West Nile virus (WNV, family Flaviviridae
, genus Flavivirus)
is maintained in a transmission cycle between birds as amplification hosts and mosquito vectors [1
]. Spillover events have significant public health and veterinary relevance [2
]. A total of 25% of the infected people develop West Nile fever (WNF) and become symptomatic (e.g., headache or muscle pain) [3
]. Severe disease progressions manifesting as WNV neuroinvasive disease (WNND) are rare (<1%) [4
]. These include syndromes of meningitis, encephalitis, and acute flaccid paralysis/poliomyelitis. Case-fatality rate of WNND is approximately 10% [5
]. Age is the most important risk factor for WNND and a fatal disease outcome [2
]. Thus, WNV circulation poses considerable risk for transfusion and organ transplantation safety [6
WNV is distributed in wide areas of Europe. The main focus of WNV circulation is in south-eastern Europe and Italy [7
]. However, low WNV activity is also observed in the neighboring countries of Germany (France, Austria, and Czech Republic). Therefore, over the last decade, different monitoring programs were implemented in Germany to screen for WNV RNA and antibodies in birds, horses, mosquitoes and chicken eggs [8
]. In 2018, an epizootic emergence of WNV was observed in Germany for the first time [13
]. All WNV-positive birds and horses were infected with the same WNV lineage 2 strain of the central European subclade II. WNV activity was detected in eastern Germany over a distance of almost 900 km (Munich to Rostock). At the same time, a large WNV outbreak was observed in south-eastern and southern Europe [7
]. However, phylogenetic analysis in combination with the wide distribution in Germany indicates that WNV may have been introduced from the Czech Republic to Germany already before 2018 [13
]. The emergence of WNV in Germany and the focus in the central part of eastern Germany was correlated with outstandingly high summer temperatures. As demonstrated for other European countries, WNV is probably predominantly transmitted by different native Culex
species. Culex pipiens
, Culex pipiens
and Culex torrentium
from Germany were experimentally proven to be susceptible to WNV infection [14
In this study, we report a WNV epidemic in Germany, 2019, triggered by an epizootic emergence among birds with spillover to horses and humans. Human and animal cases were located in the same area, showing a high WNV activity also in 2018. In both years, the region was characterized by suitable temperature conditions allowing a short extrinsic incubation period (EIP). Phylogenetic and phylogeographic analysis showed that Germany experienced several WNV introduction events. Several virus variants circulate in the affected German regions with Austria and Czech Republic as possible origins. The majority of the WNV strains involved in the German outbreak clustered together into a distinct and dominating group (Eastern German WNV clade) comprising of mosquito, bird, horse and human-related virus variants.
Globalization and climate change enhance or can lead the migration of exotic pathogens and their hosts to new environments promoting the contacts with naïve and vulnerable hosts. Thus, understanding the local ecological factors and evolutionary processes which navigate the emergence, establishment and spread of newly introduced viral diseases is critical for developing and implementing surveillance strategies for disease control. The present study aimed to elucidate the possible origins, spatiotemporal spread pattern tendencies and eco-epidemiological factors that facilitate WNV becoming an established pathogen in Germany causing neuroinvasive disease in multiple vertebrate species, including humans.
One year after the first observed autochthonous WNV transmission to birds and horses in Germany [9
], an epizootic emergence of WNV was again observed in 2019. The number of infected birds and horses was considerably higher (76 birds and 36 horses) compared to 2018 (12 birds and two horses). In contrast to the USA, WNV-associated mass mortality in birds had not been observed in Europe before [33
]. Previous hypotheses for this difference have been refuted by several research studies, e.g., demonstrating that European birds are susceptible to WNV infections and Culex
mosquitoes in Europe are competent to transmit WNV. An alternative explanation might by that the bird mortality is so low that it is not detected with current European surveillance programs. A comprehensive USUV/WNV monitoring system is in place, but we also only see the tip of the iceberg of WNV infected birds in Germany. In addition, a huge number of positive specimens were obtained from captive animals (e.g., birds kept in zoos), which have a higher probability of detection compared to wild animals. Furthermore, from these birds, a considerable proportion were birds of prey, which must be considered to have a higher susceptibility to WNV infection [34
]. This in combination with a widespread enzootic circulation of WNV and large number of equine cases—36 in 2019 in contrast to 2 in 2018—indicates an increased risk of WNV spillover into the human population.
This is reflected in the detection of five laboratory confirmed, mosquito-borne, autochthonous human WNV cases in 2019. It has to be kept in mind that less than 25% of infected humans develop noticeable symptoms [3
]. Even fewer patients (<1%) have a risk of developing WNND [4
]. The number of observed WNND cases (three of the five confirmed human WNV) gives rise to the speculation that hundreds of undetected human WNV infections in Germany occurred during the epidemic in 2019.
