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Article

Novel Amplicon-Based Sequencing Approach to West Nile Virus

by
Moussa Moïse Diagne
1,*,†,
Marie Henriette Dior Ndione
1,†,
Giulia Mencattelli
2,3,4,†,
Amadou Diallo
5,
El hadji Ndiaye
6,
Marco Di Domenico
2,
Diawo Diallo
6,
Mouhamed Kane
1,
Valentina Curini
2,
Ndeye Marieme Top
5,
Maurilia Marcacci
2,
Maïmouna Mbanne
1,
Massimo Ancora
2,
Barbara Secondini
2,
Valeria Di Lollo
2,
Liana Teodori
2,
Alessandra Leone
2,
Ilaria Puglia
2,
Alioune Gaye
6,
Amadou Alpha Sall
1,
Cheikh Loucoubar
5,
Roberto Rosà
3,
Mawlouth Diallo
6,
Federica Monaco
2,
Ousmane Faye
1,
Cesare Cammà
2,
Annapaola Rizzoli
4,
Giovanni Savini
2 and
Oumar Faye
1add Show full author list remove Hide full author list
1
Virology Department, Institut Pasteur de Dakar, Dakar BP220, Senegal
2
Istituto Zooprofilattico Sperimentale dell’Abruzzo e del Molise, 64100 Teramo, Italy
3
Centre Agriculture Food Environment, University of Trento, 38010 San Michele all’Adige, Italy
4
Research and Innovation Centre, Fondazione Edmund Mach, 38010 San Michele all’Adige, Italy
5
Epidemiology, Clinical Research and Data Science Department, Institut Pasteur de Dakar, Dakar BP220, Senegal
6
Medical Zoology Department, Institut Pasteur de Dakar, Dakar BP220, Senegal
*
Author to whom correspondence should be addressed.
†
These authors contribute equally to the work.
Viruses 2023, 15(6), 1261; https://doi.org/10.3390/v15061261
Submission received: 4 April 2023 / Revised: 14 May 2023 / Accepted: 17 May 2023 / Published: 27 May 2023
(This article belongs to the Special Issue Arbovirus Diagnostics)
Versions Notes

Abstract

:
West Nile virus is a re-emerging arbovirus whose impact on public health is increasingly important as more and more epidemics and epizootics occur, particularly in America and Europe, with evidence of active circulation in Africa. Because birds constitute the main reservoirs, migratory movements allow the diffusion of various lineages in the world. It is therefore crucial to properly control the dispersion of these lineages, especially because some have a greater health impact on public health than others. This work describes the development and validation of a novel whole-genome amplicon-based sequencing approach to West Nile virus. This study was carried out on different strains from lineage 1 and 2 from Senegal and Italy. The presented protocol/approach showed good coverage using samples derived from several vertebrate hosts and may be valuable for West Nile genomic surveillance.

1. Introduction

The threat from new re-emerging viruses has markedly increased in recent decades due to population growth, urbanization, and the expansion of global travel, facilitating the rapid spread of infection during an outbreak. West Nile virus (WNV), an arbovirus belonging to the flavivirus genus, was firstly isolated in 1937 in Uganda [1] before spreading throughout the world [2]. The enzootic cycle includes mosquitoes and several vertebrate species including birds, allowing long-distance viral spread during migratory seasons [3,4]. Humans are considered WNV dead-end hosts because no human-to-mosquito transmission has been reported yet [5]. Most WNV infections are asymptomatic or may develop into self-limited febrile illness, but a very small percentage of cases progress to neuroinvasive disease with a range of symptoms and occasionally death [6,7].
Before 1990, WNV disease was considered to have a minor public health impact with only sporadic human cases. Since the first outbreaks reported in Algeria and Romania in 1994 and 1996, the virus has diffused to cause large epidemics in North America, Northern African, and Western and Eastern European countries [7].
In Italy, areas with either proven active asymptomatic WNV circulation or high probability of human infection have been previously reported [8,9], and an increasing number of neuroinvasive human infections have been described [10,11].
In Africa, little evidence of WNV epidemics has been noted. In Senegal, where WNV was first isolated in an acute human case in 1970, the virus has also been detected in mosquitoes, birds, horses, and human samples. From 2012 to 2021, active WNV circulation in mosquitoes and humans was documented following a reintroduction event from Europe [12].
WNV exhibits great genetic diversity with currently eight different lineages (excluding Koutango virus) circulating in the world [13]. Lineages 1 (WNV-L1) and 2 (WNV-L2) are the ones causing the main public health concern [7,12]. Genetic characterization of the strains detected yield potential tracking of the routes of the introduction of viruses, which is a particular interest for public health authorities in designing surveillance and countermeasures plans.
Genome sequencing of viruses has proven to be critical in the management of epidemics. Many approaches can be used to obtain viral whole genomes: (i) propagation with cell cultures followed by nucleic acids metagenomic (mNGS); (ii) hybrid capture using specific biotinylated probes; and (iii) a multiplex PCR-based target enrichment or amplicon-based protocol. This last approach became the most used one for the SARS-CoV-2 genomic surveillance during the COVID-19 pandemic due to its applicability in a wide range of input titers, yielding directly sequenced clinical samples, as well as its high specificity and scalability under resource-limited conditions with lower costs [14,15,16,17].
Due to the wide range of WNV hosts, many One Health studies focus on WNV. As genomic data are key information for understanding the mechanisms of the emergence and circulation of this virus, it is crucial to develop a rapid, reliable, and cost-effective sequencing tool that is more accessible than isolation methods or mNGS.
We describe here the development and evaluation of a whole-genome amplicon-based sequencing approach for WNV-L1 and WNV-L2 using Illumina technology in different types of vertebrates and mammals from Senegal and Italy.

