Author Contributions
Conceptualization, M.M.D., M.H.D.N., G.M., O.F., C.C., A.R., G.S., and O.F. (Oumar Faye); methodology, M.M.D., M.H.D.N., G.M., A.D., M.D.D., C.C., A.R., G.S., and O.F. (Oumar Faye); software, A.D., M.M.D., G.M., C.C., and C.L.; validation, M.M.D., M.H.D.N., G.M., M.D.D., V.C., M.M., C.L., O.F. (Ousmane Faye), and C.C.; formal analysis, M.M.D., M.H.D.N., G.M., A.D., M.K., N.M.T., and M.M. (Maïmouna Mbanne); investigation, M.M.D., M.H.D.N., G.M., E.h.N., D.D., M.K., N.M.T., M.M. (Maïmouna Mbanne), M.A., B.S., V.D.L., L.T., A.L., I.P., and A.G.; resources, M.M.D., A.A.S., C.L., M.D., O.F. (Ousmane Faye), C.C., G.S., and O.F. (Oumar Faye); data curation, M.M.D., M.H.D.N., G.M., A.D., E.h.N., D.D., N.M.T., L.T., A.L., and I.P.; writing—original draft preparation, M.M.D.; writing—review and editing, M.H.D.N., G.M., A.D., M.D.D., E.h.N., M.D., C.C., A.R., G.S., and O.F. (Oumar Faye); visualization, M.M.D., G.M., M.D.D., V.C., M.M., M.A., B.S., V.D.L., L.T., A.L., I.P., R.R., F.M., C.C., A.R., and G.S.; supervision, M.M.D., M.H.D.N., M.D.D., V.C., C.C., G.S., and O.F. (Oumar Faye); project administration, A.R., G.S., and O.F. (Oumar Faye); funding acquisition, M.M.D., A.A.S., O.F. (Ousmane Faye), A.R., G.S., and O.F. (Oumar Faye). All authors have read and agreed to the published version of the manuscript.
Table 1.
Sequences of the West Nile virus primers sets A and B.
Table 1.
Sequences of the West Nile virus primers sets A and B.
WNV Primers Set A | WNV Primers Set B |
---|
WNV_1_LEFT | GCCTGTGTGAGCTGACAAACTT | WNV-L2_1_LEFT | GCCTGTGTGAGCTGACAAACTT |
WNV_1_RIGHT | TTCTTTTGTTTTGAGCTCCKCC | WNV-L2_1_RIGHT | TTCTTTTGTTTTGTGCTCCGGC |
WNV_2_LEFT | ACAGCGATGAAACACCTTCTGA | WNV-L2_2_LEFT | ACAGCGATGAAGCATCTCTTGA |
WNV_2_RIGHT | CGTGTCTTGGTGCATCTTCCAT | WNV-L2_2_RIGHT | GBCGDGTYTTDGTGCATCTYCC |
KOUV_2_RIGHT | TTYCCTCTGATGCATCTTCCAT | WNV-L2_3_LEFT | GTSYTRGCTGCTGGAAATGAYC |
WNV_3_LEFT | CCRGTACTGTCGGCTGGTAATG | WNV-L2_3_RIGHT | CMACCCATGTAGCTCCAGAYAC |
WNV_3_RIGHT | CVAGAACCAAATCCACCCAWGT | WNV-L2_4_LEFT | ATNCTATTGCTCCTGGTRGCA |
WNV_4_LEFT | ACAGCTTCAACTGCCTTGGAAT | WNV-L2_4_RIGHT | TCCADCCAGTTGCTTTGGTKGW |
WNV_4_RIGHT | TGRTTCTTCCTATTGCCTTGGT | WNV-L2_5_LEFT | GTRGACAGRGGATGGGGAAAYG |
WNV_5_LEFT | GGYTGCGGACTATTTGGMAAA | WNV-L2_5_RIGHT | GRTTCCTCCAHGYGGTGCTT |
WNV_5_RIGHT | CTCCACACAGTACTTCCAGCAC | WNV-L2_6_LEFT | CCTTCCTGGTYCACCGAGARTG |
WNV_6_LEFT | GGHACAAAGACGTTCTTGGTYC | WNV-L2_6_RIGHT | KGAVGAAATGGGCACYTTRCAR |
