**2. Results**

### *2.1. Arthropod Species*

A total of 10,977 hematophagous arthropods (158 mosquitoes, 10,816 sandflies and 3 biting midges) were collected and grouped into 181 pools (Table 1). Of these, sandflies were the only arthropod group collected in Intoussane, and the most abundant in Tchintabaraden (*n* = 359; 83.3%) and Tasnala (*n* = 10,428; 99.2%). *Anopheles gambiae* (58.2%) and *Cx perexiguus* (24.7%) were the most abundant among six mosquito species collected during our collection period. Sandflies were collected only by Centers for Disease Control and Prevention (CDC) light traps near herds at Intoussane, and ground pools at Tasnala, while they were found in all biotopes investigated at Tchintabaraden.


**Table 1.** List of arthropods collected in the field from three districts of Niger, 20–24 October 2016.

### *2.2. Virus Detection*

One pool of 100 sandflies collected by CDC light trap near a ground pool at Tasnala was positive for flavivirus by immunofluorescence assay IFA and West Nile virus by real time RT-PCR. The genotyping using WNV primers and probes specific to the different lineages, as well as the genome sequencing, showed the presence of WN-KOUTV in the sample.

The minimum infection rate (MIR) was 0.01 per 1000 in the ground pool where the positive pool was collected.

### *2.3. Sequencing and Evolutionary Analyses*

A genome sequence of 10,948 bp was obtained. The BLAST search showed that the sequence corresponds to WN-KOUTV and the Genbank accession number is MN057643. Phylogenetic analyses showed that the sequence of the strain isolated from sandflies belongs to the same cluster as other WN-KOUTV strains, ArD96655 (accession number KY703855.1) and Dak Ar D 5443 (accession number EU082200.2). This analysis confirmed, therefore, that the virus strain from sandflies in Niger belongs to the WN-KOUTV lineage (Figure 1).

**Figure 1.** Phylogenetic analyses using MEGA software and the maximum likelihood method. Phylogenetic analyses were conducted with sequences of the sandfly strain, other Koutango strains isolated in Senegal from ticks and rodents, and other WNV lineage strains. The accession numbers and names of the strains are mentioned. The accession number of the sandfly strain genomic sequence, PM148 from Niger, is MN057643. WNVL1: lineage 1, WNVL2: lineage 2, WNVL3: lineage 3, WNVL4: lineage 4 and WNV Put L8: putative lineage 8.

Amino acid sequence analyses of the sandfly strain and other WNV strains showed a mean genetic distance around 0.01 within the Koutango lineage (Table 2). The sandfly strain was more closed to the rodent WN-KOUTV strain, with a genetic distance of 0.005. As with other WN-KOUTV strains, the sandfly strain also showed high genetic distances with other WNV lineages, ranging from 11 to 18% (Table 2).

Amino acid sequence alignment of the new sandfly strain and other WNV strains was performed to check for mutations that have been shown to impact WNV virulence (Figure 2). Mutations already described for WN-KOUTV strains [26–28] have been detected in the pre-membrane (S to M at position 72), envelope (Y to F at position 155 of the glycosylation site), and NS5 (F to S at position 653) proteins of the new sandfly WN-KOUTV strain. In addition, the rodent WN-KOUTV strain showed a specific mutation (S to P) at position 156 of the envelope protein glycosylation site. Mutations with unknown consequence (SVA to ASS), specific to all WN-KOUTV strains analyzed here, were also detected at positions 363 to 365 of the envelope protein. The new sandfly WN-KOUTV strain shared a mutation (A to T) with WNVL1 and L2 at position 366 of the envelope protein and showed specific/unique mutations compared to other WN-KOUTV strains at position 38 (M to I) of the NS2A and 177 (V to M) of the NS3 proteins (Figure 2).


**Figure 2.** Genetic diversity of WNV lineages 1, 2 and Koutango. Alignment was conducted with sequences of the sandfly strain, other Koutango strains isolated in Senegal from ticks and rodents, and other WNV lineages 1 and 2 strains. The genomic structure of West Nile virus is shown, and the different genes are labeled. Alignments of motifs with unknown consequence and known virulence motifs are shown. Mutations specific to all Koutango strains are in bold and mutations specific to one Koutango strain are in red.

### *2.4. In Vivo Characterization*

Intra-cerebral inoculation of the new KOUTV strain isolated from sandflies to newborne mice showed 100% mortality at day two post-infection, while ArD96655 showed 100% mortality at day four post-infection. In adult mice, the Koutango sandfly strain showed 100% mortality of mice at days 7 and 10 post-infection with 100 and 1000 pfu, respectively, while ArD96655 showed 100% mortality at day six post-infection with both doses (Figure 3).


**Table 2.** Genetic distance analysis conducted using MEGA software with the Poisson correction model.

In grey, genetic distances within WN-KOUTV lineage. Red rectangle, genetic distances between sandfly strain and with other WNV lineages.

**Figure 3.** Survival curves of 5- to 6-week-old mice following intraperitoneal infection with (**A**) 100, and (**B**) 1000 pfu. Eight mice were tested for each dose. A group of mice with an injection of PBS was used as control. Mice were monitored daily for 21 days.

In both experiments, PBS-inoculated negative control groups showed no signs of disease and stayed alive throughout the experiments.
