Ixodes ricinus as potential vector for Usutu virus

Abstract

Usutu virus (USUV) is an emerging flavivirus that is maintained in an enzootic cycle with mosquitoes as vectors and birds as amplifying hosts. In Europe, the virus has caused mass mortality of wild birds, mainly among Common Blackbird (Turdus merula) populations. While mosquitoes are the primary vectors for USUV, Common Blackbirds and other avian species are exposed to other arthropod ectoparasites, such as ticks. It is unknown, however, if ticks can maintain and transmit USUV. We addressed this question using in vitro and in vivo experiments and field collected data. USUV replicated in IRE/CTVM19 Ixodes ricinus tick cells and in injected ticks. Moreover, I. ricinus nymphs acquired the virus via artificial membrane blood-feeding and maintained the virus for at least 70 days. Transstadial transmission of USUV from nymphs to adults was confirmed in 4.9% of the ticks. USUV disseminated from the midgut to the haemocoel, and was transmitted via the saliva of the tick during artificial membrane blood-feeding. We further explored the role of ticks by monitoring USUV in questing ticks and in ticks feeding on wild birds in the Netherlands between 2016 and 2019. In total, 622 wild birds and the Ixodes ticks they carried were tested for USUV RNA. Of these birds, 48 (7.7%) carried USUV-positive ticks. The presence of negative-sense USUV RNA in ticks, as confirmed via small RNA-sequencing, showed active virus replication. In contrast, we did not detect USUV in 15,381 questing ticks collected in 2017 and 2019. We conclude that I. ricinus can be infected with USUV and can transstadially and horizontally transmit USUV. However, in comparison to mosquito-borne transmission, the role of I. ricinus ticks in the epidemiology of USUV is expected to be minor.

Fig 1. Growth kinetics of tick- and mosquito-borne viruses in mammalian Vero E6 and Ixodes ricinus IRE/CTVM19 tick cell lines.

Vero E6 (A) and IRE/CTVM19 (B) cells were infected with a multiplicity of infection (MOI) of 0.1 with Usutu virus (USUV), West-Nile virus (WNV) and tick-borne encephalitis virus (TBEV). Viral titres were determined using end-point dilution assays (EPDAs). The results are shown as the mean viral titres ± standard error of three replicates. Dashed line indicates the detection limit of the EPDA at 1 x 10³ TCID₅₀/mL.

Fig 2. Viral titres of USUV injected Ixodes ricinus nymphs (A), females (B) and males (C).

Ticks were injected with USUV. Groups of ticks were sacrificed at 0, 14 and 28 (only nymphs) days post injection. Viral titres were determined using end-point dilution assays (EPDAs). Each data point represents one injected tick. Median titres are shown as horizontal black lines. A dashed line indicates the detection limit of the EPDA at 1 x 10³ TCID₅₀/mL. All samples were positive for USUV based on infectivity assay. Samples with a viral load below the detection limit of the EPDA are represented on the detection limit line. Indicated statistics show the output of GLMMs (ns = not significant, *** p < 0.001).

Fig 3. Artificial infection of Ixodes ricinus with USUV via a bloodmeal.

(A) Infection rates of USUV blood-fed nymphs at 0 (n = 152), 14 (n = 112), 28 (n = 182) and 70 (n = 318) days post engorgement. Boxplots represent four independent replicates. (B) Viral titres of USUV infected ticks at 0, 14, 28 and 70 dpe, determined using end-point dilution assays (EPDAs). Each dot represents a single tick. Median titres are shown as horizontal black lines. All samples were positive for USUV based on infectivity assay. Samples with a viral load below detection limit of the EPDA are represented on the detection limit line. (C) Engorged nymphs with legs removed. (D) Infection rate of USUV in body and legs of I. ricinus nymphs (n = 124) after 7–10 days post USUV infected blood feeding. Boxplots represent three independent experiments. Indicated statistics show the output of GLMMs. (ns = not significant, * p < 0.05, ** p < 0.01, *** p < 0.001). Dashed lines indicate the detection limit of the end-point dilution assay at 1 x 10³ TCID₅₀/mL.

Fig 4. Time series of USUV RNA detections in tick-infested wild birds and carried ticks in the Netherlands, 2016–2019.

The number of birds with ticks sampled and tested for the presence of USUV RNA is shown per month. Colors of the bars indicate the combinations of results in bird and carried ticks. One bird can carry multiple ticks.

Fig 5. Geographical distribution of USUV RNA detection in ticks collected from wild birds and in questing ticks in the Netherlands, 2016–2019.

Locations of sampling and of USUV RNA positive cases. Circles indicate sampling locations of birds carrying tick, size is proportional to the number of birds sampled. Crosses indicate collection locations of questing ticks, size is graduated by range of number of ticks collected. Provinces are labeled with two-letter abbreviation. Source administrative boundaries: CBS, Kadaster, "CBS Gebiedsindelingen 2019" (https://service.pdok.nl/cbs/gebiedsindelingen/atom/v1_0/index.xml). Created using the R packages “sp” and “rgdal” and ArcMap 10.8.1.

Fig 6. Size distribution of 18-32nt vsiRNAs and distribution of 22 nt vsiRNAs across the USUV genome.

The percentages of reads mapping to the sense (blue) or antisense (red) genome are shown. Panels A-C show the size distribution of vsiRNAs mapping to USUV genome as well as the 22 nt vsiRNAs across the USUV genome. The 3’ and 5’ untranslated regions (UTRs) as well as the structural and non-structural regions of the USUV genome are depicted together with the Capsid (C), pre-Membrane (prM) and Envelope (E) protein coding regions. Panel A and B represent two pools of artificial membrane blood-fed I. ricinus ticks. Panel C represents a pool of USUV infected Ixodes ticks collected from wild birds. vsiRNA read counts were normalized against the total small RNA library (21 million reads).

Fig 7. Phylogenetic analysis.

Maximum likelihood phylogeny of USUV complete coding sequences, subset comprising sequences from lineage Africa 3. Sequences from the Netherlands are colored in light red. Panels show close-up views into regions of the maximum likelihood phylogenetic tree, highlighting the positions of sequences generated in this study; sequences derived from birds are shown in dark red and sequences derived from ticks carried by these birds are shown in blue, all are from the Netherlands. In the overview tree, only bootstraps of major internal nodes are indicated, in the zoom, bootstraps ≥80% of the external nodes are indicated. Scale units are nucleotide substitutions per site.