Introduction
West Nile virus (WNV) and Usutu virus (USUV; genus
Flavivirus, family
Flaviviridae) are two closely related zoonotic mosquito-borne viruses. Both circulate in an enzootic cycle between mosquitoes as biological vectors and birds as primary vertebrate hosts, but can also be transmitted to other mammalian species [
1]. Infections with WNV in horses and humans can cause various clinical pictures, including severe neurological diseases [
2]. Symptomatic USUV infections with neurological disorders have been observed in humans only in individual cases, however, in recent years, the number of USUV-infected human cases in Europe has steadily increased [
3].
WNV lineage 1 strains have been circulating in Europe for several decades [
4]. In 2004, WNV lineage 2 was detected for the first time in Hungary [
5] and has since then continued to spread throughout Europe [
6]. In Germany, WNV lineage 2 has been circulating since 2018, causing infections in birds, horses and humans every year [
7‐
10]. Similarly to WNV, USUV was first detected in Europe in Austria in 2001 [
11], but retrospective analysis of historical bird tissues had shown that the virus was already present in Italy in 1996 [
12]. Since the first detection of USUV in Germany in 2010 [
13], the virus spread nationwide within a few years, causing significant numbers of bird deaths, especially in blackbirds (
Turdus merula) [
14‐
16]. Currently several USUV lineages are circulating in Germany, with USUV lineages Europe 3 and Africa 3 predominating in all federal states [
9,
14].
The distribution areas of WNV and USUV are increasingly overlapping in central European countries such as in Germany [
9] as well as in several other countries [
1,
17]. In addition to their geographical co-circulation, WNV and USUV are also epidemiologically closely-related, sharing the same vertebrate hosts and mosquito vectors [
1,
17]. The risk of co-infections with both viruses therefore exists, and indeed co-infections in birds [
18,
19] and humans [
10,
20] have already been reported.
In-vitro studies are a fundamental first step in investigating viral co-infections and their effects on virus replication. A few previously conducted
in-vitro co-infection studies examined combinations of WNV with other flaviviruses [
21,
22], but to date only one study investigated co-infections with WNV and USUV [
23]. In this study, it was shown that the replication of USUV Africa 3 was inhibited by WNV lineage 2 in mammalian, avian and mosquito cells [
23]. However, due to the co-circulation of two WNV lineages and several USUV lineages in Europe, a combination of just one lineage per virus quickly reaches its limitations in experimentally reflecting the actual situation in Europe. Further studies were necessary to investigate and understand the interactions between WNV and USUV. The aim of this study was therefore to examine growth kinetics of a range of WNV and USUV lineages and isolates and to analyse co-infections of selected viral isolates in mammalian, avian and mosquito cell lines.
Discussion
With the introduction of WNV into Germany, there is a need to understand the role that co-infections with WNV and USUV might play in the enzootic transmission cycle. The focus of this study was therefore to investigate the viral replication as well as potential interactions in co-infections of German and other European WNV and USUV strains.
All WNV and USUV isolates demonstrated a rapid viral growth followed by a steady decline of the titres due to a strong CPE in both vertebrate cell lines. Maximum titres were higher in the avian GN-R cells than in the mammalian Vero B4 cells, similar to results from previous studies [
34]. This is consistent with the more efficient viral replication of WNV and USUV in avian species compared to mammals [
35], although geese are not the primary hosts for these viruses [
36]. In contrast, there was a slower but steady viral growth in both mosquito cell lines, with limited CPE (low levels to none detected), which matches the life-long viral infectivity of mosquito vectors [
37]. The observed growth of WNV and USUV on these cell lines are in accordance to the potential vector competence of
Aedes albopictus for WNV and USUV [
38,
39] and
Culex tarsalis for WNV [
40]. Overall, the observed differences between insect and vertebrate cells were already described in previous
in-vitro experiments [
23,
41]. Decisive factors for the different viral replication kinetics might be inherent differences in viral replication in mammalian versus insect cells as well as the used incubation temperatures [
42,
43].