2. Materials and Methods

2.1. Primers Design for Tiled Amplicon-Based Sequencing Systems for West Nile Virus

Primer design was made in IPD using a web-based tool entitled Primal Scheme [18] in order to obtain two non-overlapping pools of WNV targeting primers to perform multiplexed PCR reactions, generating approximately 400 bp amplicons tiled along the targeted genome. A WNV reference genome (accession number: NC009942) was chosen as the template. An alignment of WNV whole-genome sequences available on Genbank representative of all WNV lineages in both Africa and Europe was then used to identify nucleotide mismatches for potential correction at ambiguous sites of each primer to ensure both good coverage and high specificity for diverse WNV lineages. Overall, the approach used was a two-pool multiplex amplicon-based sequencing.

2.2. West Nile Virus Primer Pools Validation

Validation of the primer sets followed several steps: (i) inclusivity test by sequencing attempts on several WNV-L1 and WNV-L2 strains; (ii) specificity and sensitivity tests by sequencing attempts on several flaviviruses and other arboviruses, as well as serial dilutions of WNV-L1 and WNV-L2 culture isolates; and (iii) final validation by sequencing confirmed positive WNV samples derived from different species of vertebrates and mosquitoes from Italy and Senegal.

2.2.1. Sequencing of WNV-L1 and WNV-L2 Isolates

The designed primer systems were challenged for amplicon-based whole-genome sequencing of well-characterized WNV-L1 and WNV-L2 isolates from Senegal and Italy. The experiments were undertaken by both the teams in Senegal and Italy with their local isolates. WNV-L1 (n = 10) and WNV-L2 (n = 8) well-characterized viral isolates from both countries were used to assess the ability of the designed primer pools for whole-genome amplicon-based sequencing. WNV strains from Senegal were obtained after infection of C6/36 monolayer cells with homogenized mosquito pools as previously described [12]. Isolates from Italy were obtained from birds’ internal organ homogenates after two to three passages on Vero monolayer cell lines, followed by an infection on C6/36 cell lines. A genome coverage of 95% and above was targeted.

2.2.2. Specificity and Sensitivity of the WNV Amplicon-Based Sequencing Systems

The second step was to assess specificity by performing the experiment on several other arboviruses: Rift Valley fever virus (RVFV); yellow fever virus (YFV); Zika virus (ZIKV); dengue 2 virus (DENV-2); Wesselsbron virus (WSLV); Kedougou virus (KDGV); Usutu virus (USUV); and chikungunya virus (CHIKV). The sensitivity of the approach was evaluated using serial dilutions of WNV-L1 and WNV-L2 culture isolates at different concentrations (106–102 RNA copy/μL). Each concentration was sequenced in triplicate.

2.2.3. Validation on Confirmed Positive WNV Samples

Finally, sequencing attempts on both WNV-L1 and WNV-L2 positive samples from mosquitoes, birds, and horses from Italy and Senegal were conducted. The CT values of the samples were confirmed by RT-qPCR using a consensus WNV assay [6] in Senegal and a molecular WNV sub-typing assay [19] in Italy, prior to proceeding to the sequencing.

2.2.4. Next-Generation Sequencing and Genome Assembly

Viral RNAs were extracted using the QIAamp viral RNA mini-kit (QIAGEN, Hilden, Germany) and were reverse-transcribed into cDNAs using the Superscript IV Reverse Transcriptase enzyme (ThermoFisher Scientific, Waltham, MA, USA). The synthesized cDNAs served as templates for direct amplification to generate approximately 400 bp amplicons tiled along the genome using two non-overlapping pools of WNV targeting primers at 10 nM and Q5® High-Fidelity 2X Master Mix (New England Biolabs) with the following thermal cycling protocol: 98 °C for 30 s; 35 cycles of 95 °C for 15 s and 65 °C for 5 min; and a final cooling step at 4 °C.
In Senegal, libraries were then synthesized by tagmentation using the Illumina DNA Prep kit and the IDT® for Illumina PCR Unique Dual Indexes. After a cleaning step with the Agencourt AMPure XP beads (Beckman Coulter, Indianapolis, IN), libraries were quantified using a Qubit 3.0 fluorometer (Invitrogen Inc., Waltham, MA, USA) for manual normalization before pooling in the sequencer. Cluster generation and sequencing were conducted with ab Illumina MiSeq instrument with 2 × 300 nt read length. Consensus genomes were generated using the nextflow-based nf-core viral reconstruction pipeline (https://github.com/nf-core/viralrecon, accessed on 20 January 2023) from the standardized nf-core pipelines [20,21]. The versions of nextflow and viralrecon used were v21.10.6 and v2.5, respectively. In Italy, amplified DNA was diluted to obtain a concentration of 100–500 ng, then used for library preparation with an Illumina DNA prep kit, and sequenced with a NextSeq 500 (Illumina Inc., San Diego, CA, USA) using a NextSeq 500/550 Mid Output Reagent Cartridge v2 for 300 cycles with standard 150 bp paired-end reads. After quality control and trimming with the Trimmomatic v0.36 (Usadellab, Düsseldorf, Germany) [22] and FastQC tool v0.11.5 (Bioinformatics Group, Babraham Institute, Cambridge, UK) [23,24], reads were de novo assembled using SPADES v3.11.1 (Algorithmic Biology Lab, St Petersburg, Russia) [25]. The contigs obtained were analyzed with BLASTn to identify the best match reference. Mapping of the trimmed reads was then performed using the iVar computational tool [26] to obtain a consensus sequence.

3. Results

3.1. West Nile Virus Oligonucleotide Primers Sets

A first multiplex primer system was designed based on a WNV-L1 reference genome (accession number: NC009942), generating a set of 35 oligonucleotide primer pairs that amplify overlapping products spanning almost the whole WNV genome.
The primers set (set A) was subsequently compared to an alignment of 15 sequences representing the different WNV lineages (Table S1). Degeneration was then added in relevant ambiguous sites on each primer in order to cover a maximum of lineages while trying to maintain a balance for specificity. The list of WNV primers in set A can be found in Table 1. We should notice that two extra primers (KOUV_2_RIGHT and KOUV_7_LEFT) were incorporated into set A to potentially extend the sequencing to Koutango virus, even if this work was not carried out in this study.
A second primer set (set B) was designed based on a WNV-L2 reference genome (accession number: MH021189) and was compared with an alignment of 82 WNV-L2 sequences (Table S2) to capture the diversity within the lineage. The list of WNV primers in set B can be found in Table 1.