WNV_6_RIGHT | GMACTTTGCAAGGTCCATCYGT | WNV-L2_7_LEFT | GCDTTYAAATTCGCYGGGACTC |
WNV_7_LEFT | AYGCTTTCAAGTTTCTTGGGACT | WNV-L2_7_RIGHT | GTGTATRGCTTTCCCYACYGAG |
KOUV_7_LEFT | AYGCTTTCAAGTTTCTTGGGCAT | WNV-L2_8_LEFT | ACTCAGAGGAGCTCAACGACTC |
WNV_7_RIGHT | AASACTTGATGGACAGCCTTCC | WNV-L2_8_RIGHT | ATTYTTGCTAGGCCTTGTGGHG |
WNV_8_LEFT | ARMGACTAGCCGCTCTAGGAGA | WNV-L2_9_LEFT | CGGTGYGGAAGTGGAGTGTTYA |
WNV_8_RIGHT | TGDGCTTTCTGAATGATCTTGGCT | WNV-L2_9_RIGHT | TRYTRTTCCATGCTCGGTTSR |
WNV_9_LEFT | AAYGATGTGGAGGCTTGGATGG | WNV-L2_10_LEFT | YGCDCCAGARCTAGCTAACAAYA |
WNV_9_RIGHT | AWRCTATTCCAAGCGCGATTSK | WNV-L2_10_RIGHT | CCCTGGYCTCCTGTTRTGRTTGC |
WNV_10_LEFT | TMTTTGCACCAGAACTCGCYAA | WNV-L2_11_LEFT | CACHYTGTGGGGTGATGGAGTT |
WNV_10_RIGHT | CWGGTCTCCGATTGTGATTGCT | WNV-L2_11_RIGHT | CAGAAGGCCCAACTGAAAAGGA |
WNV_11_LEFT | AYACCTTGTGGGGCGATGGART | WNV-L2_12_LEFT | GAYGAAAAGACCCTCGTGCA |
WNV_11_RIGHT | GGCCCAACTGAAAAGGGTCAAT | WNV-L2_12_RIGHT | CCATTTGRAAGAAAGCAGCTGCR |
WNV_12_LEFT | TGGARATCAGACCACAGAGRCA | WNV-L2_13_LEFT | CAGTCTTTCTGGTGGCTTCBTT |
WNV_12_RIGHT | TTGRAAGAAAACAGCCGCCARC | WNV-L2_13_RIGHT | YCCAGCKGCAAGTATCATBGGA |
WNV_13_LEFT | CCAGTGTTTATGGTGGCWTCGT | WNV-L2_14_LEFT | MRGTTGGAAGCYTCATCAARGARA |
WNV_13_RIGHT | CHGCAAGGATCATGGGGTTGAA | WNV-L2_14_RIGHT | TGAAAATTTCCATCRTCATCCARCC |
WNV_14_LEFT | GVAGCTTGATCAGGGAGAARAG | WNV-L2_15_LEFT | GGACRGCTGAYATYACYTGGGA |
WNV_14_RIGHT | TCCBGGATCATTCATGARCTGG | WNV-L2_15_RIGHT | RCTCATGAGAGCRGCTCCCTTR |
WNV_15_LEFT | CKGACATTTCCTGGGAAAGTGA | WNV-L2_16_LEFT | ATHATGACTCGAGGTCTGCTYG |
WNV_15_RIGHT | TCATCAAAGCGGCTCCTTTWGT | WNV-L2_16_RIGHT | MRCCATTDGGCATGATGACKCC |
WNV_16_LEFT | CTYGGCAGTTATCAAGCAGGAG | WNV-L2_17_LEFT | RGACTATCCCACYGGAACRTCA |
WNV_16_RIGHT | WATBGCGCTTATGTATGAHCCR | WNV-L2_17_RIGHT | TGGCACATGACATCAACGATYT |
WNV_17_LEFT | CMATAGTGGACAAAAACGGTGATGT | WNV-L2_18_LEFT | TKAGAGGACTTCCCATYCGGTA |
WNV_17_RIGHT | DGTGAGGGTAGCATGACACATG | WNV-L2_18_RIGHT | TTBCCCATTTTCACACTTGGAACR |
WNV_18_LEFT | ATGKCTGAAGCACTGAGRGGA | WNV-L2_19_LEFT | ACMGAGCCTGGAACACTGGVTA |
WNV_18_RIGHT | CCCATCTTGACACTAGGCACAA | WNV-L2_19_RIGHT | CCTCCATAGCAATACTCATCACCA |
WNV_19_LEFT | CGRGCTTGGAACTCTGGATAYG | WNV-L2_20_LEFT | TGATGGAAGAGTCATCCTGGGV |
WNV_19_RIGHT | TACTCATCACCAACTTGCGAYG | WNV-L2_20_RIGHT | GTGTTGGTTCGAGGTCCATCAA |
WNV_20_LEFT | GGAGAACCATCTGCAGTGACAG | WNV-L2_21_LEFT | CWGTCTGGCTCGCTTACAAAGT |
WNV_20_RIGHT | CMACTTCGTTGTTGTCTTCTAAAATTG | WNV-L2_21_RIGHT | ARGCTATTGTCTGAAGGGCRTC |
