A One-Health Approach to Investigating an Outbreak of Alimentary Tick-Borne Encephalitis in a Non-endemic Area in France (Ain, Eastern France): A Longitudinal Serological Study in Livestock, Detection in Ticks, and the First Tick-Borne Encephalitis Virus Isolation and Molecular Characterisation

Abstract

Tick-borne encephalitis virus' (TBEV) geographic range and the human incidence are increasing throughout Europe, putting a number of non-endemic regions and countries at risk of outbreaks. In spring 2020, there was an outbreak of tick-born encephalitis (TBE) in Ain, Eastern France, where the virus had never been detected before. All patients but one had consumed traditional unpasteurised raw goat cheese from a local producer. We conducted an investigation in the suspected farm using an integrative One Health approach. Our methodology included (i) the detection of virus in cheese and milk products, (ii) serological testing of all animals in the suspected farm and surrounding farms, (iii) an analysis of the landscape and localisation of wooded area, (iv) the capture of questing ticks and small mammals for virus detection and estimating enzootic hazard, and (v) virus isolation and genome sequencing. This approach allowed us to confirm the alimentary origin of the TBE outbreak and witness in real-time the seroconversion of recently exposed individuals and excretion of virus in goat milk. In addition, we identified a wooded focus area where and around which there is a risk of TBEV exposure. We provide the first TBEV isolate responsible for the first alimentary-transmitted TBE in France, obtained its full-length genome sequence, and found that it belongs to the European subtype of TBEV. TBEV is now a notifiable human disease in France, which should facilitate surveillance of its incidence and distribution throughout France.

Keywords: alimentary route; environmental investigation; one health; outbreak; tick-borne encephalitis virus.

FIGURE 1

Timeline of the food, environmental and veterinary investigations, and sampling scheme from April to November 2020.

FIGURE 2

IgG levels (A) and micro-neutralisation antibody levels (B) in goats on four dates in 2020 (May 27, June 2, June 8, and June 19) and on April 2, 2021. The threshold levels applied are indicated as dashed lines. We provide examples of seronegative individuals (green) and show results for the individual that returned seropositive via ELISA competition, but returned negative in neutralising antibody titre (grey), the three individuals that seroconverted after May 27 (yellow), the eight seropositive individuals (red), and the two individuals that returned negative IgG competition results in 2021 (blue), one of which maintains neutralising antibodies.

FIGURE 3

Localisation of the farm responsible for the tick-borne encephalitis cluster through alimentary route (star) and results of the serological survey conducted in surrounding farms. Serum samples were first tested by a competitive ELISA. Then, positive and doubtful ELISA samples were tested by specific tick-borne encephalitis virus (TBEV) micro-neutralisation tests (MNT). A serum sample was considered TBEV-seropositive when MNT was positive. Dairy cows, suckling cows, goats, suckling cows, and goats were found in farms #1 to #5, respectively.

FIGURE 4

Full-length TBEV genome sequence (GenBank accession number: OL441148) was obtained from sequencing 35 overlapping amplicons in two multiplex reactions. Read coverage varied along the sequence length, but a full-length genome sequence was successfully recovered.

FIGURE 5

Phylogenetic tree of TBEV complete open reading frames that were available in GenBank and the TBEV-Ain-France-2020 obtained in the present study (marked red) using all sequences (A) or those corresponding to the European subtype only (B). The two trees were inferred in PhyML using the Le Gascuel (LG) substitution model. Branch points indicate that results of Shimodaira-Hasgawa branch test > 0.8. Scale bar shows the number of nucleotide changes. Virus isolate names are given as follows: accession number from NCBI_ country to name_year.