3.2. WNV Primers Sets Validation

3.2.1. Validation of Set A

Inclusivity Test

After the design of set A, seven WNV-L1 and three WNV-L2 isolates from Senegal were selected, and three viral culture supernatants for each lineage from three different Italian regions were processed for amplicon-based sequencing in triplicate. Overall, tiled amplicon whole-genome sequencing undertaken on both strains from Senegal and Italy yielded 99–100% horizontal coverage with genome length between 10,961 nt and 11,018 nt for WNV-L1 and between 10,914 nt and 10,926 nt for WNV-L2 (Table 2).

Sensitivity Test

One representative isolate of each lineage, i.e., WNV 15217 (accession number: FJ483548) and WNV Thessaloniki_MC82m/2018 (accession number: MN652880) for WNV-L1 and WNV-L2, respectively, was selected to evaluate the detection limit of the set A primers under optimal conditions. Serial dilutions from 106 to 102 cp/µL were processed in triplicate for sequencing. The set A primers were able to detect more than 95% of the total WNV-L1 genome up to 104 cp/µL. At 103 cp/µL, the horizontal coverage was between 91% and 94%, while at 102 cp/µL, 80 to 82% of the WNV-L1 sequence was completed. However, poor coverage was observed in the WNV-L2 samples (between 17% and 35% completeness) as shown in Table 3.

Specificity Test

Amplicon-based whole-genome sequencing with set A primers was conducted on six flavivirus species (YFV, ZIKV, DENV-2, WSLV, KDGV, USUV), as well as RVFV and CHIKV, in order to assess the specificity of this WNV targeted approach. All the samples failed the bowtie2 1000 mapped-read threshold and no consensus genome could be assembled.

WNV Set A Primers Validation on Real Homogenates

Thirty-one (31) WNV-L1 and fifty-four (54) WNV-L2 homogenates with known Ct values by RT-qPCR were selected for targeted sequencing using the set A primers. Homogenates were obtained from mosquito pools and the internal organs of birds with low to high viral loads.
Among WNV-L1 homogenates, horizontal coverage was between 34% and 100%. A total of 35% of the samples reached above 95% horizontal coverage and about 65% of samples for 90% horizontal coverage. Most complete genomes had Ct values between 16 and 28. However, we also noted that among the least well-covered samples, Ct values ranged from 25 to 35, highlighting that factors other than the viral load could be involved. Additionally, five samples were WNV-L1/WNV-L2 co-infections, and the amplicon-based approach yielded from 87% to 96% WNV-L1 horizontal coverage, even when WNV-L2 had a higher viral load. Relatively correct coverage (between 74% and 92%) was obtained from other four samples from mosquitoes trapped in Senegal, for which viral co-infections with either alphaviruses, mesoniviruses, or flaviviruses were reported. All these results are summarized in Table 4.
Regarding WNV-L2 homogenates, experiments undertaken with the set A primers were consistent with the data from inclusivity and specificity tests. Indeed, less than 6% of the samples processed had above 95% of the genome covered (3 out 54), and 87% had ≤64% horizontal coverage, regardless of the viral load (Table 5).

3.2.2. Validation of Set B

Inclusivity Test

Five WNV-L2 isolates from Italy were selected to assess the set B primers. A total of 100% horizontal coverage was obtained for all the strains after sequencing on an Illumina MiSeq (Table 6).

Sensitivity Test

In order to identify the set B primers’ detection limit under optimal conditions, serial dilutions from 106 to102 cp/μL of the strain WNV Thessaloniki_MC82m/2018 (accession number: MN652880) were processed in triplicate for sequencing (except the to102 cp/μL concentration, which was carried out in duplicate due to insufficient volume during the experiment). A total of 100% horizontal coverage was obtained between 106 to103 cp/μL, while the two replicates for to102 cp/μL covered 93% and 95% of the genome, as shown by Table 7.

Specificity Test

Similar to the test conducted for set A, no amplification was observed using set B on the six flavivirus species mentioned above, as well as RVFV and CHIKV.

WNV Set B Primers Validation on Real Homogenates

Fifteen WNV-L2 homogenates from Italy with known CT values by RT-qPCR were selected for targeted sequencing using the set B primers. Homogenates were obtained from mosquito pools, as well as the internal organs of birds and horses with low to high viral loads. Overall, horizontal coverage between 97% and 100% was obtained on 14 out of 15 homogenates (93.3% with horizontal coverage > 95%). Only the horse sample exhibited 93% horizontal coverage. This sample was also the one with the lowest viral load (CT value: 35). All these results are summarized in Table 8.

3.2.3. Validation of Set A + B

In order to obtain a system able to efficiently sequence both WNV-L1 and WNV-L2 strains, the first set of primers (set A) was combined with the second one (set B) in equal volume. The new system, set A + B primers, was evaluated and compared in parallel with set A and set B after sequencing the WNV-L1 (n = 4) and WNV-L2 (n = 7) positive samples from internal organs of birds and horses, as well as mosquito homogenates, at different CT values (Table 9).
In WNV-L1 samples, no loss of sensitivity was observed between set A and set A + B for all the samples tested. Notably, for one sample from a yellow-legged gull at CT value 25, a gain of sensitivity was observed at 88% horizontal coverage using set A to 93% using set A + B joined. In the same way, sequencing conducted on WNV-L2 samples worked just as well with set B as with set A + B, regardless of Ct values. Indeed, almost 72% of the samples had 100% full genome (n = 5 out of 7).