WNV_21_LEFT | CYTACAAGGTTGCAGCRGCT | WNV-L2_22_LEFT | TTKGACACGATGTATGTGGTKG |
WNV_21_RIGHT | ACTCCCATGGTCATCACACWCA | WNV-L2_22_RIGHT | CAYTCTTGGTCTTGTCCAGCCA |
WNV_22_LEFT | GAGGMAGAGCTCACAGAATGGC | WNV-L2_23_LEFT | YCAGCTYGCCGTGTTTTTGATY |
WNV_22_RIGHT | CCTTGACCTCAATTCTTTGCCC | WNV-L2_23_RIGHT | CYGCAGTCACAGTCACAGTCAG |
WNV_23_LEFT | TTGTGTCATGACCCTTGTSAGC | WNV-L2_24_LEFT | YTTTGTVGACGTTGGTGTGTCA |
WNV_23_RIGHT | GGDACCATGTAGGCATAGTGGC | WNV-L2_24_RIGHT | TYGTTGCRTTCCACACTGARCT |
WNV_24_LEFT | TTYGTCGATGTTGGAGTKTCA | WNV-L2_25_LEFT | ARRACTGTCAGAGAGGCYGGAA |
WNV_24_RIGHT | GTYGTTGCGTTCCAMACWGAGC | WNV-L2_25_RIGHT | GCCTTTCCACTAACCACCGYAR |
WNV_25_LEFT | RGACHGTVCGAGAAGCYGGAAT | WNV-L2_26_LEFT | RHGCCAGGAGAGAGGGAAAYRT |
WNV_25_RIGHT | GRTCGAGGAAACBCCGTTCGAC | WNV-L2_26_RIGHT | CCARTCTTCCACCATCTCCARR |
WNV_26_LEFT | RAGAAGGCAAYRTCACYGGAGG | WNV-L2_27_LEFT | CACACTGCTCTGTGACATTGGA |
WNV_26_RIGHT | ARAATTCCCTTGGCCCTCGG | WNV-L2_27_RIGHT | AGAATTGAGGAGAGGCTTCCCY |
WNV_27_LEFT | TCAAGTGCTGAGGTTGAAGAGC | WNV-L2_28_LEFT | AAGAAAACVTGGAAGGGACCYC |
WNV_27_RIGHT | GCCTGAGTCGTTCAATCCTGTT | WNV-L2_28_RIGHT | TBGTGGTCTCATTGAGGACGTR |
WNV_28_LEFT | TGTAAACTTGGGAAGTGGAACCA | WNV-L2_29_LEFT | CTCCTTTCGGHCAACAACGRGT |
WNV_28_RIGHT | TTTWTCKCTGGCCAYAAAVGCC | WNV-L2_29_RIGHT | CYCCBAGCCACATGAACCADAT |
WNV_29_LEFT | GTGGAYACGAAAGCTCCTGARC | WNV-L2_30_LEFT | ACYTGCATCTACAACATGATGGG |
WNV_29_RIGHT | ATTGAGAAAACCSAGAGCTTCG | WNV-L2_30_RIGHT | GGBCTCATCACTTTCACGACYT |
WNV_30_LEFT | MAARGCCAARGGMAGCAGAG | WNV-L2_31_LEFT | CDAAGGTBCTTGARCTGCTDGR |
WNV_30_RIGHT | CCYCTCTGATCTTCTCTGGAGA | WNV-L2_31_RIGHT | GGACCTTTGACATGGCATTBAGR |
WNV_31_LEFT | TGAGCTCACCTATCGWCACAAA | WNV-L2_32_LEFT | GGHGATGACTGYGTGGTDAA |
WNV_31_RIGHT | CAYCCAGTTGACGGTTTCCACT | WNV-L2_32_RIGHT | GRACCCAGTTVACAGGCACA |
WNV_32_LEFT | TGGTRAAGCCCCTGGAYGAY | WNV-L2_33_LEFT | GCAGATGTGGCTGYTGCTTTAT |
WNV_32_RIGHT | TCTCCTCCTGCATGGATSGA | WNV-L2_33_RIGHT | YRTCTTCATACCTCCTCARDGA |
WNV_33_LEFT | AGWAGAGACCTGMGGYTCAT | WNV-L2_34_LEFT | GCGCHACTTGGGCTGAAAAYAT |
WNV_33_RIGHT | TCTACAAAACTGTGTCCTCAACCA | WNV-L2_34_RIGHT | MYCTTCCGAGACGGTTCTGA |
WNV_34_LEFT | AGTCAGWKCAATCATCGGRGAWG | WNV-L2_35_LEFT | GGAAGTTGAGTAGACGGTGCTG |
WNV_34_RIGHT | CACTATCGCAGACTGCACTCTC | WNV-L2_35_RIGHT | TCCCAGGTGTCAATATGCTGTT |
WNV_35_LEFT | CAGGAGGACTGGGTGAACAAAG | | |
WNV_35_RIGHT | TGGTTGTGCAGAGCAGAAGATC | | |
Table 2.
Inclusivity test of the West Nile virus set A primers.
Table 2.
Inclusivity test of the West Nile virus set A primers.
