Tracking WNV transmission with a combined dog and wild boar surveillance system

Abstract

West Nile virus (WNV) is a mosquito-borne flavivirus mainly circulating in eastern Germany, causing annually reoccurring epizootics in the avifauna as well as sporadic infections in humans and horses. WNV is closely-related to Usutu virus (USUV) and tick-borne encephalitis virus (TBEV) and co-infections thereof are becoming more frequent. To not solely be dependent on the monitoring of wild birds and horses the availability of alternative sentinel species is advantageous. The study examined the seroprevalence of WNV antibodies (Abs) in eastern Germany in readily available species: dogs, wild boars, sheep, and goats. An NS1-ELISA was implemented to ease future differentiation of cross-reacting flavivirus Abs with a sensitivity of 92.3 and 90.9% for dog and wild boar sera, respectively. Flavivirus seroprevalences were the highest in wild boars with 42.03%, followed by dogs with 7.86%, and small ruminants with 1.57%. In the wild boars and dogs, WNV Abs were most frequent (17.64 and 3.90%, respectively) while seroprevalences in small ruminants and of USUV were lower. The NS1-ELISA is cost-efficient and reliable in monitoring WNV Abs in dogs as well as wild boars and the combined testing thereof could be ideal in detecting semi-urban transmission events prior to wildlife-human spill overs.

Fig. 1

Map of sampling area in East and Central Germany: Districts in Saxony, Saxony-Anhalt, Thuringia, and Lower Saxony from which samples were collected are portrayed in light blue. Species icons display the species from which the samples originated and the number thereof per federal state. Map was created with GADM version 4.1. (https://gadm.org).

Fig. 2

Molecular detection of partial genomes from tick-borne encephalitis virus (TBEV) in wild boars from eastern Germany. (a) Map showing the origin of the two TBEV-positive serum samples (indicated by a red star) from two districts of the federal state of Saxony-Anhalt. Created with R (v4.2.2, x64)/R Studio (Version 2022.12.0 + 353)^,. (b) Phylogenetic tree of whole genome sequences available on GenBank and the partial NS5 sequences obtained from the two TBEV isolates obtained in this study (GenBank Accession nos. marked in red). Created with Geneious Prime 2021.0.1. Map was created with GADM version 4.1. (https://gadm.org).

Fig. 3

Pie charts (a–c) illustrating the distribution of the neutralizing antibodies (nAbs) based on the VNTs (Supplementary Table S5) as well as the violin plots (d–f) illustrating the antibody titer distributions for the three tested flaviviruses (West Nile virus: WNV, tick-borne encephalitis virus: TBEV, and Usutu virus: USUV) for dogs (a, d), wild boars (b, e), and small ruminants (c, f). The red dots in the violin plots denote the mean VNT titers and the red lines the standard deviation thereof. Statistical analyzes are based on non-parametric multiple pairwise-comparison Wilcoxon rank sum tests using “BH” adjustment: * = p < 0.05, ** = p < 0.01, *** = p < 0.001 (Supplementary Table S6). Created with R (v4.2.2, x64)/R Studio (Version 2022.12.0 + 353)^,.

Fig. 4

Validation of the in-house NS1-ELISA for detecting West Nile virus (WNV), tick-borne encephalitis virus (TBEV), and Usutu virus (USUV) specific antibodies in dog sera (n = 56; a–c) as well as wild boar sera (n = 145; d–f) based on the VNT-results as a gold standard. Receiver operating characteristic (ROC) curves depicting the area under the curve (AUC; shown by the blue line) for the NS1-ELISA as well as the estimated cut-offs. The statistical assessment of the performance of the NS1-ELISA is summarized in Supplementary Table S8.

Fig. 5

Seroprevalence of (a, d) West Nile virus (WNV), (b, e) tick-borne encephalitis virus (TBEV), and (c, f) Usutu virus (USUV) specific antibodies as detected by the in-house NS1-ELISA in wild boar and dog sera, respectively. Wild boar sera from the districts Saxony and Saxony-Anhalt were tested and results are depicted per district. Dog sera were tested from Saxony, Saxony-Anhalt, Lower Saxony, and Thuringia and due to the low sample size of flavivirus-positive small ruminant sera ROC-analyzes could not be performed and cut-offs not determined. Raw data and seroprevalences from the NS1-ELISA are listed in Supplementary Tables S1–S4; S7. Created with R (v4.2.2, x64)/R Studio (Version 2022.12.0 + 353)^,. Map was created with GADM version 4.1. (https://gadm.org).