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. 2023 Dec 22;16(1):23.
doi: 10.3390/v16010023.

A Multiplex Nanopore Sequencing Approach for the Detection of Multiple Arboviral Species

Joilson Xavier  1 , Vagner Fonseca  2 , Talita Adelino  3 , Felipe C M Iani  3 , Glauco C Pereira  3 , Myrian M Duarte  3 , Mauricio Lima  4 , Emerson Castro  4 , Carla Oliveira  5 , Hegger Fritsch  1 , Natalia Guimarães  3 , Ludmila O Lamounier  3 , Fernanda Khouri Barreto  6 , Camilo M M Braga de Oliveira  7 , Crhistinne C Maymone Gonçalves  8 , Danielle Malta Lima  9 , Elaine C de Oliveira  10 , Gislene G de Castro Lichs  11 , Iago Gomes  12 , Janaina Mazaro  13 , Janete T N Rodrigues  7 , Jayra Abrantes  12 , Jeová K B Colares  9 , Kleber G Luz  14 , Luana Barbosa da Silva  10 , Luiz Demarchi  11 , Magaly C B Câmara  12 , Marina C S Umaki Zardin  11 , Rafaela Sabatini Mello Pinheiro  15 , Rutilene Barbosa Souza  7 , Simone K Haddad  16 , Stephanni Figueiredo da Silva  10 , Svetoslav N Slavov  16 , Themis Rocha  12 , Noelia Morel  17 , Hector Chiparelli  17 , Analía Burgueño  17 , Victoria Bórmida  17 , María N Cortinas  17 , Rosario S Martín  17 , Allan C Pereira  18 , Marcelo F Dos Santos  18 , Walter André Júnior  18 , Jairo Mendez Rico  19 , Leticia Franco  19 , Alexander Rosewell  2 , Rodrigo F do Carmo Said  2 , Carlos F C de Albuquerque  2 , Ethel L Noia Maciel  20 , Marília Santini de Oliveira  21 , Rivaldo Venâncio da Cunha  22 , Livia C Vinhal Frutuoso  23 , Ana M B de Filippis  5 , Marta Giovanetti  4   24   25 , Luiz Carlos Junior Alcantara  4   25
Affiliations

A Multiplex Nanopore Sequencing Approach for the Detection of Multiple Arboviral Species

Joilson Xavier et al. Viruses. .

Abstract

The emergence and continued geographic expansion of arboviruses and the growing number of infected people have highlighted the need to develop and improve multiplex methods for rapid and specific detection of pathogens. Sequencing technologies are promising tools that can help in the laboratory diagnosis of conditions that share common symptoms, such as pathologies caused by emerging arboviruses. In this study, we integrated nanopore sequencing and the advantages of reverse transcription polymerase chain reaction (RT-PCR) to develop a multiplex RT-PCR protocol for the detection of Chikungunya virus (CHIKV) and several orthoflaviviruses (such as dengue (Orthoflavivirus dengue), Zika (Orthoflavivirus zikaense), yellow fever (Orthoflavivirus flavi), and West Nile (Orthoflavivirus nilense) viruses) in a single reaction, which provides data for sequence-based differentiation of arbovirus lineages.

Keywords: Chikungunya virus; arbovirus; genotyping; nanopore sequencing; orthoflaviviruses.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
A multiplex RT-PCR protocol for amplification and sequencing of nucleic acids of Chikungunya virus and orthoflaviviruses (Ampli-FlaCk). (A) Organization of orthoflaviviruses and Chikungunya virus (genus Alphavirus) genomes. Primers (small orange lines) are depicted near their expected binding positions. Genome representations are based on the following references for orthoflaviviruses and Chikungunya virus, respectively: NC_002031 and KP164568.1. (B) Schematic representation of the Ampli-FlaCk workflow.
Figure 2
Figure 2
Sequencing performance of the Ampli-FlaCk protocol. (A) Electrophoresis analysis of RT-PCR products from cultured dengue virus type 2 (DENV-2, Ct = 22) and Chikungunya virus (CHIKV, Ct = 33), in duplicates (top image). The bottom image displays electrophoresis results for cultured dengue virus type 1 (DENV-1, Ct = 25) and West Nile virus (WNV, numbers in parentheses represent samples having different Ct values, 1 = 21, and 2 = 33). M = Marker. Images were cropped and converted to grayscale for clarity; see the Data Availability Statement for original, unedited images. (B) Distribution of the number of reads on target and the RT-qPCR Ct values from the clinical specimens (n = 45) used for testing the protocol. (C) Distribution of the mean depth of coverage and the RT-qPCR Ct values from the tested clinical specimens (n = 45). (D) Distribution of the reads/NTC ratio and the RT-qPCR Ct values from the tested clinical specimens (n = 45). (B–D) The dark blue line represents a smooth local regression line (method = LOESS), and the light grey area around the trend line represents the 95% confidence interval.
Figure 3
Figure 3
Reconstructed genotyping phylogeny of dengue virus type 2 (Orthoflavivirus denguei) and Chikungunya virus. (A) Maximum likelihood phylogenetic reconstruction of DENV-2 using partial sequences (n = 58) of the NS5 gene, including sequences (n = 22, red tips) generated in this study from clinical samples. (B) Maximum likelihood phylogenetic reconstruction of CHIKV using partial sequences (n = 36) of the envelope gene, including sequences (n = 11, pink tips) generated in this study from clinical samples. Numbers along branches represent Ultrafast bootstrap values (only for the main branches, to maintain clarity).
See this image and copyright information in PMC

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