4. Discussion

NGS is now an essential tool in the study of infectious diseases, both at the fundamental level and in its application to public health. The COVID-19 pandemic has thus been a patent example of the importance of being able to obtain information on the genetic signature of pathogens in real time. However, it should be noted that sequencing technology, and in particular whole-genome sequencing, remains an expensive approach with significant experimental constraints (for instance, the host genome background with a relatively lower amount of genetic material of the pathogen of interest in clinical specimens) in order to have some quality of data generated. A multiplex PCR-based target enrichment or amplicon-based protocol [14] was mostly used to overcome these challenges during SARS-CoV-2 genomic surveillance, yielding more than 14 million genomes in the GISAID platform at the time of writing this manuscript [27].
WNV is becoming a major health problem in Europe and cases have also recently been detected in Africa [7,12].
WNV cases are mainly due to lineages 1 and 2. The mechanisms of diffusion of viral strains, in particular by the migratory movements of birds, are actively studied. The genetic characterization of the identified strains allow better control of the dissemination routes for effective sanitary measures. NGS showed the persistence of a WNV strain after winter in Andalusia in Spain, suggesting endemicity with potential future epidemics in the area [28]. Another recent genomic study evidenced continuous WNV-L2 circulation in Italy throughout the year [29], while a reintroduction event was identified from Europe to Senegal, highlighting a potential threat [12].
Genomic characterization is even more important because it has been shown that West African lineages have higher virulence and replicative efficiency in vitro and in vivo compared to similar lineages circulating in the United States and Europe [6]. Genomic surveillance is thus essential as it allows a better understanding of the dissemination and dynamic of WNV strains.
In order to ensure the sustainability of this type of surveillance, we describe here the development and evaluation of a whole-genome amplicon-based sequencing approach for WNV-L1 and WNV-L2 by Illumina technology in different types of vertebrate and mosquito species from Senegal and Italy.
Three sets of primers were then designed and assessed with WNV-L1 and WNV-L2 strains. Set A and set B are specific to WNV-L1 and WNV-L2 strains, respectively, while the third one, a mixture of the two previous sets, is able to amplify both lineages.
Thus, the use of one set or another depends on the context. Indeed, in the case where the lineage is already well defined, it is appropriate to use the specific sets, whereas set A + B fits more in a context where no lineage characterization could be made before sequencing.
The evaluation in this study could only be carried out with the WNV-L1 and WNV-L2 strains. Because set A was designed from at least one representative of all the WNV lineages, it would be appropriate to undertake a similar evaluation with at least set A and set A + B on other lineages than WNV-L1 and WNV-L2. Moreover, the repetition of these experiments by other groups allows the observed results to be refined, particularly in terms of correlation with Ct values. Indeed, even if this work was carried out with rigor and with two teams in Senegal and Italy, external factors such as the sample quality after long-term storage or the sample type may have impacted the outputs of the results.
In any case, the approach presented in this manuscript could be a valuable tool for any WNV genomic investigation.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/v15061261/s1, Table S1: WNV Sequences aligned for set A primers design, Table S2: WNV-L2 Sequences aligned for set B primers design.

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.

Funding

This research was partly funded by the Africa Pathogen Genomics Initiative (Africa PGI) through the Bill & Melinda Gates Foundation (4306-22-EIPHLSS-GENOMICS), the Institut Pasteur de Dakar internal funds, and an international PhD initiative including Fondazione Edmund Mach, University of Trento, and Istituto Zooprofilattico of Teramo.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The data presented in this study are available in the results section of the article as well as in the supplementary materials. No new data were created or analyzed in this study.

Acknowledgments

We are grateful to all the logistic and administrative teams in Senegal and Italy who have indirectly contributed to the completion of this work.

Conflicts of Interest

The authors declare no conflict of interest. All authors have read and agreed to the published version of the manuscript.