RT-PCR Ct Value | Total Number of Trimmate Reads | Number of WNV Reads | % HCoverage | VCoverage | Consensus Sequence Length |
---|
Viral strain WNV L1 Italy | 14 | 2369.723 | 649.022 | 99% | 5802.67 | 10.969 |
14 | 1922.532 | 581.215 | 99% | 5346.85 | 10.966 |
13 | 2715.830 | 754.081 | 99% | 6244.36 | 10.966 |
No. of replicates with coverage ≥ 95% | 3/3 (100%) | |
Viral strain WNV L1 Senegal | 25 | 623.535 | 238.259 | 99% | 7997 | 10.961 |
28 | 5034.151 | 1035.983 | 99% | 6862.12 | 10.965 |
16 | 5034.151 | 547.775 | 99% | 5183.83 | 10.966 |
19 | 810.906 | 327.395 | 100% | 3971.4 | 11.018 |
17 | 924.142 | 363.236 | 99% | 4412.7 | 10.963 |
17 | 899.818 | 342.133 | 99% | 3851.34 | 10.966 |
17 | 819.552 | 358.631 | 99% | 4306.94 | 10.961 |
No. of replicates with coverage ≥ 95% | 7/7 (100%) | |
Viral strain WNV L2 Italy | 18 | 2607.185 | 546.249 | 100% | 3607.99 | 10.926 |
18.71 | 2466.682 | 511.381 | 100% | 3488.1 | 10.926 |
18.13 | 2061.961 | 465.844 | 100% | 3445.55 | 10.926 |
No. of replicates with coverage ≥ 95% | 3/3 (100%) | |
Viral strain WNV L2 Senegal | 14 | 4861.644 | 706.114 | 100% | 3519.51 | 10.914 |
14 | 4087.737 | 835.098 | 100% | 5593.58 | 10.914 |
14 | 4750.250 | 885.595 | 100% | 5609.34 | 10.914 |
No. of replicates with coverage ≥ 95% | 3/3 (100%) | |
Table 3.
Sensitivity test of the West Nile virus set A primers.
Table 3.
Sensitivity test of the West Nile virus set A primers.
Viral Strain | Quantity Value (cp/μL) | Quantity Mean Value (Ct) | Total Number of Trimmate Reads | Number of WNV Reads | % HCoverage | VCoverage | Consensus Sequence Length |
---|
WNV L1 (reference used for the mapping on Genpat: WNV L1 FJ483548 | 106 | 18.68 | 1457.278 | 503.152 | 99% | 5043.56 | 10.959 |
106 | 1371.693 | 476.605 | 99% | 4997.04 | 10.964 |
106 | 938.406 | 375.653 | 99% | 4372.01 | 10.959 |
No. of replicates with coverage ≥ 95% | 3/3 (100%) | | |
105 | 23.45 | 1342.407 | 426.914 | 99% | 4587.33 | 10.960 |
105 | 1174.155 | 397.215 | 99% | 4435.79 | 10.959 |
105 | 825.135 | 315.219 | 99% | 3776.67 | 10.956 |
No. of replicates with coverage ≥ 95% | 3/3 (100%) | | |
104 | 27 | 906.690 | 292.718 | 96% | 3456.26 | 10.964 |
104 | 984.952 | 297.351 | 97% | 3414.38 | 10.959 |
104 | 1247.127 | 327.095 | 96% | 3570.22 | 10.959 |
No. of replicates with coverage ≥ 95% | 3/3 (100%) | | |
103 | 30 | 1096.401 | 249.798 | 94% | 2735.41 | 10.962 |
103 | 506.380 | 153.284 | 93% | 1853.42 | 10.955 |
103 | 569.039 | 170.272 | 91% | 2099.54 | 10.955 |
No. of replicates with coverage ≥ 95% | 0/3 (0%) | | |
102 | 33 | 57.229 | 20.115 | 82% | 295.76 | 10.366 |
102 | 53.435 | 18.515 | 81% | 281.027 | 10.958 |
102 | 41426 | 15.149 | 80% | 227.617 | 10.951 |
No. of replicates with coverage ≥ 95% | 0/3 (0%) | | |
WNV L2 (reference used for the mapping: WNV L2 MN652880) | 106 | 18.68 | 1262.167 | 12.484 | 35% | 391.547 | 10.028 |
106 | 1315.568 | 11.576 | 33% | 383.276 | 10.928 |
106 | 1103.838 | 16.652 | 26% | 706.146 | 10.928 |
No. of replicates with coverage ≥ 95% | 0/3 (0%) | | |
105 | 22.87 | 1210.495 | 11.251 | 36% | 347.197 | 10.928 |
105 | 823.025 | 10.243 | 33% | 345.101 | 10.928 |
105 | 1478.160 | 12.253 | 34% | 394.675 | 10.928 |
No. of replicates with coverage ≥ 95% | 0/3 (0%) | | |
104 | 26.41 | 1228.945 | 9.715 | 32% | 337.862 | 10.928 |
104 | 1090.849 | 9.547 | 32% | 327.57 | 10.928 |
104 | 947.964 | 4.405 | 30% | 159.122 | 10.928 |
No. of replicates with coverage ≥ 95% | 0/3 (0%) | | |
103 | 30 | 442.063 | 2.796 | 26% | 121.744 | 10.928 |
103 | 577.277 | 3.500 | 27% | 143.439 | 10.926 |
103 | 369.041 | 1.615 | 22% | 79.118 | 10.245 |
No. of replicates with coverage ≥ 95% | 0/3 (0%) | | |
102 | 33.14 | 35.426 | 1.362 | 17% | 789.157 | 10.924 |
102 | 68.307 | 627 | 17% | 360.495 | 10.926 |
102 | 63.643 | 731 | 18% | 571.297 | 10.926 |
No. of replicates with coverage ≥ 95% | 0/3 (0%) | | |
Table 4.
