Investigation of the association of Rhinovirus and Respiratory Syncytial Virus with severe acute respiratory illness in South Africa

Abstract

Pneumonia is a major cause of morbidity and mortality in children worldwide and causes 18% of all deaths in children less than 5 years of age. In order to best understand the full burden of acute respiratory infection in South Africa, it is important to conduct surveillance and measure the burden of mild, outpatient disease as well as more severe, inpatient disease. By comparing the prevalence of these respiratory viruses detected in patients with influenza-like illness (ILI) and severe acute respiratory illness (SARI) to control patients the contribution of these respiratory viruses to SARI maybe better understood. The aim of this study was to determine the contribution of the major respiratory viruses to severe acute respiratory illness in South Africa both during and after the pandemic and compare molecular epidemiology and host innate immune responses as factors that may influence the role of the two most frequently detected viruses, respiratory syncytial virus (RSV) and rhinovirus as pathogens in a population with a high HIV sero-prevalence. To facilitate the detection of these viruses, a multiplex reverse transcription real-time polymerase chain reaction (rRT-PCR) assay that can detect the ten major respiratory viruses influenza A and B, RSV, enterovirus, human metapneumovirus, adenovirus, rhinovirus and parainfluenza 1, 2 and 3, was developed. This assay allowed us to determine the prevalence of these respiratory viruses in patients with SARI, ILI and controls, as well as to determine the attributable fraction of each of these viruses in SARI and ILI relative to controls. We were also able to determine the seasonality of each of these viruses in South Africa. While RSV was detected most frequently in children, rhinovirus was by far the most prevalent virus detected over all age groups in the study. We described the molecular epidemiology of rhinovirus in SARI, ILI and control cases to determine whether a specific rhinovirus species could be linked to severe disease. All 3 species of rhinovirus was identified in South Africa, although RV-A and RV-C were more common than RV-B. While rhinovirus was associated with symptomatic respiratory illness; there was no association between RV species and disease severity. In order to determine if RSV genotypes could be identified in more severe cases and how the virus re-establish epidemics over time, we described the molecular epidemiology of RSV over 16 years in South Africa. Positive selection drove both RSV-A and B genotypes to evolve, resulting in replacement of all genotypes over the 16-year period in South Africa. We were unable to attribute more severe disease to either RSV-A or B; rather that it was linked to the predominant strain of the season, which was likely driven by herd immunity in previous seasons. In a previous study in South Africa RSV-B deletion mutants were identified in HIV-infected patients in South Africa. To determine the frequency of this phenomena and the possible role in disease in these patients the surveillance program was used to address these questions in a larger population group. Deletions in the G-protein of both RSV-A and RSV-B strains were identified in HIV-infected SARI patients. Whole genome analysis of the deletion mutants revealed that even though there were unique non-synonymous mutations for the deletions mutants and the cumulative effect of these substitutions might affect RSV fitness, no changes were observed that could result in altered virus-host attachment, changes in immune evasion strategies or distorted fusion abilities. The specimens obtained from the patients carrying the RSV strains containing the G-protein deletions contained significantly lower levels of pro-and anti-inflammatory cytokines, suggesting that the absence of the G-protein inhibited the virus s ability to activate the normal RSV anti-viral response. The inflammatory responses of children infected by RSV, rhinovirus or both showed that rhinovirus infection elicits a strong Th1 response, while RSV infection skews the balance towards a Th2 response. Co-infections did not result in more severe disease and the effect that these 2 viruses have on the innate immune response may results in a further Th1/Th2 imbalance. Understanding the contribution of RSV and RV to severe respiratory disease will allow for informed decision making when selecting and setting criteria for vaccine development and implementation as well as therapeutic interventions. To conclude, this study provided a useful tool for defining the major respiratory viruses in SARI and confirmed the importance of Influenza, RSV, rhinovirus, and human metapneumovirus as pathogens. Changes in the circulating strains of RSV may overcome herd immunity and re-establish infections. RSV G-protein deletion mutants appeared only in HIV-infected patients and had a much reduced immune-modulatory effect relative to wild type strains. Results suggest that a co-infection between RSV and rhinovirus will further imbalance the innate immune response and may affect the pathogenesis in HIV infected and young patients.