Presence of sandfly-borne phleboviruses of two antigenic complexes (Sandfly fever Naples virus and Sandfly fever Sicilian virus) in two different bio-geographical regions of Tunisia demonstrated by a microneutralisation-based seroprevalence study in dogs

Sonia Sakhria 4 Sulaf Alwassouf 0 1 2 3 Wasfi Fares 4 Laurence Bichaud 0 1 2 3 Khalil Dachraoui 4 Cigdem Alkan 0 1 2 3 Ziad Zoghlami 4 Xavier de Lamballerie 0 1 2 3 Elyes Zhioua 4 Remi N Charrel 0 1 2 3 0 IHU Mediterranee Infection, APHM Public Hospitals of Marseille , 13385 Marseille , France 1 Aix Marseille Universite, IRD French Institute of Research for Development, EHESP French School of Public Health, EPV UMR_D 190 Emergence des Pathologies Virales , 13385 Marseille , France 2 IHU Mediterranee Infection, APHM Public Hospitals of Marseille , 13385 Marseille , France 3 Aix Marseille Universite, IRD French Institute of Research for Development, EHESP French School of Public Health, EPV UMR_D 190 Emergence des Pathologies Virales , 13385 Marseille , France 4 Institut Pasteur de Tunis, Laboratory of Vector Ecology , Tunis , Tunisia Background: Sandfly-borne phleboviruses are present in North Africa where they can infect humans in regions where Leishmania infantum, the causative agent of zoonotic visceral leishmaniasis in the Western Mediterranean basin is present affecting both humans and dogs. We investigated the capacity of dogs to be used as sentinels for sandfly-borne phleboviruses as previously shown for leishmaniasis. Findings: A total of 312 sera were collected from guard dogs in two different bioclimatic regions (governorates of Kairouan and Bizerte) of Tunisia where zoonotic visceral leishmaniasis has been reported. These sera were tested for the presence of neutralising antibodies against 3 phleboviruses: Toscana virus, Punique virus and Sicilian virus. In the governorate of Kairouan, seroprevalence rates of 7.5%, 43.5%, and 38.1% were observed for Toscana, Punique and Sicilian virus, respectively. A high proportion of sera from the governorate of Bizerte were hemolyzed and showed high cytotoxicity for the cells and subsequently precluded detailed interpretation of this batch. However, validated results for 27 sera were in agreement with data observed in the governorate of Kairouan. Conclusions: Toscana virus is present in the governorate of Kairouan but at a lower rate compared to Punique and Sicilian viruses. These three sandfly-borne phleboviruses can infect dogs. Direct detection and isolation of the viruses are now to be attempted in animals as well as in humans. Our findings showed that guard dogs are good sentinels for virus transmitted by sandflies and strongly suggested that the high seroprevalence rates observed in dogs merit further attention. - Background Recent studies have indicated that sandfly-borne phleboviruses (genus Phlebovirus, family Bunyaviridae) were not only geographically restricted to southern Europe, but were also present in North Africa [1-6]. At least, viruses of 3 different antigenic complexes are transmitted by sandflies in the Old World: Salehabad complex, Sandfly fever Sicilian complex and Sandfly fever Naples complex. Two phleboviruses of the Sandfly fever Naples complex were reported to be present in Tunisia, namely Toscana virus and Punique virus as assessed by virus isolation [2,7]. Another virus, provisionally named Utique virus that belongs to the Sandfly fever Sicilian complex was genetically detected in Tunisia but it has not been isolated yet [7]. A recent sero-epidemiological study conducted in the governorate of Bizerte (northern Tunisia) showed that both Toscana and Punique viruses could infect human populations although Toscana virus was much more prevalent than Punique virus [5]. In Tunisia, Toscana, Punique and Utique viruses were detected and/or isolated from Phlebotomus pernicious and Phlebotomus longicuspis, which are considered as possible vectors of phleboviruses [2,7]. Both sandfly species are also the main vectors of Leishmania infantum, etiologic agent of zoonotic visceral leishmaniasis (ZVL) in Tunisia [8-10]. Dogs are the main reservoir host of L. infantum and therefore they are used as sentinels to assess the risk of ZVL and other zoonotic vector-borne diseases [11]. Phlebotomus pernicious and P. longicuspis are widely distributed in Tunisia [12], and subsequently, we hypothesized a large distribution of sandfly-borne phleboviruses. It is important to point out that the governorate of Kairouan is the most endemic foci for ZVL [10]; in addition approximately 40% of the rural human population living in the governorate of Bizerte possesses antibodies neutralizing Toscana virus (TOSV-NT-Ab) [5]. Therefore, human populations are exposed to sandflyborne diseases in both governorates. Findings The study took place in the governorate of Bizerte located in Northern Tunisia and in the governorate of Kairouan located in Central Tunisia corresponding to two different bio-geographical areas (Figure 1). To assess the circulation of sandfly-borne phleboviruses, dogs were used as sentinel. A retrospective study on dogs was undertaken in several districts of the governorates of Bizerte and Kairouan during the fall of 2013. Sampling was performed in five locations belonging to different bio-climatic zones varying from humid to arid (Sejnane: 3656 N, 921 E, humid; Mateur: 37 03 N, 9 28E, Sub-humid; Borj Youssef; 3656N, 1007E, semi-arid; Haffouz: 3451 N, 929E, arid; Bouhajla, 3524N, 956 E, arid) (Figure 1). The selected sites were restricted to previously surveyed areas characterized by the abundance of phlebotomine species of the subgenus Larroussius [2,10,12]. The typical setting of a house in endemic areas for ZVL includes the guard dog, attached for his entire life close to the house, and to the sheep shed and chicken henhouse. Houses are always surrounded by cactus to provide protection against trespassing and cactus peers are highly appreciated by villagers. This ecological setting offers suitable biotope for sandflies. Sheep sheds made usually with mud walls are breeding sites for sandflies (Zhioua, unpublished data). Flowers of the cactus are the only sugar source available around. Animals located in the peridomestic areas are the main source of blood meal including humans for sandflies. Dogs are the main source of L. infantum infection to sandflies. We organized door-to-door visits with a local veterinarian and a health worker who introduced the team to the local population. Information regarding age, sex, race was obtained after interviewing dog owners who gave their consent to be involved in the study. After filling out the questionnaire, each dog was examined clinically by the veterinarian and a 2-ml blood sample was collected by venepuncture of the forelimb. This study was performed following approval from the IACUC of Pasteur Institute of Tunis, Tunisia IPT/UESV/19/2010. Dog sera were tested by using a microneutralisation assay performed comparatively with (i) two viruses belonging to the Sandfly fever Naples species (Toscana virus and Punique virus), (ii) and one virus belonging to the Sandfly fever Sicilian species (Sicilian virus) as described previously [5]. Neutralisation is the most dis (...truncated)