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.

Keywords: RSV; bronchiolitis; mucin; patient-derived virus.

Figure 1

Phylogenetic trees for RSV-A and RSV-B clinical isolates. The phylogenetic trees were constructed with maximum-likelihood with 1000 bootstrap replicates using MEGA X software. The trees are based on a 342nt and 330nt fragment of the G protein of RSV-A (A) and RSV-B (B) strains respectively, consisting of the second hypervariable region. Nucleotide sequences of the clinical isolates (indicated with •) were compared to reference strains found on GenBank (indicated with genotype and accession number). The outgroups are represented by prototype strains M11486 for RSV-A and M17213 for RSV-B. Bootstrap values greater than 70% are indicated at the branch nodes and the scale bar represents the number of substitutions per site.

Figure 2

Replication kinetics and infectious virus production in HEp-2 cells. (A,B) HEp-2 cells were infected with clinical isolates and RSV reference strains A2 and B1. Cultures were fixed after 24 h, 48 h and 72 h, permeabilized and stained with polyclonal antibody (pAb) goat-anti-RSV antibody and AF488 donkey-anti-goat (IgG). Nuclei were visualized with DAPI and cultures were analysed with fluorescence microscopy. RSV positive cells were counted and calculated to the total number of nuclei to reach a percentage of RSV infected cells. (A) Replication kinetics of RSV-A clinical isolates and (B) Replication kinetics of RSV-B clinical isolates. (C,D) HEp-2 cells were infected with clinical isolates and RSV reference strains A2 and B1. After 24 h, 48 h and 72 h, supernatants were collected and used for quantification by conventional plaque assay. Data represents mean values ± SEM (n = 3), significant differences compared to the reference strains are indicated by *p < 0.05; ***p < 0.001 (two-way ANOVA).

Figure 3

Replication kinetics and infectious virus production in A549 cells. (A,B) A549 cells were infected with clinical isolates and RSV reference strains A2 and B1. Cultures were fixed after 24 h, 48 h and 72 h, permeabilized and stained with pAb goat-anti-RSV antibody and AF488 donkey-anti-goat (IgG). Nuclei were visualized with DAPI and cultures were analysed with fluorescence microscopy. RSV positive cells were counted and calculated to the total number of nuclei to reach a percentage of RSV infected cells. (A) Replication kinetics of RSV-A clinical isolates and (B) Replication kinetics of RSV-B clinical isolates. (C,D) A549 cells were infected with clinical isolates and RSV reference strains A2 and B1. After 24 h, 48 h and 72 h, supernatants were collected and used for quantification by conventional plaque assay. Data represents mean values ± SEM (n = 3), significant differences compared to the reference strains are indicated by *p < 0.05; ***p < 0.001 (two-way ANOVA).

Figure 4

Replication kinetics and infectious virus production in BEAS-2B cells. (A,B) BEAS-2B cells were infected with clinical isolates and RSV reference strains A2 and B1. Cultures were fixed after 24 h, 48 h and 72 h, permeabilized and stained with pAb goat-anti-RSV antibody and AF488 donkey-anti-goat (IgG). Nuclei were visualized with DAPI and cultures were analysed with fluorescence microscopy. RSV positive cells were counted and calculated to the total number of nuclei to reach a percentage of RSV infected cells. (A) Replication kinetics of RSV-A clinical isolates and (B) Replication kinetics of RSV-B clinical isolates. (C,D) BEAS-2B cells were infected with clinical isolates and RSV reference strains A2 and B1. After 24 h, 48 h and 72 h, supernatants were collected and used for quantification by conventional plaque assay. Data represents mean values ± SEM (n = 3), significant differences compared to the reference strains are indicated by *p < 0.05; **p < 0.01 ***; p < 0.001 (two-way ANOVA).

Figure 5

Thermal stability profiles at 37 °C, 32 °C and 4 °C. Clinical isolates, RSV A2 and RSV B1 were aliquoted and exposed to 37 °C (A,B), 32 °C (C,D) or 4°C (E,F). One aliquot of each was snap frozen at 0 h, 24 h, 48 h and 72 h. Aliquots were used for quantification by conventional plaque assay and calculated to the amount at 0 h. Data represents mean values ± SEM (n = 3), significant differences compared to the reference strains are indicated by *p < 0.05;**p < 0.01; ***p < 0.001 (two-way ANOVA).

Figure 6

The capacity for syncytium formation of clinical isolates. HEp-2 cells were infected with clinical isolates and RSV reference strains A2 and B1 for 2 h, inoculum was replaced by DMEM-10 containing 0.6% Avicel^®® and incubated for 48 h at 37 °C. Afterwards, cells were fixed, permeabilized and stained with pAb goat-anti-RSV and AF488 donkey-anti-goat. Nuclei were visualized with DAPI and cultures were analysed with fluorescence microscopy. (A, B) Mean syncytium size was calculated by counting the number of nuclei in syncytia in three pictures taken at 10× magnification. (C,D) Mean syncytium frequency was calculated by dividing the number of syncytial cells by the total number of infected cells. Data represents mean values ± SEM (n = 3), significant differences compared to the reference strains are indicated by *p < 0.05; ***p < 0.001 (one-way ANOVA).

Figure 7

Plaque reduction of the clinical isolates with palivizumab. HEp-2 cells were infected for 2h with clinical isolates and reference strains that were pre-incubated for 1h with a palivizumab dilution series. Inoculum was replaced with DMEM-10 containing 0.6% Avicel^®® and incubated for three days at 37°C. Afterwards, the cells were fixed, stained with palivizumab as primary antibody and goat-anti-human conjugated with HRP, plaques were visualized with chloronapthol. Individual values are plotted as 2log ED50, data represents mean values ± SEM (n = 3).

Figure 8

mRNA levels of mucins 1, 4, 5AC and 5B in infected A549 cells. A549 cells were infected with a MOI of 0.1 of clinical isolates and reference strains for 2 h at 37 °C. Inoculum was replaced with DMEM-10 and cells were incubated for 48h at 37 °C. Afterwards, cells were lysed, total RNA was extracted and the expression of MUC1 (A), MUC4 (B), MUC5AC (C) and MUC5B (D) was determined by qRT-PCR. Data represents mean values ± SEM (n = 3), statistically significant differences compared to the reference strains are indicated with ***p < 0.001 (one-way ANOVA).