Isolation and Characterization of Clinical RSV Isolates in Belgium during the Winters of 2016–2018

Abstract

Respiratory Syncytial Virus (RSV) is a very important viral pathogen in children, immunocompromised and cardiopulmonary diseased patients and the elderly. Most of the published research with RSV was performed on RSV Long and RSV A2, isolated in 1956 and 1961, yet recent RSV isolates differ from these prototype strains. Additionally, these viruses have been serially passaged in cell culture, which may result in adaptations that affect virus–host interactions. We have isolated RSV from mucosal secretions of 12 patients in the winters 2016–2017 and 2017–2018, of which eight RSV-A subtypes and four RSV-B subtypes. Passage 3 of the isolates was assessed for viral replication kinetics and infectious virus production in HEp-2, A549 and BEAS-2B cells, thermal stability at 37 °C, 32 °C and 4 °C, syncytia formation, neutralization by palivizumab and mucin mRNA expression in infected A549 cells. We observed that viruses isolated in one RSV season show differences on the tested assays. Furthermore, comparison with RSV A2 and RSV B1 reveals for some RSV isolates differences in viral replication kinetics, thermal stability and fusion capacity. Major differences are, however, not observed and differences between the recent isolates and reference strains is, overall, similar to the observed variation in between the recent isolates. One clinical isolate (BE/ANT-A11/17) replicated very efficiently in all cell lines, and remarkably, even better than RSV A2 in the HEp-2 cell line.

Reverse genetics systems for contemporary isolates of respiratory syncytial virus enable rapid evaluation of antibody escape mutants

“…Research into RSV has faced many technical challenges since the initial discovery of this virus in October 1955 following an outbreak of an acute respiratory disease in a chimpanzee colony (48). These include the necessity for stabilization of virus stocks (49), large variation in growth and plaque phenotype of wild-type strains upon isolation (27,28), lack of an animal model that fully recapitulates the disease observed in humans (50), difficulties with viral glycoprotein expression (51,52), and instability of cloned RSV sequences in standard plasmid vectors (40). In this study, we have attempted to moderate some of these impediments to research by generating stable reverse genetics systems for representative strains of the currently predominant RSV ON1 and BA genotypes and characterizing individual palivizumab escape mutations in the context of quantitative cellfusion assays and recombinant viruses.…”

“…Observed variation in plaque phenotype and titers of wild-type RSV strains following isolation in vitro and characterization of phenotypic differences with older prototypic RSV strains are topics of increasing interest in contemporary RSV research (27,28,30,56). Although few studies have systemically compared the in vitro growth of prototypic RSV-A and -B strains, it is wellestablished that the prototypic A2 or Long strains of RSV-A grow to higher titers and are more fusogenic than prototypic RSV-B strains such as B1 or CH-18537 (28,29,57). These observations are also replicated in studies performed using clinical isolates in which RSV-A strains produced significantly larger plaques and grew to higher titers than RSV-B strains upon isolation from patient samples (27,58).…”

“…Major differences have been previously observed in the properties of RSV strains during propagation in cell culture, including syncytium size and phenotype, virus titers, and thermal stability (26)(27)(28). In general, RSV-A strains have been shown to attain higher titers in cell culture with increased plaque size in comparison with RSV-B strains (29).…”

“…This has resulted in a preponderance of studies using older laboratory-adapted subtype A strains such as A2 or Long, which both present fewer technical challenges with respect to in vitro virus culture. In recent years, however, an increased use of clinical isolates in RSV studies has become apparent (28,30) which minimizes the potential confounding factor of acquisition of mutations during prolonged passage in transformed cell lines (31).…”

The Clinical Characteristics and Outcomes of Adult Patients With Pneumonia Related to Three Paramyxoviruses

“…Furthermore, the outcomes of infectious diseases were influenced by hosts, pathogens, and environments ( 31 ). In some studies, clinical outcomes were only compared directly with disease severity of the three PMV infections and were not controlled for confounders ( 10 , 21 , 23 , 28 , 29 , 32 ). In our study, after adjustment for numerous potential confounders, we found that, compared with hMPV-P and hPIV-p, RSV-p was associated with increased risks for invasive ventilation, ICU admission, and 30-day mortality.…”