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Table
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 AWNV Primers Set B
WNV_1_LEFTGCCTGTGTGAGCTGACAAACTTWNV-L2_1_LEFTGCCTGTGTGAGCTGACAAACTT
WNV_1_RIGHTTTCTTTTGTTTTGAGCTCCKCCWNV-L2_1_RIGHTTTCTTTTGTTTTGTGCTCCGGC
WNV_2_LEFTACAGCGATGAAACACCTTCTGAWNV-L2_2_LEFTACAGCGATGAAGCATCTCTTGA
WNV_2_RIGHTCGTGTCTTGGTGCATCTTCCATWNV-L2_2_RIGHTGBCGDGTYTTDGTGCATCTYCC
KOUV_2_RIGHTTTYCCTCTGATGCATCTTCCATWNV-L2_3_LEFTGTSYTRGCTGCTGGAAATGAYC
WNV_3_LEFTCCRGTACTGTCGGCTGGTAATGWNV-L2_3_RIGHTCMACCCATGTAGCTCCAGAYAC
WNV_3_RIGHTCVAGAACCAAATCCACCCAWGTWNV-L2_4_LEFTATNCTATTGCTCCTGGTRGCA
WNV_4_LEFTACAGCTTCAACTGCCTTGGAATWNV-L2_4_RIGHTTCCADCCAGTTGCTTTGGTKGW
WNV_4_RIGHTTGRTTCTTCCTATTGCCTTGGTWNV-L2_5_LEFTGTRGACAGRGGATGGGGAAAYG
WNV_5_LEFTGGYTGCGGACTATTTGGMAAAWNV-L2_5_RIGHTGRTTCCTCCAHGYGGTGCTT
WNV_5_RIGHTCTCCACACAGTACTTCCAGCACWNV-L2_6_LEFTCCTTCCTGGTYCACCGAGARTG
WNV_6_LEFTGGHACAAAGACGTTCTTGGTYCWNV-L2_6_RIGHTKGAVGAAATGGGCACYTTRCAR
WNV_6_RIGHTGMACTTTGCAAGGTCCATCYGTWNV-L2_7_LEFTGCDTTYAAATTCGCYGGGACTC
WNV_7_LEFTAYGCTTTCAAGTTTCTTGGGACTWNV-L2_7_RIGHTGTGTATRGCTTTCCCYACYGAG
KOUV_7_LEFTAYGCTTTCAAGTTTCTTGGGCATWNV-L2_8_LEFTACTCAGAGGAGCTCAACGACTC
WNV_7_RIGHTAASACTTGATGGACAGCCTTCCWNV-L2_8_RIGHTATTYTTGCTAGGCCTTGTGGHG
WNV_8_LEFTARMGACTAGCCGCTCTAGGAGAWNV-L2_9_LEFTCGGTGYGGAAGTGGAGTGTTYA
WNV_8_RIGHTTGDGCTTTCTGAATGATCTTGGCTWNV-L2_9_RIGHTTRYTRTTCCATGCTCGGTTSR
WNV_9_LEFTAAYGATGTGGAGGCTTGGATGGWNV-L2_10_LEFTYGCDCCAGARCTAGCTAACAAYA
WNV_9_RIGHTAWRCTATTCCAAGCGCGATTSKWNV-L2_10_RIGHTCCCTGGYCTCCTGTTRTGRTTGC
WNV_10_LEFTTMTTTGCACCAGAACTCGCYAAWNV-L2_11_LEFTCACHYTGTGGGGTGATGGAGTT
WNV_10_RIGHTCWGGTCTCCGATTGTGATTGCTWNV-L2_11_RIGHTCAGAAGGCCCAACTGAAAAGGA
WNV_11_LEFTAYACCTTGTGGGGCGATGGARTWNV-L2_12_LEFTGAYGAAAAGACCCTCGTGCA
WNV_11_RIGHTGGCCCAACTGAAAAGGGTCAATWNV-L2_12_RIGHTCCATTTGRAAGAAAGCAGCTGCR
WNV_12_LEFTTGGARATCAGACCACAGAGRCAWNV-L2_13_LEFTCAGTCTTTCTGGTGGCTTCBTT
WNV_12_RIGHTTTGRAAGAAAACAGCCGCCARCWNV-L2_13_RIGHTYCCAGCKGCAAGTATCATBGGA
WNV_13_LEFTCCAGTGTTTATGGTGGCWTCGTWNV-L2_14_LEFTMRGTTGGAAGCYTCATCAARGARA
WNV_13_RIGHTCHGCAAGGATCATGGGGTTGAAWNV-L2_14_RIGHTTGAAAATTTCCATCRTCATCCARCC
WNV_14_LEFTGVAGCTTGATCAGGGAGAARAGWNV-L2_15_LEFTGGACRGCTGAYATYACYTGGGA
WNV_14_RIGHTTCCBGGATCATTCATGARCTGGWNV-L2_15_RIGHTRCTCATGAGAGCRGCTCCCTTR
WNV_15_LEFTCKGACATTTCCTGGGAAAGTGAWNV-L2_16_LEFTATHATGACTCGAGGTCTGCTYG
WNV_15_RIGHTTCATCAAAGCGGCTCCTTTWGTWNV-L2_16_RIGHTMRCCATTDGGCATGATGACKCC
WNV_16_LEFTCTYGGCAGTTATCAAGCAGGAGWNV-L2_17_LEFTRGACTATCCCACYGGAACRTCA
WNV_16_RIGHTWATBGCGCTTATGTATGAHCCRWNV-L2_17_RIGHTTGGCACATGACATCAACGATYT
WNV_17_LEFTCMATAGTGGACAAAAACGGTGATGTWNV-L2_18_LEFTTKAGAGGACTTCCCATYCGGTA
WNV_17_RIGHTDGTGAGGGTAGCATGACACATGWNV-L2_18_RIGHTTTBCCCATTTTCACACTTGGAACR
WNV_18_LEFTATGKCTGAAGCACTGAGRGGAWNV-L2_19_LEFTACMGAGCCTGGAACACTGGVTA
WNV_18_RIGHTCCCATCTTGACACTAGGCACAAWNV-L2_19_RIGHTCCTCCATAGCAATACTCATCACCA
WNV_19_LEFTCGRGCTTGGAACTCTGGATAYGWNV-L2_20_LEFTTGATGGAAGAGTCATCCTGGGV
WNV_19_RIGHTTACTCATCACCAACTTGCGAYGWNV-L2_20_RIGHTGTGTTGGTTCGAGGTCCATCAA
WNV_20_LEFTGGAGAACCATCTGCAGTGACAGWNV-L2_21_LEFTCWGTCTGGCTCGCTTACAAAGT
WNV_20_RIGHTCMACTTCGTTGTTGTCTTCTAAAATTGWNV-L2_21_RIGHTARGCTATTGTCTGAAGGGCRTC
WNV_21_LEFTCYTACAAGGTTGCAGCRGCTWNV-L2_22_LEFTTTKGACACGATGTATGTGGTKG
WNV_21_RIGHTACTCCCATGGTCATCACACWCAWNV-L2_22_RIGHTCAYTCTTGGTCTTGTCCAGCCA
WNV_22_LEFTGAGGMAGAGCTCACAGAATGGCWNV-L2_23_LEFTYCAGCTYGCCGTGTTTTTGATY
WNV_22_RIGHTCCTTGACCTCAATTCTTTGCCCWNV-L2_23_RIGHTCYGCAGTCACAGTCACAGTCAG
WNV_23_LEFTTTGTGTCATGACCCTTGTSAGCWNV-L2_24_LEFTYTTTGTVGACGTTGGTGTGTCA
WNV_23_RIGHTGGDACCATGTAGGCATAGTGGCWNV-L2_24_RIGHTTYGTTGCRTTCCACACTGARCT
WNV_24_LEFTTTYGTCGATGTTGGAGTKTCAWNV-L2_25_LEFTARRACTGTCAGAGAGGCYGGAA
WNV_24_RIGHTGTYGTTGCGTTCCAMACWGAGCWNV-L2_25_RIGHTGCCTTTCCACTAACCACCGYAR
WNV_25_LEFTRGACHGTVCGAGAAGCYGGAATWNV-L2_26_LEFTRHGCCAGGAGAGAGGGAAAYRT
WNV_25_RIGHTGRTCGAGGAAACBCCGTTCGACWNV-L2_26_RIGHTCCARTCTTCCACCATCTCCARR
WNV_26_LEFTRAGAAGGCAAYRTCACYGGAGGWNV-L2_27_LEFTCACACTGCTCTGTGACATTGGA
WNV_26_RIGHTARAATTCCCTTGGCCCTCGGWNV-L2_27_RIGHTAGAATTGAGGAGAGGCTTCCCY
WNV_27_LEFTTCAAGTGCTGAGGTTGAAGAGCWNV-L2_28_LEFTAAGAAAACVTGGAAGGGACCYC
WNV_27_RIGHTGCCTGAGTCGTTCAATCCTGTTWNV-L2_28_RIGHTTBGTGGTCTCATTGAGGACGTR
WNV_28_LEFTTGTAAACTTGGGAAGTGGAACCAWNV-L2_29_LEFTCTCCTTTCGGHCAACAACGRGT
WNV_28_RIGHTTTTWTCKCTGGCCAYAAAVGCCWNV-L2_29_RIGHTCYCCBAGCCACATGAACCADAT
WNV_29_LEFTGTGGAYACGAAAGCTCCTGARCWNV-L2_30_LEFTACYTGCATCTACAACATGATGGG
WNV_29_RIGHTATTGAGAAAACCSAGAGCTTCGWNV-L2_30_RIGHTGGBCTCATCACTTTCACGACYT
