An amplicon-based sequencing approach for Usutu virus characterization

Usutu virus (USUV), an arbovirus from the Flaviviridae family, genus Flavivirus, has recently gained increasing attention because of its potential for emergence. After his discovery in South Africa, USUV spread to other African countries, then emerged in Europe where it was responsible for epizootics. The virus has recently been found in Asia. USUV infection in humans is considered to be most often asymptomatic or to cause mild clinical signs. However, a few cases of neurological complications such as encephalitis or meningo-encephalitis have been reported in both immunocompromised and immunocompetent patients. USUV natural life cycle involves Culex mosquitoes as its main vector, and multiple bird species as natural viral reservoirs or amplifying hosts, humans and horses can be incidental hosts. Phylogenetic studies carried out showed eight lineages, showing an increasing genetic diversity for USUV. This work describes the development and validation of a novel whole-genome amplicon-based sequencing approach to Usutu virus. This study was carried out on different strains from Senegal and Italy. The new approach showed good coverage using samples derived from several vertebrate hosts and may be valuable for Usutu virus genomic surveillance to better understand the dynamics of evolution and transmission of the virus. Supplementary Information The online version contains supplementary material available at 10.1186/s12985-024-02426-7.


Introduction
An increasing number of Infectious diseases (re-)emerge annually due to a combination of ecological and socioeconomical drivers such as increase in human population density, ageing, travel, urbanization, biodiversity loss and climate change, promoting the evolution and spread of new pathogens [1].Among them, arboviruses have a significantly impact on human and animal health through their emergence and re-emergence in these populations, representing a significant threat for new epidemics worldwide.[2,3].
Usutu virus (USUV), an arbovirus from the Flaviviridae family, genus Flavivirus, has recently gained significant scientific attention, particularly since its emergence in Europe [3].It was first isolated in 1959 from a Culex neavei mosquito caught near the Usutu river in Swaziland, South Africa [4].The virus belongs to the antigenic serocomplex of Japanese encephalitis, together with Japanese encephalitis virus (JEV), West Nile virus (WNV), and Murray Valley encephalitis virus (MVEV).It is a (+)-strand RNA genome of 11,064 nucleotides encoding a single polyprotein of 3,434 amino acids that is subsequently cleaved into structural (C, prM and E) and nonstructural (NS1, NS2A, NS2B, NS3, NS4A, NS4B and NS5) proteins [5].
Similar to other flaviviruses, the natural life cycle of USUV involves Culex mosquitoes as its main vector, and multiple bird species as natural viral reservoirs or amplifying hosts [3].Humans and horses can be incidental dead-end hosts.USUV isolation, detection or serological evidence were reported in various animal species including rodents, shrews, bats, dogs, squirrels, wild boars, roedeers, and lizards, expending the incidental host range .
After its discovery in South Africa, USUV spread to other African countries including Senegal, Uganda, Central African Republic, Nigeria, Burkina Faso, and Côte d'Ivoire, causing mild sporadic infections in the continent [32].Outside Africa, it was observed in Europe for the first time in Vienna in 2001 [9], where it was responsible for massive outbreaks in blackbirds (Turdus merula) and gray owls (Strix nebulosa).A retrospective study analysing dead birds conducted in the Tuscany region of Italy showed that USUV has been circulating in Europe since 1996 [10].Between 2014 and 2015, the virus was first isolated in Israel from mosquito pools, marking the first isolation in the Asian continent [33].Recently, USUV has also caused neurological cases in immunocompetent patients in Croatia, Italy, Germany, Austria and France [31,[34][35][36][37]. Indeed, there is increasing evidence that the virus is pathogenic for humans, and might become a growing potential public health issue.
USUV is found in a broad spectrum of animals and mosquitoes, sometimes in co-infection with insect specific viruses or other arboviruses sharing the same reservoirs or vectors [38,IPD unpublished data].This poses a diagnostic challenge for both serology and molecular biology tests.We therefore set up an amplicon-based system for the specific sequencing of USUV.The sequencing approach, used extensively during the COVID-19 pandemic, allowed health authorities and the scientific community to quickly monitor SARS-CoV-2 introductions and identify new variants for appropriate countermeasures and better management of the pandemic [39].This tool will enable the acquisition of complete genomes of USUV strains circulating at known and unknown vectors, reservoirs, or hosts, allowing for better genetic diversity assessment and genomic surveillance in Africa, Europe and other parts of the world where the virus can emerge.

Primers design for USUV tiled amplicons-based sequencing systems
Two non-overlapping pools of USUV targeting primers were designed in IPD to perform multiplexed PCR reactions, using the web-based tool entitled Primal Scheme [40] and the USUV reference genome (accession number: MT188658.1)as template.Approximately, 400 bp tiled amplicons were generated along the targeted genome.An alignment of USUV sequences available on Genbank were then used to identify nucleotide mismatches for potential correction on ambiguous sites of each primer to ensure both good coverage and high specificity to USUV lineages.

