The circulation of West Nile virus (WNV), Usutu virus (USUV), and tick-borne encephalitis virus (TBEV) was investigated in south-western France during the first six months of 2023, following the emergence of WNV in equids in Gironde, a département in south-western France, in 2022. Blood samples were collected from 494 horses located in the Gironde département and divided into three zones: the Confluence zone, the Intermediate zone and the Arcachon Basin. Samples were tested for WNV-, USUV- and TBEV-specific antibodies. An overall seroprevalence of 14% (95% CI [11–18%]) for orthoflavivirus antibodies was detected in Gironde. The highest seroprevalence rates for WNV and USUV were observed in the Confluence Zone (9%, 95% CI [6–13%] and 5%, 95% CI [3–8%], respectively), where the type of housing (animals kept in pasture only) and proximity to a special bird protection area were identified as risk factors for WNV seropositivity. This study presents the first seroprevalence investigation of WNV, USUV and TBEV infections in equids located on the Atlantic coast of France and demonstrates intense circulation of WNV in this region, as well as evidence of equine USUV-specific infection.

Tick-borne viruses are responsible for various symptoms in humans and animals, ranging from simple fever to neurological disorders or haemorrhagic fevers. The Kemerovo virus (KEMV) is a tick-borne orbivirus, and it has been suspected to be responsible for human encephalitis cases in Russia and central Europe. It has been isolated from Ixodes persulcatus and Ixodes ricinus ticks. In a previous study, we assessed the vector competence of I. ricinus larvae from Slovakia for KEMV, using an artificial feeding system. In the current study, we used the same system to infect different tick population/species, including I. ricinus larvae from France and nymphs from Slovakia, and I. persulcatus larvae from Russia. We successfully confirmed the first two criteria of vector competence, namely, virus acquisition and trans-stadial transmission, for both tick species that we tested. The estimated infection rates of engorged and moulted ticks suggest specificities between viral strains and tick species/developmental stages.

Kemerovo virus (KEMV) is a tick-borne orbivirus transmitted by ticks of the genus Ixodes. Previous animal experimentation studies with orbiviruses, in particular the interferon receptor double knock-out (IFNAR^(−/−)) mouse model, did not indicate bias that is related to age or sex. We endeavoured to assess the effect of serial and alternated passages of KEMV in mammalian or Ixodes cells on virus replication and potential virulence in male or female IFNAR^(−/−) mice, with important age differences: younger males (4–5 months old), older males (14–15 months old), and old females (14–15 months old). After 30 serial passages in mammalian or tick cells, or alternated passages in the two cell types, older female mice which were inoculated with the resulting virus strains were the first to show clinical signs and die. Younger males behaved differently from older males whether they were inoculated with the parental strain of KEMV or with any of the cell culture-passaged strains. The groups of male and female mice inoculated with the mammalian cell culture-adapted KEMV showed the lowest viraemia. While older female and younger male mice died by day 6 post-inoculation, surprisingly, the older males survived until the end of the experiment, which lasted 10 days. RNA extracted from blood and organs of the various mice was tested by probe-based KEMV real-time RT-PCR. Ct values of the RNA extracts were comparable between older females and younger males, while the values for older males were >5 Ct units higher for the various organs, indicating lower levels of replication. It is noteworthy that the hearts of the old males were the only organs that were negative for KEMV RNA. These results suggest, for the first time, an intriguing age- and sex-related bias for an orbivirus in this animal model. Changes in the amino acid sequence of the RNA-dependent RNA polymerase of Kemerovo virus, derived from the first serial passage in Ixodes cells (KEMV Ps.IRE1), were identified in the vicinity of the active polymerase site. This finding suggests that selection of a subpopulation of KEMV with better replication fitness in tick cells occurred.

The circulation of West Nile virus (WNV), Usutu virus (USUV) and Tick-borne encephalitis virus (TBEV) was investigated in southwestern France within the first six months of 2023 following the emergence of WNV in equids in Gironde county in 2022. Blood samples were collected from 494 horses located in Gironde County divided into three zones: the Confluence, the Intermediate zone and the Arcachon basin and tested for WNV, USUV and TBEV specific antibodies. An overall seroprevalence of 14% (95% CI [11%-18%]) for orthoflavivirus antibody was detected in Gironde county. The highest seroprevalence rate for WNV and USUV were detected in the Confluence zone (9%, 95% CI, [6%-13%] and 5%, 95% CI, [3%-8%] respectively). The type of housing (animals kept in pasture only) and the distance to the nearest bird special protection area (SPA) were identified as risk factors for WNV seropositivity. This paper presents the first seroprevalence study investigating WNV, USUV and TBEV infections, conducted in equids located on the Atlantic coast of France and demonstrates an intense circulation of WNV in this region of France as well as equine USUV specific infection.