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Rapid Amplicon Nanopore Sequencing (RANS) for the Differential Diagnosis of Monkeypox Virus and Other Vesicle-Forming Pathogens

As of July 2022, more than 16,000 laboratory-confirmed monkeypox (MPX) cases have been reported worldwide. Until recently, MPX was a rare viral disease seldom detected outside Africa. MPX virus (MPXV) belongs to the Orthopoxvirus (OPV) genus and is a genetically close relative of the Variola virus (...

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Autores principales: Israeli, Ofir, Guedj-Dana, Yehoudit, Shifman, Ohad, Lazar, Shirley, Cohen-Gihon, Inbar, Amit, Sharon, Ben-Ami, Ronen, Paran, Nir, Schuster, Ofir, Weiss, Shay, Zvi, Anat, Beth-Din, Adi
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9416277/
https://www.ncbi.nlm.nih.gov/pubmed/36016439
http://dx.doi.org/10.3390/v14081817
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author Israeli, Ofir
Guedj-Dana, Yehoudit
Shifman, Ohad
Lazar, Shirley
Cohen-Gihon, Inbar
Amit, Sharon
Ben-Ami, Ronen
Paran, Nir
Schuster, Ofir
Weiss, Shay
Zvi, Anat
Beth-Din, Adi
author_facet Israeli, Ofir
Guedj-Dana, Yehoudit
Shifman, Ohad
Lazar, Shirley
Cohen-Gihon, Inbar
Amit, Sharon
Ben-Ami, Ronen
Paran, Nir
Schuster, Ofir
Weiss, Shay
Zvi, Anat
Beth-Din, Adi
author_sort Israeli, Ofir
collection PubMed
description As of July 2022, more than 16,000 laboratory-confirmed monkeypox (MPX) cases have been reported worldwide. Until recently, MPX was a rare viral disease seldom detected outside Africa. MPX virus (MPXV) belongs to the Orthopoxvirus (OPV) genus and is a genetically close relative of the Variola virus (the causative agent of smallpox). Following the eradication of smallpox, there was a significant decrease in smallpox-related morbidity and the population’s immunity to other OPV-related diseases such as MPX. In parallel, there was a need for differential diagnosis between the different OPVs’ clinical manifestations and diseases with similar symptoms (i.e., chickenpox, herpes simplex). The current study aimed to provide a rapid genetic-based diagnostic tool for accurate and specific identification of MPXV and additional related vesicle-forming pathogens. We initially assembled a list of 14 relevant viral pathogens, causing infectious diseases associated with vesicles, prone to be misdiagnosed as MPX. Next, we developed an approach that we termed rapid amplicon nanopore sequencing (RANS). The RANS approach uses diagnostic regions that harbor high homology in their boundaries and internal diagnostic SNPs that, when sequenced, aid the discrimination of each pathogen within a group. During a multiplex PCR amplification, a dA tail and a 5′-phosphonate were simultaneously added, thus making the PCR product ligation ready for nanopore sequencing. Following rapid sequencing (a few minutes), the reads were compared to a reference database and the nearest strain was identified. We first tested our approach using samples of known viruses cultured in cell lines. All the samples were identified correctly and swiftly. Next, we examined a variety of clinical samples from the 2022 MPX outbreak. Our RANS approach identified correctly all the PCR-positive MPXV samples and mapped them to strains that were sequenced during the 2022 outbreak. For the subset of samples that were negative for MPXV by PCR, we obtained definite results, identifying other vesicle-forming viruses: Human herpesvirus 3, Human herpesvirus 2, and Molluscum contagiosum virus. This work was a proof-of-concept study, demonstrating the potential of the RANS approach for rapid and discriminatory identification of a panel of closely related pathogens. The simplicity and affordability of our approach makes it straightforward to implement in any genetics lab. Moreover, other differential diagnostics panels might benefit from the implementation of the RANS approach into their diagnostics pipelines.