Sequencing of USUV isolates
The designed primer systems were challenged for amplicon-based whole genome sequencing of well characterized USUV isolates from Senegal and Italy.The sequencing experiments were undertaken both by the teams in Senegal and Italy with their local isolates.USUV strains from Senegal were obtained by infecting C6/36 cell monolayers with homogenized mosquito pools, as previously described [41].Isolates from Italy were obtained from birds' internal organs and mosquito homogenates after two to three passages on Vero monolayer cell lines followed by an infection on C6/36 cell lines.A cut-off of 95% horizontal coverage was chosen as a good metric for inclusivity.

Validation on USUV field samples
Sequencing efforts were conducted on USUV positive samples from mosquitoes and birds collected in both Italy and Senegal.CT values of the different samples were determined by RT-qPCR using a consensus USUV assay in Senegal [43] and a molecular USUV assay [44] in Italy before sequencing.Briefly, in Senegal viral RNA extraction was performed with the QIAamp viral RNA mini kit (Qiagen, Heiden, Germany) according to the manufacturer's instructions.Viral RNAs were amplified by qRT-PCR using the Quantitect Reverse Transcription Kit (Qiagen, Heiden, Germany) according to the manufacturer's instructions and a specific real-time RT-PCR assay to Identify Usutu Virus [43].In Italy, Viral RNA was extracted by using the MagMAX CORE Nucleic Acid Purification KIT (Applied Biosystem, Thermo Fisher Scientific, Life Technologies Corporation, TX, USA) and amplified by a rapid and specific real-time RT-PCR assay to Identify Usutu Virus [44], by using the Superscript III Platinum OneStep qRT-PCR System (Invitrogen) according to the manufacturer's instructions.

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 USUV targeting primers at 10 nM and Q5® High-Fidelity 2X Master Mix (NEB, NEW ENGLAND, Biolabs).
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), libraries were quantified using a Qubit 3.0 fluorometer (Invitrogen Inc., Waltham, MA) for manual normalization before pooling in the sequencer.Clusters generation and sequencing were performed on an Illumina MiSeq instrument with 2 × 300-nt read-length.Consensus genomes were generated using the nextflow (v21.10.6) based nf-core viral reconstruction (v2.5) pipeline (https://github.com/nf-core/viralrecon)from the standardized nf-core pipelines [44,45].
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, 300 cycles, and standard 150 bp paired-end reads.After quality control and trimming with Trimmomatic v0.36 (Usadellab, Düsseldorf, Germany) (Bolger et al., 2014) and FastQC tool v0.11.5 (Bioinformatics Group, Babraham Institute, Cambridge, UK) [47,48] reads were de novo assembled using SPADES v3.11.1 (Algorithmic Biology Lab, St Petersburg, Russia) [49].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 [50] to obtain a consensus sequence.

USUV phylogenetic tree
A phylogenetic tree The newly generated USUV genomes were analysed with other whole genomes available in Genbank.A phylogenetic tree was generated with the strains obtained during this study (horizontal coverage greater than or equal to 95%) with complete genomes available on Genbank.Indeed, complete genomes of strains of each lineage described for USUV (Africa 1, 2, 3 et Europe 1, 2, 3, 4, 5) [30][31][32] were chosen according to availability.Sequences were aligned using MAFFT [51], and the alignment was run under the best model in IQ-TREE [52].The maximum-likelihood (ML) phylogenetic tree was visualized using Figtree V1.4.4 [53].

USUV oligonucleotide primer pools
Overall, thirty-five overlapping oligonucleotide primer pairs covering almost the whole genome of USUV were obtained after the design based on a USUV reference genome (accession number: MT188658.1).
The primers set was subsequently compared to an alignment of twenty-one sequences representing the different USUV lineages (supplementary material S1).In order to cover a maximum of lineages while maintaining a balance for specificity, degeneration was then added in relevant ambiguous sites on each primer.The list of USUV primers can be found in Table 1.

Inclusivity test
Overall, three strains from Italy and ten for Senegal, with Ct values varying from 10 to 33, were used to perform the
All the samples failed bowtie2 1000 mapped read threshold and no consensus genome could be assembled.

