Zika virus isolation, propagation, and quantification using multiple methods.

Zika virus (ZIKV) was isolated from the archival urine, serum, and autopsy specimens by intrathoracic inoculation of Toxorhynchitis splendens and followed by three blind sub-passaging in C6/36 mosquito cells. The virus isolates were identified using an immunofluorescence assay and real-time reverse...

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Autores principales:	Worawat Dangsagul, Kriengsak Ruchusatsawat, Apiwat Tawatsin, Don Changsom, Pirom Noisumdaeng, Sukontip Putchakarn, Chayawat Phatihattakorn, Prasert Auewarakul, Pilaipan Puthavathana
Formato:	article
Lenguaje:	EN
Publicado:	Public Library of Science (PLoS) 2021
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/b3e9228075e54ba9adffe9f354264a7a
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spelling	oai:doaj.org-article:b3e9228075e54ba9adffe9f354264a7a2021-12-02T20:08:52ZZika virus isolation, propagation, and quantification using multiple methods.1932-620310.1371/journal.pone.0255314https://doaj.org/article/b3e9228075e54ba9adffe9f354264a7a2021-01-01T00:00:00Zhttps://doi.org/10.1371/journal.pone.0255314https://doaj.org/toc/1932-6203Zika virus (ZIKV) was isolated from the archival urine, serum, and autopsy specimens by intrathoracic inoculation of Toxorhynchitis splendens and followed by three blind sub-passaging in C6/36 mosquito cells. The virus isolates were identified using an immunofluorescence assay and real-time reverse transcription-polymerase chain reaction (real-time RT-PCR). This study analyzed 11 ZIKV isolates. One isolate (0.6%) was obtained from 171 urine samples, eight (8.7%) from 92 serum samples and two from tissues of an abortive fetus. After propagation in C6/36 cells, ZIKV was titrated by plaque and focus forming unit (FFU) assays in Vero cell monolayers, and viral genomes were determined via real-time and digital RT-PCR. Plaque and FFU assay quantitations were comparable, with the amount of infectious viruses averaging 106-107 PFU or FFU/ml. Real-time RT-PCR semi-quantified the viral genome numbers, with Ct values varying from 12 to 14. Digital RT-PCR, which precisely determines the numbers of the viral genomes, consistently averaged 10-100 times higher than the number of infectious units. There was good correlation between the results of these titration methods. Therefore, the selection of a method should be based on the objectives of each research studies.Worawat DangsagulKriengsak RuchusatsawatApiwat TawatsinDon ChangsomPirom NoisumdaengSukontip PutchakarnChayawat PhatihattakornPrasert AuewarakulPilaipan PuthavathanaPublic Library of Science (PLoS)articleMedicineRScienceQENPLoS ONE, Vol 16, Iss 7, p e0255314 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Worawat Dangsagul Kriengsak Ruchusatsawat Apiwat Tawatsin Don Changsom Pirom Noisumdaeng Sukontip Putchakarn Chayawat Phatihattakorn Prasert Auewarakul Pilaipan Puthavathana Zika virus isolation, propagation, and quantification using multiple methods.
description	Zika virus (ZIKV) was isolated from the archival urine, serum, and autopsy specimens by intrathoracic inoculation of Toxorhynchitis splendens and followed by three blind sub-passaging in C6/36 mosquito cells. The virus isolates were identified using an immunofluorescence assay and real-time reverse transcription-polymerase chain reaction (real-time RT-PCR). This study analyzed 11 ZIKV isolates. One isolate (0.6%) was obtained from 171 urine samples, eight (8.7%) from 92 serum samples and two from tissues of an abortive fetus. After propagation in C6/36 cells, ZIKV was titrated by plaque and focus forming unit (FFU) assays in Vero cell monolayers, and viral genomes were determined via real-time and digital RT-PCR. Plaque and FFU assay quantitations were comparable, with the amount of infectious viruses averaging 106-107 PFU or FFU/ml. Real-time RT-PCR semi-quantified the viral genome numbers, with Ct values varying from 12 to 14. Digital RT-PCR, which precisely determines the numbers of the viral genomes, consistently averaged 10-100 times higher than the number of infectious units. There was good correlation between the results of these titration methods. Therefore, the selection of a method should be based on the objectives of each research studies.
format	article
author	Worawat Dangsagul Kriengsak Ruchusatsawat Apiwat Tawatsin Don Changsom Pirom Noisumdaeng Sukontip Putchakarn Chayawat Phatihattakorn Prasert Auewarakul Pilaipan Puthavathana
author_facet	Worawat Dangsagul Kriengsak Ruchusatsawat Apiwat Tawatsin Don Changsom Pirom Noisumdaeng Sukontip Putchakarn Chayawat Phatihattakorn Prasert Auewarakul Pilaipan Puthavathana
author_sort	Worawat Dangsagul
title	Zika virus isolation, propagation, and quantification using multiple methods.
title_short	Zika virus isolation, propagation, and quantification using multiple methods.
title_full	Zika virus isolation, propagation, and quantification using multiple methods.
title_fullStr	Zika virus isolation, propagation, and quantification using multiple methods.
title_full_unstemmed	Zika virus isolation, propagation, and quantification using multiple methods.
title_sort	zika virus isolation, propagation, and quantification using multiple methods.
publisher	Public Library of Science (PLoS)
publishDate	2021
url	https://doaj.org/article/b3e9228075e54ba9adffe9f354264a7a
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Zika virus isolation, propagation, and quantification using multiple methods.

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