WNV_30_LEFTMAARGCCAARGGMAGCAGAGWNV-L2_31_LEFTCDAAGGTBCTTGARCTGCTDGR
WNV_30_RIGHTCCYCTCTGATCTTCTCTGGAGAWNV-L2_31_RIGHTGGACCTTTGACATGGCATTBAGR
WNV_31_LEFTTGAGCTCACCTATCGWCACAAAWNV-L2_32_LEFTGGHGATGACTGYGTGGTDAA
WNV_31_RIGHTCAYCCAGTTGACGGTTTCCACTWNV-L2_32_RIGHTGRACCCAGTTVACAGGCACA
WNV_32_LEFTTGGTRAAGCCCCTGGAYGAYWNV-L2_33_LEFTGCAGATGTGGCTGYTGCTTTAT
WNV_32_RIGHTTCTCCTCCTGCATGGATSGAWNV-L2_33_RIGHTYRTCTTCATACCTCCTCARDGA
WNV_33_LEFTAGWAGAGACCTGMGGYTCATWNV-L2_34_LEFTGCGCHACTTGGGCTGAAAAYAT
WNV_33_RIGHTTCTACAAAACTGTGTCCTCAACCAWNV-L2_34_RIGHTMYCTTCCGAGACGGTTCTGA
WNV_34_LEFTAGTCAGWKCAATCATCGGRGAWGWNV-L2_35_LEFTGGAAGTTGAGTAGACGGTGCTG
WNV_34_RIGHTCACTATCGCAGACTGCACTCTCWNV-L2_35_RIGHTTCCCAGGTGTCAATATGCTGTT
WNV_35_LEFTCAGGAGGACTGGGTGAACAAAG
WNV_35_RIGHTTGGTTGTGCAGAGCAGAAGATC
Table
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 ValueTotal Number of Trimmate ReadsNumber of WNV Reads% HCoverageVCoverageConsensus Sequence Length
Viral strain WNV L1 Italy142369.723649.02299%5802.6710.969
141922.532581.21599%5346.8510.966
132715.830754.08199%6244.3610.966
No. of replicates with coverage ≥ 95%3/3 (100%)
Viral strain WNV L1 Senegal25623.535238.25999%799710.961
285034.1511035.98399%6862.1210.965
165034.151547.77599%5183.8310.966
19810.906327.395100%3971.411.018
17924.142363.23699%4412.710.963
17899.818342.13399%3851.3410.966
17819.552358.63199%4306.9410.961
No. of replicates with coverage ≥ 95%7/7 (100%)
Viral strain WNV L2 Italy182607.185546.249100%3607.9910.926
18.712466.682511.381100%3488.110.926
18.132061.961465.844100%3445.5510.926
No. of replicates with coverage ≥ 95%3/3 (100%)
Viral strain WNV L2 Senegal144861.644706.114100%3519.5110.914
144087.737835.098100%5593.5810.914
144750.250885.595100%5609.3410.914
No. of replicates with coverage ≥ 95%3/3 (100%)
Table
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 StrainQuantity Value (cp/μL)Quantity Mean Value (Ct)Total Number of Trimmate ReadsNumber of WNV Reads% HCoverageVCoverageConsensus Sequence Length
WNV L1 (reference used for the mapping on Genpat: WNV L1 FJ48354810618.681457.278503.15299%5043.5610.959
1061371.693476.60599%4997.0410.964
106938.406375.65399%4372.0110.959
No. of replicates with coverage ≥ 95%3/3 (100%)
10523.451342.407426.91499%4587.3310.960
1051174.155397.21599%4435.7910.959
105825.135315.21999%3776.6710.956
No. of replicates with coverage ≥ 95%3/3 (100%)
10427906.690292.71896%3456.2610.964
104984.952297.35197%3414.3810.959
1041247.127327.09596%3570.2210.959
No. of replicates with coverage ≥ 95%3/3 (100%)
103301096.401249.79894%2735.4110.962
103506.380153.28493%1853.4210.955
103569.039170.27291%2099.5410.955
No. of replicates with coverage ≥ 95%0/3 (0%)
1023357.22920.11582%295.7610.366
10253.43518.51581%281.02710.958
1024142615.14980%227.61710.951
No. of replicates with coverage ≥ 95%0/3 (0%)
WNV L2 (reference used for the mapping: WNV L2 MN652880)10618.681262.16712.48435%391.54710.028
1061315.56811.57633%383.27610.928
1061103.83816.65226%706.14610.928
No. of replicates with coverage ≥ 95%0/3 (0%)
10522.871210.49511.25136%347.19710.928
105823.02510.24333%345.10110.928
1051478.16012.25334%394.67510.928
No. of replicates with coverage ≥ 95%0/3 (0%)
10426.411228.9459.71532%337.86210.928
1041090.8499.54732%327.5710.928
104947.9644.40530%159.12210.928
No. of replicates with coverage ≥ 95%0/3 (0%)
10330442.0632.79626%121.74410.928
103577.2773.50027%143.43910.926
103369.0411.61522%79.11810.245
No. of replicates with coverage ≥ 95%0/3 (0%)
10233.1435.4261.36217%789.15710.924
10268.30762717%360.49510.926
10263.64373118%571.29710.926
No. of replicates with coverage ≥ 95%0/3 (0%)
Table
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 NumberHostRT-PCR Ct ValueCo-InfectionCt Value# Total Trimmate Reads# WNV L1 Reads% HCoverageVCoverageConsensus Sequence Length
1Accipiter gentilis15-2,037,215591,954100%6362.0411,027
2Pica pica16-14,648,0251,333,187100%6381.6711,016
3Corvus cornix16-6,447,209657,94899%5135.4510,966
4Pica pica18-2,923,237387,98699%4264.5610,966
5Phalacrocorax carbo19-11,441,5181,107,29199%5888.4910,961
6Corvus cornix19-2,347,380351,29299%4177.6910,963
7Culex pipiens20-3,495,685744,56299%5560.6810,968
8Culex pipiens22-7,571,826597,49998%3973.210,967
9Corvus cornix22-5,382,363629,36499%4806.1110,960
10Passer domesticus22-3,942,375304,21597%3084.410,962