Test of the West Nile virus set A primers with WNV-L1 homogenates. (* Samples with multiple viral species/WNV lineage).
Table 4.
Test of the West Nile virus set A primers with WNV-L1 homogenates. (* Samples with multiple viral species/WNV lineage).
Viral Homogenate WNV L1—Sample Number | Host | RT-PCR Ct Value | Co-InfectionCt Value | # Total Trimmate Reads | # WNV L1 Reads | % HCoverage | VCoverage | Consensus Sequence Length |
---|
1 | Accipiter gentilis | 15 | - | 2,037,215 | 591,954 | 100% | 6362.04 | 11,027 |
2 | Pica pica | 16 | - | 14,648,025 | 1,333,187 | 100% | 6381.67 | 11,016 |
3 | Corvus cornix | 16 | - | 6,447,209 | 657,948 | 99% | 5135.45 | 10,966 |
4 | Pica pica | 18 | - | 2,923,237 | 387,986 | 99% | 4264.56 | 10,966 |
5 | Phalacrocorax carbo | 19 | - | 11,441,518 | 1,107,291 | 99% | 5888.49 | 10,961 |
6 | Corvus cornix | 19 | - | 2,347,380 | 351,292 | 99% | 4177.69 | 10,963 |
7 | Culex pipiens | 20 | - | 3,495,685 | 744,562 | 99% | 5560.68 | 10,968 |
8 | Culex pipiens | 22 | - | 7,571,826 | 597,499 | 98% | 3973.2 | 10,967 |
9 | Corvus cornix | 22 | - | 5,382,363 | 629,364 | 99% | 4806.11 | 10,960 |
10 | Passer domesticus | 22 | - | 3,942,375 | 304,215 | 97% | 3084.4 | 10,962 |
11 | Culex pipiens | 23 | - | 4,560,711 | 283,771 | 93% | 2899.47 | 10,960 |
12 | Corvus Cornix | 24 | - | 3,052,530 | 342,271 | 93% | 3508.25 | 10,966 |
13 * | Culex pipiens | 25 | L2 Ct 28 | 3,576,787 | 403,166 | 94% | 3761.48 | 10,966 |
14 * | Culex pipiens | 25 | L2 Ct 28 | 1,557,373 | 263,149 | 90% | 2471.3 | 10,954 |
15 | Larus michahellis | 25 | - | 1,813,567 | 129,834 | 88% | 1805.95 | 10,952 |
16 | Streptopelia decaocto | 26 | - | 1,439,897 | 203,172 | 91% | 2592.09 | 10,961 |
17 | Pica pica | 26 | - | 7,677,172 | 251,982 | 81% | 2920.03 | 10,963 |
18 | Parus major | 26 | - | 755,951 | 38,780 | 69% | 747.61 | 9389 |
19 * | Culex pipiens | 27 | L2 Ct 32 | 3,371,541 | 369,610 | 96% | 3287.31 | 10,956 |
20 | Turdus merula | 27 | - | 2,710,572 | 66,229 | 82% | 1039.08 | 10,954 |
21 | Culex pipiens | 28 | - | 6,865,408 | 269,727 | 94% | 2618.84 | 10,962 |
22 * | Culex pipiens | 28 | L2 Ct 31 | 3,378,716 | 190,102 | 87% | 2201.29 | 10,966 |
23 | Streptopelia decaocto | 28 | - | 1,842,010 | 22,006 | 68% | 430.494 | 10,946 |
24 | Equus caballus | 28 | - | 1,805,957 | 159,703 | 92% | 2101.31 | 10,960 |
25 | Athene noctua | 29 | - | 3,394,989 | 15,767 | 34% | 404 | 10,802 |
26 | Columba palumbus | 31 | - | 835,362 | 9948 | 57% | 211.706 | 10,960 |
27 * | Culex pipiens | 33 | L2 Ct 25 | 4,662,340 | 220,508 | 91% | 2381.21 | 10,963 |
28 * (Alphavirus, Mesonivirus) | Culex neavei | 28.5 | - | 391,494 | 1,135,180 | 92.42% | 2514.3 | 10,194 |
29 * (Barkedji, Mesonivirus) | Culex poicilipes | 35.59 | - | 64,716 | 616,500 | 82.35% | 1789.36 | 9083 |
30 * (Barkedji) | Culex neavei | 29.52 | - | 302,175 | 1,394,466 | 89% | 2001.3 | 9819 |
31 * (Alphavirus, Barkedji, Usutu) | Culex neavei | 25.03 | - | 180,791 | 484,221 | 73.89% | 822.02 | 8150 |
Table 5.
