR in mice inoculated with inactivated virus.11 Whether this correlation only reflects a similar time course of these 2 variables, or indeed suggests a possible association between the persistence of RSV RNA and long-term airway disease, is an intriguing possibility that will require further studies.
Gene Expression Profiles Correlate With Disease Severity in Experimental RSV-Induced Chronic Airway Disease.
We have recently applied microarray analysis to further understand and characterize the impact of RSV infection at the gene expression level. Whole lung RNA was extracted from RSV-infected mice and sham-inoculated controls and analyzed during the acute and chronic phases of the disease.19 RSV induced significant changes in lung gene expression compared with sham-inoculated controls. We observed that the gene expression patterns identified changed over time. Indeed, on day 42, which represents the chronic phase of the disease, statistical analysis identified 13 genes significantly overexpressed in RSV-infected mice compared with controls that strongly correlated with the AHR observed.19 This initial analysis provides additional evidence of the ability of RSV to induce chronic changes in the lower respiratory tract. It also shows the value of a genomics approach to identify markers that correlate with disease severity and at the same time to potentially provide novel insights into the pathogenesis of this infection.19
Among the many complex factors that contribute to the development of asthma in children, viral respiratory infections, in particular infections with RSV, have been clearly associated with an increased risk for recurrent wheezing.2,3
Despite the significant efforts aimed to facilitate the understanding of the pathogenesis of RSV-induced chronic airway disease, many questions remain unresolved. Studies in humans and in animal models have shown that interventions directed at reducing viral replication with anti-RSV neutralizing antibodies resulted in improvement of acute disease severity and long-term pulmonary abnormalities. Recent observations in the mouse model have demonstrated the persistence of RSV RNA in the lower respiratory tract, which coincides in time with chronic pulmonary findings. It is possible that the recently recognized RSV persistence could also occur in children. If that is the case, it may contribute to the long-term pulmonary abnormalities observed in children after RSV LRTI and open the possibility of new preventive and/or therapeutic interventions for those patients with viral-associated recurrent wheezing.
1. Bont L, Aalderen WM, Kimpen JL. Long-term consequences of respiratory syncytial virus (RSV) bronchiolitis. Paediatr Respir Rev
2. Sigurs N, Gustafsson PM, Bjarnason R, et al. Severe respiratory syncytial virus bronchiolitis in infancy and asthma and allergy at age 13. Am J Respir Crit Care Med
3. Stein RT, Sherrill D, Morgan WJ, et al. Respiratory syncytial virus in early life and risk of wheeze and allergy by age 13 years. Lancet
4. Simoes EA, Groothuis JR, Carbonell-Estrany X, et al. Palivizumab prophylaxis, respiratory syncytial virus, and subsequent recurrent wheezing. J Pediatr
5. Korppi M, Piippo-Savolainen E, Korhonen K, Remes S. Respiratory morbidity 20 years after RSV infection in infancy. Pediatr Pulmonol
6. Ermers MJ, Hoebee B, Hodemaekers HM, Kimman TG, Kimpen JL, Bont L. IL-13 genetic polymorphism identifies children with late wheezing after respiratory syncytial virus infection. J Allergy Clin Immunol
7. Awomoyi AA, Rallabhandi P, Pollin TI, et al. Association of TLR4 polymorphisms with symptomatic respiratory syncytial virus infection in high-risk infants and young children. J Immunol
8. Guerrero-Plata A, Ortega E, Ortiz-Navarrete V, Gomez B. Antigen presentation by a macrophage-like cell line persistently infected with respiratory syncytial virus. Virus Res
9. Chavez-Bueno S, Mejias A, Gomez AM, et al. Respiratory syncytial virus-induced acute and chronic airway disease is independent of genetic background: an experimental murine model. Virol J
10. Mejias A, Chavez-Bueno S, Rios AM, et al. Comparative effects of two neutralizing anti-respiratory syncytial virus (RSV) monoclonal antibodies in the RSV murine model: time versus potency. Antimicrob Agents Chemother
11. Estripeaut D, Somers CS, Mejias A, et al. Mice inoculated with live respiratory syncytial virus (L-RSV) but not inactivated RSV demonstrate long-term RNA persistence and pulmonary function abnormalities. Presented at the Pediatric Academic Societies Annual Meeting; May 5–8, 2007; Toronto, Canada. Abstract No. 8220.
12. Schwarze J, O'Donnell DR, Rohwedder A, Openshaw PJ. Latency and persistence of respiratory syncytial virus despite T cell immunity. Am J Respir Crit Care Med
13. Hegele RG, Hayashi S, Bramley AM, Hogg JC. Persistence of respiratory syncytial virus genome and protein after acute bronchiolitis in guinea pigs. Chest
14. Seemungal T, Harper-Owen R, Bhowmik A, et al. Respiratory viruses, symptoms, and inflammatory markers in acute exacerbations and stable chronic obstructive pulmonary disease. Am J Respir Crit Care Med
15. Wilkinson TM, Donaldson GC, Johnston SL, Openshaw PJ, Wedzicha JA. Respiratory syncytial virus, airway inflammation, and FEV1 decline in patients with chronic obstructive pulmonary disease. Am J Respir Crit Care Med
16. Kling S, Donninger H, Williams Z, et al. Persistence of rhinovirus RNA after asthma exacerbation in children. Clin Exp Allergy
17. Jafri HS, Chavez-Bueno S, Mejias A, et al. Respiratory syncytial virus induces pneumonia, cytokine response, airway obstruction, and chronic inflammatory infiltrates associated with long-term airway hyperresponsiveness in mice. J Infect Dis
18. Mejias A, Chavez-Bueno S, Rios AM, et al. Anti-respiratory syncytial virus (RSV) neutralizing antibody decreases lung inflammation, airway obstruction, and airway hyperresponsiveness in a murine RSV model. Antimicrob Agents Chemother
19. Mejias A, Rios AM, Chavez-Bueno S, et al. Lung gene expression profiling correlates with pulmonary function abnormalities induced by RSV in the mouse model. Presented at the Pediatric Academic Societies Annual Meeting; May 14–17, 2005; Washington, DC. Abstract No. 707.