11Culex pipiens23-4,560,711283,77193%2899.4710,960
12Corvus Cornix24-3,052,530342,27193%3508.2510,966
13 *Culex pipiens25L2 Ct 283,576,787403,16694%3761.4810,966
14 *Culex pipiens25L2 Ct 281,557,373263,14990%2471.310,954
15Larus michahellis25-1,813,567129,83488%1805.9510,952
16Streptopelia decaocto26-1,439,897203,17291%2592.0910,961
17Pica pica26-7,677,172251,98281%2920.0310,963
18Parus major26-755,95138,78069%747.619389
19 *Culex pipiens27L2 Ct 323,371,541369,61096%3287.3110,956
20Turdus merula27-2,710,57266,22982%1039.0810,954
21Culex pipiens28-6,865,408269,72794%2618.8410,962
22 *Culex pipiens28L2 Ct 313,378,716190,10287%2201.2910,966
23Streptopelia decaocto28-1,842,01022,00668%430.49410,946
24Equus caballus28-1,805,957159,70392%2101.3110,960
25Athene noctua29-3,394,98915,76734%40410,802
26Columba palumbus31-835,362994857%211.70610,960
27 *Culex pipiens33L2 Ct 254,662,340220,50891%2381.2110,963
28 * (Alphavirus, Mesonivirus)Culex neavei28.5-391,4941,135,18092.42%2514.310,194
29 * (Barkedji, Mesonivirus)Culex poicilipes35.59-64,716616,50082.35%1789.369083
30 * (Barkedji)Culex neavei29.52-302,1751,394,46689%2001.39819
31 * (Alphavirus, Barkedji, Usutu)Culex neavei25.03-180,791484,22173.89%822.028150
Table
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 NumberHostRT-PCR Ct ValueCo-Infection Ct Value# Total Trimmate Reads# WNV Reads% HCoverageVCoverageConsensus Sequence Length
1Accipiter gentilis16-3336.278450.617100%3304.610.926
2Accipiter gentilis16-1773.333393.12599%2982.3810.926
3Accipiter gentilis19-2753.185225.09672%1533.6310.926
4Garrulus glandarius20-3540.566304.09496%1588.4410.923
5Culex pipiens22-1092.527517.4458%779.91210.921
6Culex pipiens23-568.56254.65859%905.15510.834
7Corvus cornix23-1383.83185.88062%1168.9510.922
8Passer italiae24-476.12045.64447%1089.7910.923
9Columba palumbus26-1313.90076.77364%908.82210.923
10Columba palumbus27-5.3777.31925%257.25610.868
11Columba palumbus28-545.944507323%281.0098.425
12Turdus merula31-514.655724%26.336375
13Pica pica31-330.3742805%685.1974.128
14Phasianus colchicus32-243.6562425%543.4637.510
15Pica pica33-341.1536139%760.2038.413
16Pica pica33-376.1851284%307.7153.779
17Egretta garzetta34-616.7813956%715.3967.923
18Culex pipiens29-1537.44824.51733%676.5019.393
19Culex pipiens28-588.31332.43749%749.22410.924
20Culex pipiens27-606.88628.18152%494.55110.923
21Culex pipiens25-884.75643.65752%864.51710.891
22Culex pipiens30-259.1732.5723%989.276381
23Culex pipiens28-436.46114.47329%630.09710.923
24Culex pipiens24-2172.95245.56845%716.97410.923
25 *Culex pipiens31L1 Ct 283378.7169.13227%447.98610.928
26Culex pipiens25-339.61733.68142%827.22910.790
27Culex pipiens21-1223.42235.45123%1197.7410.921
28Culex pipiens23-1832.34138.13415%1569.3410.555
29Culex pipiens25-686.60225.50918%1178.5510.609
30Culex pipiens27-502.1628.2533%3113.1388
31Corvus cornix28-1357.3217.15931%312.30710.475
32Pica pica20-6417.489198.61394%1616.1110.927
33 *Culex pipiens25L1 Ct 334662.34033.60860%534.95510.926
34 *Culex pipiens24USUV Ct 275133.18377.02261%888.53210.925
35Culex pipiens24-153.6011047%275.337556
36Corvus cornix29-877.21800%00
37Culex pipiens23-199.55099.49088%1126.1410.890
38Culex pipiens24-323.1155.04823%308.1364.395
39 *Culex pipiens27L1 Ct 2535767879.46151%239.31210.928
40Culex pipiens22-78.37842.63763%824.46110.922
41Larus marinus23-546.215142.17881%1460.9710.923
42 *Culex pipiens27L1 Ct 323371.5415.60347%154.3210.928
43Culex pipiens29-153.4134.36731%187.099.364
44Culex pipiens28-66.2027.78636%290.74210.018
45 *Culex pipiens28L1 Ct 251557.3738.94548%243.94110.926
46Culex pipiens28-118.3962.42015%219.7337.809
47 *Culex pipiens27USUV Ct 271516.32423044%985.1947.469
48Corvus cornix28-470.1136.19440%207.82710.923
49 *Culex pipiens23USUV Ct 214272.99424829%941.93610.806
50Culex pipiens15-327.23888.89661%1398.2210.922
51Ochlerotatus caspius25-361.19416.43632%684.57610.844
52Culex pipiens24-401.40825.09142%804.5979.592
53Pica pica23-899.5972.77124%150.2158.402
54 *Culex pipiens29USUV Ct 26117.76818.20454%451.54610.907
Table
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% HCoverageVCoverageConsensus Sequence Length
Viral strain WNV L2 Italy151218.086284.232100%3820.7710.926
151792.478402.082100%5234.3610.926
151440.061338.706100%4543.8110.926
171711.005328.182100%4374.4110.926
18941.641224.716100%3023.7710.926
N of replicates with Coverage ≥ 95%5/5 (100%)
