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

Van der Gucht, Winke; Stobbelaar, Kim; Govaerts, Matthias; Mangodt, Thomas; Barbezange, Cyril; Leemans, Annelies; De Winter, Benedicte; Van Gucht, Steven; Caljon, Guy; Maes, Louis; De Dooy, Jozef; Jorens, Philippe; Smet, Annemieke; Cos, Paul; Verhulst, Stijn; Delputte, Peter L.

To study the dynamics of virus infection, both viral replication kinetics and infectious virus production were assessed in HEp-2, A549 and BEAS-2B cells. BE/ANT-B15/17) and one strain grown on Vero cells (BE/ANT-B20/17) that resulted in significantly more infected cells at 72 h than the reference RSV B1, whereas just one strain (BE/ANT-B2/17) seemed to result in comparable infection as the RSV B1. Infectious virus production of RSV-B strains shows that even though the BE/ANT-B20/17 and BE/ANT-B15/17 reach a very high percentage of infected cells, less infectious particles are produced compared to BE/ANT-B13/17, suggesting either that the particles may not be efficiently released in the supernatant and remain more cell-associated or that the particles may efficiently attach but replicate at lower levels. The same experiment was repeated in the A549 (Figure 3 ) cell line in which for the RSV-A isolates ( Figure 3A ), the RSV A2 shows the highest percentage of infected cells, followed closely by the BE/ANT-A11/17, reaching only slightly lower percentages of infected cells than in the HEp-2 cells after 72 h. This strain also produced the highest amounts of infectious virus in A549 cells ( Figure 3C ). In HEp-2 cells both the RSV-B subtype isolates BE/ANT-B13/17 and BE/ANT-B20/17 reach a higher percentage of infected cells than the RSV B1, whereas results of A549 replication kinetics suggest that the BE/ANT-B13/17 and BE/ANT-B2/17 strains reach similar infection rates ( Figure 3B ). The BE/ANT-B20/17 reached about 50% infection after 48 h but the infection then seemed to flatten out towards 72 h, resulting in a significant difference with infection rates of the RSV B1. Interestingly, the isolate BE/ANT-B2/17, which did not efficiently infect HEp-2 cells now reached a near 100% infected number in A549 cells in 72 h. Unsurprisingly, the BE/ANT-B15/17 achieved again the lowest number of infected cells and levels of virus production in A549 cells ( Figure 3D ). As the BEAS-2B cell line is also a highly permissive cell line for RSV infection and widely used as well, we assessed viral growth and production kinetics in this cell line (Figure 4 ). For all RSV-A clinical isolates, no major differences were observed after 48 h and 72 h of infection in percentage of infected cells ( Figure 4A ). After 72h of infection, the amount of infectious virus released by the cells was the highest for RSV A2 and clinical isolate BE/ANT-A11/17. Larger differences were observed between the clinical isolates of the RSV-B subtype ( Figure 4B ). BE/ANT-B13/17 reached percentages and infectious virus production that were comparable to RSV B1 ( Figure 4B,D) . Isolates BE/ANT-B2/17 and BE/ANT-B15/17 had the lowest infection rates and infectious virus production in both this cell line as well as in the HEp-2 cells (Figure 4B,D) .