In-vitro attenuations of the virus isolates to certain cell lines must also not be disregarded.
When comparing the viral growth kinetics within one virus species, USUV Europe 3 demonstrated slower viral growth than USUV Africa 3 on CT cells but not on C6/36 cells. A possible explanation might be the presence of a functional RNA interference pathway, which is the main antiviral pathway in mosquitoes [
44], and proved to be sufficient in CT but not C6/36 cells [
37,
45]. Apart from that, there were no differences in the viral replication kinetics. This is in accordance with
in-vivo observations in geese where two different WNV strains, whose isolates were also used in the current study, caused comparable pathology [
25,
26]. In contrast, another
in-vitro study reported differences in viral replication of USUV strains in cell culture as well as virulence in mice [
46]. Overall, all WNV strains replicated faster and to higher maximum titres than the USUV strains on all tested cell lines. This result has also been reported from other cell experiments [
23,
47] and possibly explains the higher number of WNV deceased birds [
9] as well as the higher disease severity of WNV in humans [
10].
Other
in-vitro co-infection studies with flaviviruses mostly reported a competition between both viruses, resulting in the inhibition of at least one virus [
23,
48,
49]. Similarly, a competition between WNV and USUV could be observed in this study, with a decreased replication of USUV in all cell lines. The suppression of USUV was most evident in the avian cells. The faster replication of WNV observed in the mono-infections appears to have caused a competitive advantage of WNV over USUV. Due to their genetic and phylogenetic relationship, WNV and USUV likely use the same cell receptors and/or components for their replication [
37], resulting in a competition for these resources in both host and vector cells. Similarly, closely-related viruses can activate identical cellular defences, in turn cross-protecting cells against an additional infection [
50]. This is also supported by the fact that the suppression of USUV was dependent on WNV MOI, where a lower concentration of WNV particles might enable USUV to initially infect more cells, resulting in a higher maximum titre.
The viral interference appeared not to be strain or lineage dependent as similar results were found for combinations with other virus isolates. This was not surprising as almost all strains had similar viral kinetics. It must be noted that, even though marked differences in virulence were not observed between the virus strains used in this study, it is not uncommon for WNV to show variances in its efficiency to replicate and become neuroinvasive, as shown for Australian strains in cells and an established mouse model [
51]. Interestingly, however, even the different growth kinetics of both USUV lineages on CT cells did not have an impact on the outcome. Therefore, competition for resources seems to be more likely than a potential impact of RNA interference. In the vertebrate cells (Vero B4 and GN-R), the growth of WNV was also reduced when the cells were co-infected with USUV. However, it remains unclear if this WNV-reduction was caused by USUV or by the general loss of viable vertebrate cells over time. Since WNV growth did not appear to be affected in the co-infected insect cell lines (CT and C6/36) the latter explanation is more likely.
Overall, WNV appears to have an advantage over USUV, possibly due to the observed different replication kinetics in host and vector cells. This is in accordance to
in-vivo findings in birds and mosquitoes. Birds that were co-infected with both viruses had higher viral loads of WNV than USUV [
18], and USUV infection was reduced in
Cx. pipiens biotype
pipiens that were simultaneously infected with WNV [
23,
52]. Taken together with the results of this study, WNV proves to be a virus with a high viral fitness, possessing the ability to replicate rapidly and efficiently in a broad range of host and vector cells. It can outcompete closely related viruses such as USUV. This might also be one of the reasons for its unprecedented worldwide distribution to date. However, there are still some unanswered questions. Although the viral interference between WNV and USUV was confirmed in all mosquito cell lines, the suppression of USUV in mosquitoes
in-vivo could not be confirmed for every mosquito species [
52]. Similarly, co-infections in mammalian and avian species might lead to unpredictable outcomes. The exact cellular mechanisms underlying the interactions between WNV and USUV remain unexplained and should be targeted by future investigations.
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