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spelling pubmed-94162772022-08-27 Rapid Amplicon Nanopore Sequencing (RANS) for the Differential Diagnosis of Monkeypox Virus and Other Vesicle-Forming Pathogens Israeli, Ofir Guedj-Dana, Yehoudit Shifman, Ohad Lazar, Shirley Cohen-Gihon, Inbar Amit, Sharon Ben-Ami, Ronen Paran, Nir Schuster, Ofir Weiss, Shay Zvi, Anat Beth-Din, Adi Viruses Communication As of July 2022, more than 16,000 laboratory-confirmed monkeypox (MPX) cases have been reported worldwide. Until recently, MPX was a rare viral disease seldom detected outside Africa. MPX virus (MPXV) belongs to the Orthopoxvirus (OPV) genus and is a genetically close relative of the Variola virus (the causative agent of smallpox). Following the eradication of smallpox, there was a significant decrease in smallpox-related morbidity and the population’s immunity to other OPV-related diseases such as MPX. In parallel, there was a need for differential diagnosis between the different OPVs’ clinical manifestations and diseases with similar symptoms (i.e., chickenpox, herpes simplex). The current study aimed to provide a rapid genetic-based diagnostic tool for accurate and specific identification of MPXV and additional related vesicle-forming pathogens. We initially assembled a list of 14 relevant viral pathogens, causing infectious diseases associated with vesicles, prone to be misdiagnosed as MPX. Next, we developed an approach that we termed rapid amplicon nanopore sequencing (RANS). The RANS approach uses diagnostic regions that harbor high homology in their boundaries and internal diagnostic SNPs that, when sequenced, aid the discrimination of each pathogen within a group. During a multiplex PCR amplification, a dA tail and a 5′-phosphonate were simultaneously added, thus making the PCR product ligation ready for nanopore sequencing. Following rapid sequencing (a few minutes), the reads were compared to a reference database and the nearest strain was identified. We first tested our approach using samples of known viruses cultured in cell lines. All the samples were identified correctly and swiftly. Next, we examined a variety of clinical samples from the 2022 MPX outbreak. Our RANS approach identified correctly all the PCR-positive MPXV samples and mapped them to strains that were sequenced during the 2022 outbreak. For the subset of samples that were negative for MPXV by PCR, we obtained definite results, identifying other vesicle-forming viruses: Human herpesvirus 3, Human herpesvirus 2, and Molluscum contagiosum virus. This work was a proof-of-concept study, demonstrating the potential of the RANS approach for rapid and discriminatory identification of a panel of closely related pathogens. The simplicity and affordability of our approach makes it straightforward to implement in any genetics lab. Moreover, other differential diagnostics panels might benefit from the implementation of the RANS approach into their diagnostics pipelines. MDPI 2022-08-18 /pmc/articles/PMC9416277/ /pubmed/36016439 http://dx.doi.org/10.3390/v14081817 Text en © 2022 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
spellingShingle Communication
Israeli, Ofir
Guedj-Dana, Yehoudit
Shifman, Ohad
Lazar, Shirley
Cohen-Gihon, Inbar
Amit, Sharon
Ben-Ami, Ronen
Paran, Nir
Schuster, Ofir
Weiss, Shay
Zvi, Anat
Beth-Din, Adi
Rapid Amplicon Nanopore Sequencing (RANS) for the Differential Diagnosis of Monkeypox Virus and Other Vesicle-Forming Pathogens
title Rapid Amplicon Nanopore Sequencing (RANS) for the Differential Diagnosis of Monkeypox Virus and Other Vesicle-Forming Pathogens
title_full Rapid Amplicon Nanopore Sequencing (RANS) for the Differential Diagnosis of Monkeypox Virus and Other Vesicle-Forming Pathogens
title_fullStr Rapid Amplicon Nanopore Sequencing (RANS) for the Differential Diagnosis of Monkeypox Virus and Other Vesicle-Forming Pathogens
title_full_unstemmed Rapid Amplicon Nanopore Sequencing (RANS) for the Differential Diagnosis of Monkeypox Virus and Other Vesicle-Forming Pathogens
title_short Rapid Amplicon Nanopore Sequencing (RANS) for the Differential Diagnosis of Monkeypox Virus and Other Vesicle-Forming Pathogens
title_sort rapid amplicon nanopore sequencing (rans) for the differential diagnosis of monkeypox virus and other vesicle-forming pathogens
topic Communication
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9416277/
https://www.ncbi.nlm.nih.gov/pubmed/36016439
http://dx.doi.org/10.3390/v14081817
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