**1. Introduction**

West Nile virus (WNV) is flavivirus maintained in nature through an enzootic transmission cycle between *Culex* spp. mosquitoes including *Cx. pipiens*, *Cx. quinquefasciatus*, *Cx. neavei* and birds [1–3]. WN fever outbreaks occur essentially in humans and horses, considered as dead-end hosts [4]. Clinical symptoms range from asymptomatic or flu-like illness to severe neurological and meningoencephalitis syndromes [3]. WNV is one of

**Citation:** Fall, G.; Diallo, D.; Soumaila, H.; Ndiaye, E.H.; Lagare, A.; Sadio, B.D.; Ndione, M.H.D.; Wiley, M.; Dia, M.; Diop, M.; et al. First Detection of the West Nile Virus Koutango Lineage in Sandflies in Niger. *Pathogens* **2021**, *10*, 257. https://doi.org/10.3390/ pathogens10030257

4

5

8

9

**\***

Academic Editor: Andrew Taylor-Robinson

Received: 15 January 2021 Accepted: 19 February 2021 Published: 24 February 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 World Health Organization; Licensee MDPI, Basel, Switzerland. This is an open access article distributed under the terms of the Creative Commons Attribution IGO License (http://creativecommons.org/licenses/ by/3.0/igo/legalcode), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. In any reproduction of this article there should not be any suggestion that WHO or this article endorse any specific organisation or products. The use of the WHO logo is not permitted.

the most widespread flaviviruses worldwide, has caused massive human and animal infections, and some fatal cases, particularly in America and Europe [5–8]. WNV has a grea<sup>t</sup> genetic diversity with at least eight different lineages, and of them four (lineages 1, 2, Koutango and putative new lineage 8) are present in Africa [9]. The WN lineage 1 is distributed worldwide and has been responsible for all major WN outbreaks [6,8,10,11]. The WN lineage 2 was exclusively present in Africa until 2004, when it emerged in Europe and replaced lineage 1 [12]. Migratory birds that overwintered in Africa were the most likely source of introduction of WN lineage 2 into Europe. The putative new lineage was isolated from *Cx. perfuscus* in the south-east of Senegal in 1992 and was never found associated with animals or humans [9]. The Koutango lineage of West Nile virus (WN-KOUTV) was first isolated in 1968 from the wild rodent *Tatera kempi* in Senegal [13] and in 1974 from gerbils in Somalia [14]. WN-KOUTV was initially classified as a distinct virus and later, based on phylogenetic studies, considered a WNV lineage [15,16]. WN-KOUTV is exclusively detected in Africa, and unlike other WNV lineages, it was once isolated from mosquitoes and mainly from ticks and rodents, particularly in Senegal [17]. In humans, serological evidence in Gabon [18] and a report of an accident where a Senegalese laboratory worker was symptomatically infected with WN-KOUTV [19] have been shown. Different symptoms such as two-day fever accompanied by achiness and retrobulbar headache, to erythematous eruption on the flanks, were detected [13,19]. Patients with acute febrile illness ruled out for malaria and Lassa fever in Sierra Leone were found to present neutralizing antibodies to WN-KOUTV [20]. This unpublished study shows that natural human infections with WN-KOUTV are occurring in Africa and suggests that this virus is likely the etiological agen<sup>t</sup> of at least some of the fevers with unknown origin.

In animal models, intra-cerebral inoculation of the virus to new-borne mice causes death on days three to four post-infection [13], and in adult mice, WN-KOUTV showed higher virulence compared to all other WNV lineages [17,21,22]. Currently, the transmission cycle remains unclear, and the roles of ticks and mosquitoes are not ye<sup>t</sup> known. Indeed, vector competence studies showed that *Cx. quinquefasciatus* and *Cx. neavei*, proven vectors for other WNV lineages, were not competent for WN-KOUTV lineage [9]. Another vector competence study showed that *Aedes aegypti* was found to carry the virus and disseminated the infection after a blood meal with high viral dose [23], suggesting that this mosquito species could probably transmit WN-KOUTV lineage to humans. It has also been shown that *Ae. aegypti* was able to vertically transmit the virus [24]. Vector competence of ticks, mostly associated with the virus in the wild, has never been tested.

In the context of Rift Valley fever (RVF) outbreak investigations in Niger, in 2016 [25], mosquitoes and sandflies were collected for arbovirus detection. The laboratory analyses revealed the presence of the WN-KOUTV lineage in a pool of sandflies. Here, we describe this first detection of WN-KOUTV in sandflies, the virus isolation, viral genome sequencing and analyses, and in vivo characterization in mice.