Test of the West Nile virus set A primers with WNV-L2 homogenates. (* Samples with multiple viral species/WNV lineage).
Table 5.
Test of the West Nile virus set A primers with WNV-L2 homogenates. (* Samples with multiple viral species/WNV lineage).
Viral Homogenate WNV L2—Sample Number | Host | RT-PCR Ct Value | Co-Infection Ct Value | # Total Trimmate Reads | # WNV Reads | % HCoverage | VCoverage | Consensus Sequence Length |
---|
1 | Accipiter gentilis | 16 | - | 3336.278 | 450.617 | 100% | 3304.6 | 10.926 |
2 | Accipiter gentilis | 16 | - | 1773.333 | 393.125 | 99% | 2982.38 | 10.926 |
3 | Accipiter gentilis | 19 | - | 2753.185 | 225.096 | 72% | 1533.63 | 10.926 |
4 | Garrulus glandarius | 20 | - | 3540.566 | 304.094 | 96% | 1588.44 | 10.923 |
5 | Culex pipiens | 22 | - | 1092.527 | 517.44 | 58% | 779.912 | 10.921 |
6 | Culex pipiens | 23 | - | 568.562 | 54.658 | 59% | 905.155 | 10.834 |
7 | Corvus cornix | 23 | - | 1383.831 | 85.880 | 62% | 1168.95 | 10.922 |
8 | Passer italiae | 24 | - | 476.120 | 45.644 | 47% | 1089.79 | 10.923 |
9 | Columba palumbus | 26 | - | 1313.900 | 76.773 | 64% | 908.822 | 10.923 |
10 | Columba palumbus | 27 | - | 5.377 | 7.319 | 25% | 257.256 | 10.868 |
11 | Columba palumbus | 28 | - | 545.944 | 5073 | 23% | 281.009 | 8.425 |
12 | Turdus merula | 31 | - | 514.655 | 72 | 4% | 26.336 | 375 |
13 | Pica pica | 31 | - | 330.374 | 280 | 5% | 685.197 | 4.128 |
14 | Phasianus colchicus | 32 | - | 243.656 | 242 | 5% | 543.463 | 7.510 |
15 | Pica pica | 33 | - | 341.153 | 613 | 9% | 760.203 | 8.413 |
16 | Pica pica | 33 | - | 376.185 | 128 | 4% | 307.715 | 3.779 |
17 | Egretta garzetta | 34 | - | 616.781 | 395 | 6% | 715.396 | 7.923 |
18 | Culex pipiens | 29 | - | 1537.448 | 24.517 | 33% | 676.501 | 9.393 |
19 | Culex pipiens | 28 | - | 588.313 | 32.437 | 49% | 749.224 | 10.924 |
20 | Culex pipiens | 27 | - | 606.886 | 28.181 | 52% | 494.551 | 10.923 |
21 | Culex pipiens | 25 | - | 884.756 | 43.657 | 52% | 864.517 | 10.891 |
22 | Culex pipiens | 30 | - | 259.173 | 2.572 | 3% | 989.276 | 381 |
23 | Culex pipiens | 28 | - | 436.461 | 14.473 | 29% | 630.097 | 10.923 |
24 | Culex pipiens | 24 | - | 2172.952 | 45.568 | 45% | 716.974 | 10.923 |
25 * | Culex pipiens | 31 | L1 Ct 28 | 3378.716 | 9.132 | 27% | 447.986 | 10.928 |
26 | Culex pipiens | 25 | - | 339.617 | 33.681 | 42% | 827.229 | 10.790 |
27 | Culex pipiens | 21 | - | 1223.422 | 35.451 | 23% | 1197.74 | 10.921 |
28 | Culex pipiens | 23 | - | 1832.341 | 38.134 | 15% | 1569.34 | 10.555 |
29 | Culex pipiens | 25 | - | 686.602 | 25.509 | 18% | 1178.55 | 10.609 |
30 | Culex pipiens | 27 | - | 502.162 | 8.253 | 3% | 3113.1 | 388 |
31 | Corvus cornix | 28 | - | 1357.321 | 7.159 | 31% | 312.307 | 10.475 |
32 | Pica pica | 20 | - | 6417.489 | 198.613 | 94% | 1616.11 | 10.927 |
33 * | Culex pipiens | 25 | L1 Ct 33 | 4662.340 | 33.608 | 60% | 534.955 | 10.926 |
34 * | Culex pipiens | 24 | USUV Ct 27 | 5133.183 | 77.022 | 61% | 888.532 | 10.925 |
35 | Culex pipiens | 24 | - | 153.601 | 10 | 47% | 275.337 | 556 |
36 | Corvus cornix | 29 | - | 877.218 | 0 | 0% | 0 | 0 |
37 | Culex pipiens | 23 | - | 199.550 | 99.490 | 88% | 1126.14 | 10.890 |
38 | Culex pipiens | 24 | - | 323.115 | 5.048 | 23% | 308.136 | 4.395 |
39 * | Culex pipiens | 27 | L1 Ct 25 | 3576787 | 9.461 | 51% | 239.312 | 10.928 |
