Cellular co-infections of West Nile virus and Usutu virus influence virus growth kinetics

Abstract

The mosquito-borne flaviviruses West Nile virus (WNV) and Usutu virus (USUV) pose a significant threat to the health of humans and animals. Both viruses co-circulate in numerous European countries including Germany. Due to their overlapping host and vector ranges, there is a high risk of co-infections. However, it is largely unknown if WNV and USUV interact and how this might influence their epidemiology. Therefore, in-vitro infection experiments in mammalian (Vero B4), goose (GN-R) and mosquito cell lines (C6/36, CT) were performed to investigate potential effects of co-infections in vectors and vertebrate hosts. The growth kinetics of German and other European WNV and USUV strains were determined and compared. Subsequently, simultaneous co-infections were performed with selected WNV and USUV strains. The results show that the growth of USUV was suppressed by WNV in all cell lines. This effect was independent of the virus lineage but depended on the set WNV titre. The replication of WNV also decreased in co-infection scenarios on vertebrate cells. Overall, co-infections might lead to a decreased growth of USUV in mosquitoes and of both viruses in vertebrate hosts. These interactions can strongly affect the epidemiology of USUV and WNV in areas where they co-circulate.

Keywords: Co-infection; Flaviviruses; In-vitro; Usutu virus; Viral interference; West Nile virus.

Fig. 1

Workflow of in-vitro infections and performed mono- and co-infections in this study. (a) Workflow of mono- and co-infections (simultaneous) using Vero B4, GN-R, C6/36 and CT cells. (b) Display of the performed infection experiments with focus on virus combinations (WNV versus USUV). Boxes with a grey background display the mono-infections, boxes with a blue background display the co-infections and boxes framed in red show where two different MOIs for WNV were used (1 or 0.1). Created with Biorender.com

Fig. 2

Growth kinetics of West Nile viruses (WNV) and Usutu viruses (USUV). All mono-infections were performed with a multiplicity of infection (MOI) of 1. The solid lines are drawn through the mean values of the three biological replicates for all tested time points measured by virus titration. The error bars represent the standard deviation (± SD). Incomplete error bars occur when y-min of the error bars is negative and therefore not displayed in the logarithmic y-scale

Fig. 3

Virus secretion in mono- and co-infections of West Nile virus (WNV) and Usutu virus (USUV). WNV lineage 2 isolated in Germany in 2018 and USUV Europe 3 isolated in Germany in 2011 were used for co-infections in vertebrate (Vero B4 GN-R) as well mosquito cell lines (C6/36 and CT). Co-infections were performed with a multiplicity of infection (MOI) of 1 for USUV and either 1 or 0.1 for WNV. The solid and dashed lines are drawn through the mean values of the three biological replicates for all tested time points measured by RT-qPCR based on a relative and absolute standard curve running in parallel. The error bars represent the standard deviation (± SD). Incomplete error bars occur when y-min of the error bars is negative and therefore not displayed in the logarithmic y-scale

Fig. 4

Virus secretion in co-infections of different West Nile virus (WNV) and Usutu virus (USUV) lineages. Co-infections were performed with WNV lineage 1 (Italy, 2009) and 2 (Germany, 2018) and with USUV Europe 3 (Germany, 2011) and Africa 3 (Germany, 2016) in vertebrate (Vero B4) as well as mosquito cell lines (C6/36). Co-infections were performed with a multiplicity of infection (MOI) of 1 for both viruses. The solid and dashed lines are drawn through the mean values of the three biological replicates for all tested time points measured by RT-qPCR based on a relative and absolute standard curve running in parallel. The error bars represent the standard deviation (± SD). Incomplete error bars occur when y-min of the error bars is negative and therefore not displayed in the logarithmic y-scale