Table
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 StrainQuantity Value (cp/5 μL)Quantity Mean Value (Ct)# Total Trimmate Reads# WNV Reads% HCoverageVCoverageConsensus Sequence Length
WNV L2 (reference used for the mapping: WNV L2 MN652880)106191808.185381.930100%5038.7310.913
1064928.502673.900100%6845.5510.926
1063099.180511.446100%6107.3810.913
No. of replicates with coverage ≥ 95%3/3 (100%)
105222665.260409.989100%5157.7410.914
1051049.020237.471100%3194.3610.926
1052820.387429.662100%5335.9910.912
No. of replicates with coverage ≥ 95%3/3 (100%)
104261651.945261.024100%3450.1210.913
1042483.786337.982100%4234.9110.914
1042681.807356.233100%4334.7110.926
No. of replicates with coverage ≥ 95%3/3 (100%)
103301570.036236.060100%3029.5310.894
1031153.288196.257100%2614.9110.904
103782.424285.13699%3070.4710.912
No. of replicates with coverage ≥ 95%3/3 (100%)
102331764.307159.59195%2189.3110.597
1022082.360177.21293%2389.5410.800
102NANANANANA
No. of replicates with coverage ≥ 95%1/2 (50%)
Table
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 NumberRT-PCR Ct ValueHostTotal Number of Trimmate ReadsNumber of WNV Reads% HCoverageVCoverageConsensus Sequence Length
117Pica pica1134.961295.649100%3977.5810.892
227Corvus cornix1448.962284.947100%3818.1510.912
321Culex pipiens1733.553385.088100%5042.3510.913
423Culex pipiens1528.111339.207100%4525.7210.912
521Athene noctua1801.390376.213100%4946.5310.878
622Culex pipiens1407.784312.37699%4201.8910.879
719Passer domesticus1515.878205.605100%2770.4510.914
830Corvus cornix1470.367209.31698%2799.5710.872
930Pica pica2150.396205.46699%2662.6110.868
1027Sylvia atricapilla3649.208281.10299%3426.0710.880
1125Culex pipiens2094.249349.710100%4076.3510.878
1229Anopheles maculipennis3435.959563176100%5709.1410.912
1325Culex pipiens3120.601281.102100%4601.8110.912
1427Culex pipiens2193.040259.48397%3231.7410.904
1535Equus ferus caballus1623.442133.08893%1814.7410.936
Table
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 L1RT-PCR Ct ValueHostUsed PrimersTotal Number of Trimmate ReadsNumber of WNV Reads% HCoverageVCoverageConsensus Sequence Length
1L1 19Corvus cornixSet A2347.380351.29299%4177.6910.963
Set A + B1118.615287.67299%3987.1210.963
2L1 25Larus michahellisSet A1813.567129.83488%1805.9510.952
Set A + B651.72391.64293%1305.8310.960
3L1 18Pica picaSet A2923.237387.98699%4264.5610.966
Set A + B4965.722512.72599%4562.2810.966
4L1 28Equus ferus caballusSet A1805.957159.70392%2101.3110.960
Set A + B2319.560165.37692%2200.3910.960
Viral Homogenate WNV L2RT-PCR Ct ValueHostUsed PrimersTotal Number of Trimmate ReadsNumber of WNV Reads% HCoverageVCoverageConsensus Sequence Length
1L2 17Pica picaSet B2059.659351.670100%4232.7810.892
Set A + B1134.961295.649100%3977.5810.892
2L2 27Corvus cornixSet B1793.046187.826100%2156.8110.926
Set A + B1448.962284.947100%3818.1510.912
3L2 21Culex pipiensSet B2219.785312.851100%3515.0510.926
Set A + B1733.553385.088100%5042.3510.913
4L2 23Culex pipiensSet B2045.957275.354100%2943.7710.926
Set A + B1528.111339.207100%4525.7210.912
5L2 21Athene noctuaSet B3413.467359.916100%4106.3910.892
Set A + B1801.390376.213100%4946.5310.878
6L2 22Culex pipiensSet B3109.597239.79595%2761.2310.892
Set A + B1407.784312.37699%4201.8910.879
7L2 19Passer domesticusSet B1621.442123.81389%1836.8510.924
Set A + B1515.878205.605100%2770.4510.914
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Diagne, M.M.; Ndione, M.H.D.; Mencattelli, G.; Diallo, A.; Ndiaye, E.h.; Di Domenico, M.; Diallo, D.; Kane, M.; Curini, V.; Top, N.M.; et al. Novel Amplicon-Based Sequencing Approach to West Nile Virus. Viruses 2023, 15, 1261. https://doi.org/10.3390/v15061261

AMA Style

Diagne MM, Ndione MHD, Mencattelli G, Diallo A, Ndiaye Eh, Di Domenico M, Diallo D, Kane M, Curini V, Top NM, et al. Novel Amplicon-Based Sequencing Approach to West Nile Virus. Viruses. 2023; 15(6):1261. https://doi.org/10.3390/v15061261

Chicago/Turabian Style

Diagne, Moussa Moïse, Marie Henriette Dior Ndione, Giulia Mencattelli, Amadou Diallo, El hadji Ndiaye, Marco Di Domenico, Diawo Diallo, Mouhamed Kane, Valentina Curini, Ndeye Marieme Top, and et al. 2023. "Novel Amplicon-Based Sequencing Approach to West Nile Virus" Viruses 15, no. 6: 1261. https://doi.org/10.3390/v15061261

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