40 | Culex pipiens | 22 | - | 78.378 | 42.637 | 63% | 824.461 | 10.922 |
41 | Larus marinus | 23 | - | 546.215 | 142.178 | 81% | 1460.97 | 10.923 |
42 * | Culex pipiens | 27 | L1 Ct 32 | 3371.541 | 5.603 | 47% | 154.32 | 10.928 |
43 | Culex pipiens | 29 | - | 153.413 | 4.367 | 31% | 187.09 | 9.364 |
44 | Culex pipiens | 28 | - | 66.202 | 7.786 | 36% | 290.742 | 10.018 |
45 * | Culex pipiens | 28 | L1 Ct 25 | 1557.373 | 8.945 | 48% | 243.941 | 10.926 |
46 | Culex pipiens | 28 | - | 118.396 | 2.420 | 15% | 219.733 | 7.809 |
47 * | Culex pipiens | 27 | USUV Ct 27 | 1516.324 | 230 | 44% | 985.194 | 7.469 |
48 | Corvus cornix | 28 | - | 470.113 | 6.194 | 40% | 207.827 | 10.923 |
49 * | Culex pipiens | 23 | USUV Ct 21 | 4272.994 | 248 | 29% | 941.936 | 10.806 |
50 | Culex pipiens | 15 | - | 327.238 | 88.896 | 61% | 1398.22 | 10.922 |
51 | Ochlerotatus caspius | 25 | - | 361.194 | 16.436 | 32% | 684.576 | 10.844 |
52 | Culex pipiens | 24 | - | 401.408 | 25.091 | 42% | 804.597 | 9.592 |
53 | Pica pica | 23 | - | 899.597 | 2.771 | 24% | 150.215 | 8.402 |
54 * | Culex pipiens | 29 | USUV Ct 26 | 117.768 | 18.204 | 54% | 451.546 | 10.907 |
Table 6.
Inclusivity test of the West Nile virus set B primers.
Table 6.
Inclusivity test of the West Nile virus set B primers.
RT-PCR Ct Value | # Total Trimmate Reads | # WNV Reads | % HCoverage | VCoverage | Consensus Sequence Length |
---|
Viral strain WNV L2 Italy | 15 | 1218.086 | 284.232 | 100% | 3820.77 | 10.926 |
15 | 1792.478 | 402.082 | 100% | 5234.36 | 10.926 |
15 | 1440.061 | 338.706 | 100% | 4543.81 | 10.926 |
17 | 1711.005 | 328.182 | 100% | 4374.41 | 10.926 |
18 | 941.641 | 224.716 | 100% | 3023.77 | 10.926 |
N of replicates with Coverage ≥ 95% | 5/5 (100%) | |
Table 7.
Sensitivity test of the West Nile virus set B primers.
Table 7.
Sensitivity test of the West Nile virus set B primers.
Viral Strain | Quantity Value (cp/5 μL) | Quantity Mean Value (Ct) | # Total Trimmate Reads | # WNV Reads | % HCoverage | VCoverage | Consensus Sequence Length |
---|
WNV L2 (reference used for the mapping: WNV L2 MN652880) | 106 | 19 | 1808.185 | 381.930 | 100% | 5038.73 | 10.913 |
106 | 4928.502 | 673.900 | 100% | 6845.55 | 10.926 |
106 | 3099.180 | 511.446 | 100% | 6107.38 | 10.913 |
No. of replicates with coverage ≥ 95% | 3/3 (100%) | | |
105 | 22 | 2665.260 | 409.989 | 100% | 5157.74 | 10.914 |
105 | 1049.020 | 237.471 | 100% | 3194.36 | 10.926 |
105 | 2820.387 | 429.662 | 100% | 5335.99 | 10.912 |
No. of replicates with coverage ≥ 95% | 3/3 (100%) | | |
104 | 26 | 1651.945 | 261.024 | 100% | 3450.12 | 10.913 |
104 | 2483.786 | 337.982 | 100% | 4234.91 | 10.914 |
104 | 2681.807 | 356.233 | 100% | 4334.71 | 10.926 |
No. of replicates with coverage ≥ 95% | 3/3 (100%) | | |
103 | 30 | 1570.036 | 236.060 | 100% | 3029.53 | 10.894 |
103 | 1153.288 | 196.257 | 100% | 2614.91 | 10.904 |
103 | 782.424 | 285.136 | 99% | 3070.47 | 10.912 |
No. of replicates with coverage ≥ 95% | 3/3 (100%) | | |
102 | 33 | 1764.307 | 159.591 | 95% | 2189.31 | 10.597 |
102 | 2082.360 | 177.212 | 93% | 2389.54 | 10.800 |
102 | NA | NA | NA | NA | NA |
No. of replicates with coverage ≥ 95% | 1/2 (50%) | | |
Table 8.
Test of the West Nile virus set B primers with WNV-L2 homogenates.
Table 8.
Test of the West Nile virus set B primers with WNV-L2 homogenates.