Sensitivity test
One representative USUV isolate (accession number: ON032476) was chosen to assess the primers set limit of detection under optimal conditions.Serial dilutions from 10^6 to10^2 cp/µl were processed in triplicate for sequencing.The system was able to capture more than 95% of USUV whole genome until 10^4 cp/µl.At 10^3 cp/µl, horizontal coverage was between 92 and 96% while 83-88% of the USUV sequence was completed at 10^2 cp/µl (Table 3).The Table 2 shows the results obtained after sequencing of three and ten USUV strains from Italy and Senegal respectively, for inclusivity test of the USUV primers.Ct values of tested strains, number of total reads and specific USUV reads, horizontal and vertical coverages and consensus sequence length obtained after sequencing for each strain were presented in this table VCoverage = vertical coverage HCoverage = horizontal coverage

T G T G G T T G A T G T T G G A A
The Table 1 shows the sequences of the USUV primers set designed in this study  4 and 5.

Usutu virus phylogenetic tree
The phylogenetic tree obtained after analysis of newly USUV strains and USUV available complete genomes in Genbank, confirms that the complete genomes sequences obtained belong to USUV and that these strains have a resemblance to the old strains (Fig. 1).

Discussion
Arboviruses constitute a broad group of viruses with a strong impact on human and animal health.In order to prevent the underreporting and underestimation of these viruses, there is a strong need of a reliable integrated surveillance system at the animal, human and vector interface.High-throughput sequencing approaches, as the nucleic acid metagenomics; the hybrid capture using specific biotinylated probes; the multiplex PCR-based target enrichment; or the amplicon-based protocol might be crucial to improve viral diagnosis and better understand the genetic diversity of viruses, enabling the implementation of appropriate countermeasures [42].Specifically, the amplicon-based sequencing approach, allowing targeted-sequencing, has been extensively used in the recent past for SARS-CoV-2 and WNV genomic surveillance to monitor the virus introductions and local transmission, aiding in understanding the global diffusion network and viral evolution [54].This method offers a solution to sequencing constraints such as the host genomic background and low viral loads [55].
USUV, an arbovirus causing neurological disease in human, has become widespread in Africa and Europe [32,56] and has emerged in Asia [33].Several detections of the virus in humans, mosquitoes and animals have been recorded in recent years across these three continents.USUV genetic lineages are geographically specific, enabling genomic surveillance to track viral strain diffusion patterns.In this study, we propose a whole genome amplicon-based sequencing approach for Illumina technology for USUV, to establish an efficient genomic surveillance system for monitoring the emergence and re-emergence of this arbovirus that, widespread in Africa and Europe, is causing neurological disease in humans and which evolution and expansion might represent a potential public health future threat worldwide [32,56].The method was validated in vertebrates and mosquitoes from Senegal and Italy using specific primers designed based on different USUV lineages.Nearly complete genomes sequences of USUV strains from both countries were obtained with good primer sensitivity and specificity.In addition, USUV samples co-infected with other viruses were sequenced with good horizontal and vertical coverages.In view of these results, this sequencing

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The Table 3 shows the evaluation of sensitivity of USUV primers set.The number of total reads and specific USUV reads, horizontal and vertical coverages and consensus sequence length obtained after sequencing of serial dilutions of an USUV strain were presented in this table VCoverage = vertical coverage HCoverage = horizontal coverage method can be used for USUV monitoring, directly from mosquito and bird homogenates.However, additional comparative studies could be carried out to assess the described USUV primers set and refine the observed results.
In the literature, many phylogenetic studies which have shown that USUV can have several lineages including Africa 1 to 3 and Europe 1 to 5 have been essentially based on the NS5 gene of the virus [28][29][30][31]57].New phylogenetic studies based on complete genomes of all detected strains in African and European countries using this tool could allow to better determine the diversity of this virus.
Overall, this novel amplicon based sequencing tool can support USUV genomic surveillance worldwide.

Fig. 1
Fig.1USUV phylogenetic tree.Newly obtained USUV (in red) strains and available complete genomes (in black) of USUV in Genbank were analysed to obtain a phylogenetic tree to compare sequences each others

Table 1
Sequences of the USUV primers set

Table 2
Inclusivity test of the USUV primers

Table 3
Sensitivity test of the USUV primers

Table 4
Test of the USUV primers with USUV homogenatesThe Table4shows the results obtained after sequencing of USUV homogenates (mosquitoes and birds) with the USUV primers set.The hosts species, Ct values of tested strains, number of total reads and specific USUV reads, horizontal and vertical coverages and consensus sequence length obtained after sequencing were presented in this table

Table 5
Quantitative and conventional RT-PCR results of samples with multiple viral speciesThe Table5shows the quantitative and conventional RT-PCR results of samples with multiple viral species including USUV.Host species, Ct values and/or results of conventional RT-PCR of different viruses detected in each strain were presented in this table (-): Negative (+): Detected by conventional RT-PCR SINDV: Sindbis virus; MESOV: Mesonivirus virus; BAGV: Bagaza virus; WNV: West nile virus; BARKV: Barkedji virus; ONNV: O'nyong'nyong virus