WNV transmission and spread is significantly influenced by climatic conditions, e.g., shaping phenology and abundance of the vector. Temperature is one of the most important factors directly affecting the EIP in different mosquito vector species [14
]. High daily average temperatures (> 20 °C) over several days are required to allow for WNV transmission, which is correlated to the main distribution of WNV in south-eastern Europe. This also matches the spatial pattern of WNV in Germany. The summers in 2018 and 2019 were both characterized by extraordinarily high temperatures allowing low EIP values. The area in central-eastern Germany as the main focus of WNV circulation in both years was characterized by shorter EIP compared to previous years and most other areas in Germany [13
]. Furthermore, these areas in Germany are directly neighboring countries reporting several years of WNV circulation (e.g., Czech Republic), leading to a high risk of short distance introductions e.g., by infected birds. The analysis also indicated that the areas along the Upper Rhine Valley in south-west Germany had a high suitability for WNV circulation, but no WNV activity was observed in all previous surveillance programs [8
]. Most likely, no WNV introduction and circulation occurred yet, which underpins the thesis of the entries over short distances. Future studies are needed to understand if the virus did not yet spread to this area or if there are other factors reducing the risk of virus circulation (e.g., distribution of suitable vector or host species). The phylogenetic analyses indicated that Germany experienced at least six distinct WNV introduction events, with Austria and Czech Republic as possible origin for the progenitors of the German WNV epizootic strain variants. The majority of these strains clustered together into a distinct subclade (EGC).
The ongoing circulation and dominance of the EGC detected in 2019 indicates successful overwintering of WNV in Germany, e.g., through WNV persistence in hibernating mosquitoes throughout the winter season [35
]. The virus variants of the EGC at multiple sites detected in the epidemic in 2019 are descendants of a common ancestor in the wider central European environment which dates back to the time span 2010–2012. Where and when the subsequent virus evolution to the current variants took place and how descendants were eventually transferred to Germany remains elusive. However, such a translocation and subsequent virus amplification may have been fostered by the extremely favorable climatic conditions for mosquitos in Germany in spring/summer 2018, and the short distance transmission with infected birds from neighboring countries.
There has been a comprehensive USUV/WNV monitoring system in place in Germany for over a decade which involves ornithologists, zoological gardens and bird clinics supplying thousands of zoo and wild bird samples for WNV antibody and genome analysis. Moreover, there has been an exhaustive mosquito surveillance in place in Germany since 2009. By both surveillance approaches a variety of viruses were found, such as Sindbis virus, Batai virus and USUV, but not WNV [9
]. At the same time, different long-distance, partial and short-distance migratory birds showed neutralizing antibodies against WNV before 2018 [9
]. Although any such monitoring scheme has its predictive limitations due to sampling size constraints, all the negative WNV monitoring results from birds, horses and mosquitos before 2018 and the proximity to a larger region with active WNV circulation supports a recent introduction of multiple WNV descendants e.g., from Czech Republic to Germany. However, sequencing a larger number of more current WNV strains from e.g., Austria or the Czech Republic would help to answer the circumstances of when and what in regard to the development of the East German Clade variants. Overall, the number of recent whole genome sequences is limited and should be markedly increased using NGS-based approaches.
Most of the singleton WNV variants in Germany do not contain the monophyletic Lys2114Arg mutation located in the C terminus of the NS3 gene, even if these strains circulate in the central-eastern part of the country with very high WNV activity and rapid expansion of the EGC. Although these singletons have circulated and evolved under the same ecological conditions as members of EGC, it seems that these variants were not able to perpetuate and establish a stable enzootic cycle leading to a similar epizootic/epidemic scenario as for the EGC group. In case of the EGC, the adaptation to naïve vector and host populations leads to the emergence of local virus variants. The most likely scenario for EGC might be enzootic maintenance similar to that observed for WNV in the United States [38
]. This hypothesis is supported by the observation that EGC form a star-like structure (population expansion after a single viral introduction) in which the variant viral strains accumulate changes during the rapid adaptation to the local ecological conditions (adaptation of the virus to the host populations and its enzootic maintenance), as observed for Usutu virus [40
We found evidence that the phylogenetic structure of EGC and virus genetic population growth is shaped by the geographic location and average extrinsic incubation period, which likely facilitated rapid short-distance virus dispersal in 2018/2019. This demonstrates that local ecological factors (e.g., average temperature profile during the vector season) could predict the local and regional dispersal patterns of WNV in our data sets.
The purifying and negative selections observed for WNV in Germany were expected given the transmission and infection modes of arboviruses, leading to the accumulation of synonymous mutations [41
]. Mutation observed at amino acid position Lys2114Arg has been found to be involved in the formation of EGC, while Val1493Ile (NS2b), Pro1754His (NS3), Ser2287Gly and Ala2322Thr (NS4b), Ala2827Thr and Lys3056Arg (NS5) are specific for the CEC (convergent evolution). Similar patterns of parallel or convergent evolution have been observed for WNV. This suggests that a limited number of residue changes are permitted due to functional constraints [42
]. Viral adaptation in vector and vertebrate hosts by local overwintering or reintroduction of the virus and local ecological conditions (e.g., high average EIP) could be considered key determinants in the spatial dispersal and establishment of WNV. It is interesting to note that the Lys2114Arg mutation is specific for the newly described EGC. The impact of this mutation is unclear; a similar change in the WNV NS3 helicase (Thr1754Pro) generated a highly virulent phenotype to American crows [43
]. In vitro and in vivo experiments with strains from the EGC might show the role of fitness and pathogenicity in the future.