First cases of European bat lyssavirus type 1 in Iberian serotine bats: Implications for the molecular epidemiology of bat rabies in Europe

Abstract

Previous studies have shown that EBLV-1 strains exclusively hosted by Eptesicus isabellinus bats in the Iberian Peninsula cluster in a specific monophyletic group that is related to the EBLV-1b lineage found in the rest of Europe. More recently, enhanced passive surveillance has allowed the detection of the first EBLV-1 strains associated to Eptesicus serotinus south of the Pyrenees. The aim of this study is the reconstruction of the EBLV-1 phylogeny and phylodynamics in the Iberian Peninsula in the context of the European continent. We have sequenced 23 EBLV-1 strains detected on nine E. serotinus and 14 E. isabellinus. Phylogenetic analyses were performed on the first 400-bp-5' fragment of the Nucleoprotein (N) gene together with other 162 sequences from Europe. Besides, fragments of the variable region of the phosphoprotein (P) gene and the glycoprotein-polymerase (G-L) intergenic region were studied on Spanish samples. Phylogenies show that two of the new EBLV-1a strains from Iberian E. serotinus clustered together with French strains from the North of the Pyrenees, suggesting a recent expansion southwards of this subtype. The remaining seven Iberian strains from E. serotinus grouped, instead, within the cluster linked, so far, to E. isabellinus, indicating that spatial distribution prevails over species specificity in explaining rabies distribution and supporting interspecific transmission. The structure found within the Iberian Peninsula for EBLV-1b is in concordance with that described previously for E. isabellinus. Finally, we have found that the current EBLV-1 European strains could have emerged only 175 years ago according to our evolutionary dynamics analyses.

Fig 1. EBLV-1 in Europe.

1A: Geographical distribution of the lineages described within the EBLV-1 rabies virus across Western Europe: EBLV-1a (red dots) along Northern, Central Europe. EBLV-1b (yellow dots) along Central Europe and the Iberian EBLV-1b, with a further subdivision between Southwestern major (purple diamonds), Southwestern minor (blue diamonds) Southeastern (black diamonds) and Northwestern. (green diamonds) strains. Two strains from the same location (R76, 292R07) not grouping in any of the previous lineages are represented by a dotted diamond. Colored circles correspond to the main groups identified in the phylogenetic reconstruction shown in Fig 1B. The base layer has been taken from https://mapmaker.ecdc.europa.eu/#, map produced on: 1 Mar 2018. Administrative boundaries: Eurogeographics, UN-FAO. 1B: Midpoint-rooted Bayesian phylogenetic consensus reconstruction of European EBLV-1 based on a 400-bp-5' first fragment of the Nucleoprotein gene. Tree nodes show posterior probability (black) and also Maximum-Parsimony bootstrap support (red), Neighbor-Joining bootstrap support (blue) and Maximum-Likelihood bootstrap support (green). Colored circles correspond to the main phylogenetic groups.

Fig 2. Viral genetic geographic structure across Europe through median-joining haplotype network.

2A: EBLV-1a lineage. Circle’s sizes are proportional to the number of individuals sharing this particular haplotype and color code the regions of origin of haplotypes, Red: Germany; Dark Blue: France; Brown: Denmark; Grey: Hungary; Purple: Poland; Orange: The Netherlands; Light Green: Spain. Reconstructed haplotypes are represented as small red squares. 2B: EBLV-1b lineage. Circle’s sizes are proportional to the number of individuals sharing this particular haplotype and colors code the regions of origin of haplotypes, Red: Central France; Dark Blue: North-west France; Yellow: North-east France; Black: Germany; Orange: The Netherlands; Light Green: Southern Spain; Dark Green: Northern Spain. Reconstructed haplotypes are represented as small red squares.

Fig 3. Iberian EBLV-1b midpoint rooted bayesian phylogenetic reconstruction based on 1,809 bp sequences of the concatenated fragments of nucleoprotein, phosphoprotein and glycoprotein-polymerase’s intergenic region.

Tree nodes show posterior probability (black) and Maximum-Parsimony (red), Neighbor-Joining (blue) and Maximum-Likelihood (green) bootstrap support values. Two Iberian EBLV-1a sequences (2011_Riglos, 201539226_HU) have been included as outgroup. Color code of the diamonds associated to the main groups is the same than in Fig 1A. Two Iberian EBLV-1a sequences (2011_Riglos, 201539226_HU) are included as outgroup. The sequence 69R00_SE, marked with a black dot within the north-east-E. serotinus group, was obtained from an E. isabellinus from the south-west (see text).