Viral Homogenate WNV L2—Sample Number | RT-PCR Ct Value | Host | Total Number of Trimmate Reads | Number of WNV Reads | % HCoverage | VCoverage | Consensus Sequence Length |
---|
1 | 17 | Pica pica | 1134.961 | 295.649 | 100% | 3977.58 | 10.892 |
2 | 27 | Corvus cornix | 1448.962 | 284.947 | 100% | 3818.15 | 10.912 |
3 | 21 | Culex pipiens | 1733.553 | 385.088 | 100% | 5042.35 | 10.913 |
4 | 23 | Culex pipiens | 1528.111 | 339.207 | 100% | 4525.72 | 10.912 |
5 | 21 | Athene noctua | 1801.390 | 376.213 | 100% | 4946.53 | 10.878 |
6 | 22 | Culex pipiens | 1407.784 | 312.376 | 99% | 4201.89 | 10.879 |
7 | 19 | Passer domesticus | 1515.878 | 205.605 | 100% | 2770.45 | 10.914 |
8 | 30 | Corvus cornix | 1470.367 | 209.316 | 98% | 2799.57 | 10.872 |
9 | 30 | Pica pica | 2150.396 | 205.466 | 99% | 2662.61 | 10.868 |
10 | 27 | Sylvia atricapilla | 3649.208 | 281.102 | 99% | 3426.07 | 10.880 |
11 | 25 | Culex pipiens | 2094.249 | 349.710 | 100% | 4076.35 | 10.878 |
12 | 29 | Anopheles maculipennis | 3435.959 | 563176 | 100% | 5709.14 | 10.912 |
13 | 25 | Culex pipiens | 3120.601 | 281.102 | 100% | 4601.81 | 10.912 |
14 | 27 | Culex pipiens | 2193.040 | 259.483 | 97% | 3231.74 | 10.904 |
15 | 35 | Equus ferus caballus | 1623.442 | 133.088 | 93% | 1814.74 | 10.936 |
Table 9.
Test of the West Nile virus set A + B primers with WNV-L1 and WNV-L2 homogenates.
Table 9.
Test of the West Nile virus set A + B primers with WNV-L1 and WNV-L2 homogenates.
Viral Homogenate WNV L1 | RT-PCR Ct Value | Host | Used Primers | Total Number of Trimmate Reads | Number of WNV Reads | % HCoverage | VCoverage | Consensus Sequence Length |
---|
1 | L1 19 | Corvus cornix | Set A | 2347.380 | 351.292 | 99% | 4177.69 | 10.963 |
Set A + B | 1118.615 | 287.672 | 99% | 3987.12 | 10.963 |
2 | L1 25 | Larus michahellis | Set A | 1813.567 | 129.834 | 88% | 1805.95 | 10.952 |
Set A + B | 651.723 | 91.642 | 93% | 1305.83 | 10.960 |
3 | L1 18 | Pica pica | Set A | 2923.237 | 387.986 | 99% | 4264.56 | 10.966 |
Set A + B | 4965.722 | 512.725 | 99% | 4562.28 | 10.966 |
4 | L1 28 | Equus ferus caballus | Set A | 1805.957 | 159.703 | 92% | 2101.31 | 10.960 |
Set A + B | 2319.560 | 165.376 | 92% | 2200.39 | 10.960 |
Viral Homogenate WNV L2 | RT-PCR Ct Value | Host | Used Primers | Total Number of Trimmate Reads | Number of WNV Reads | % HCoverage | VCoverage | Consensus Sequence Length |
1 | L2 17 | Pica pica | Set B | 2059.659 | 351.670 | 100% | 4232.78 | 10.892 |
Set A + B | 1134.961 | 295.649 | 100% | 3977.58 | 10.892 |
2 | L2 27 | Corvus cornix | Set B | 1793.046 | 187.826 | 100% | 2156.81 | 10.926 |
Set A + B | 1448.962 | 284.947 | 100% | 3818.15 | 10.912 |
3 | L2 21 | Culex pipiens | Set B | 2219.785 | 312.851 | 100% | 3515.05 | 10.926 |
Set A + B | 1733.553 | 385.088 | 100% | 5042.35 | 10.913 |
4 | L2 23 | Culex pipiens | Set B | 2045.957 | 275.354 | 100% | 2943.77 | 10.926 |
Set A + B | 1528.111 | 339.207 | 100% | 4525.72 | 10.912 |
5 | L2 21 | Athene noctua | Set B | 3413.467 | 359.916 | 100% | 4106.39 | 10.892 |
Set A + B | 1801.390 | 376.213 | 100% | 4946.53 | 10.878 |
6 | L2 22 | Culex pipiens | Set B | 3109.597 | 239.795 | 95% | 2761.23 | 10.892 |
Set A + B | 1407.784 | 312.376 | 99% | 4201.89 | 10.879 |
7 | L2 19 | Passer domesticus | Set B | 1621.442 | 123.813 | 89% | 1836.85 | 10.924 |
Set A + B | 1515.878 | 205.605 | 100% | 2770.45 | 10.914 |