Construction of an Influenza D Virus with an Eight-Segmented Genome

Influenza D virus (IDV) may cause the bovine respiratory disease complex, which is the most common and costly disease affecting the cattle industry. Previously, we revealed that eight segments could be actively packaged in its single virion, suggesting that IDV with the seven-segmented genome shows...

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Autores principales:	Hiroho Ishida, Shin Murakami, Haruhiko Kamiki, Hiromichi Matsugo, Misa Katayama, Wataru Sekine, Kosuke Ohira, Akiko Takenaka-Uema, Taisuke Horimoto
Formato:	article
Lenguaje:	EN
Publicado:	MDPI AG 2021
Materias:	bovine respiratory disease complex influenza D virus mutant recombinant virus reverse genetics nonstructural protein Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/5acac4dbed744e5b9926fe325cd4c656
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spelling	oai:doaj.org-article:5acac4dbed744e5b9926fe325cd4c6562021-11-25T19:12:58ZConstruction of an Influenza D Virus with an Eight-Segmented Genome10.3390/v131121661999-4915https://doaj.org/article/5acac4dbed744e5b9926fe325cd4c6562021-10-01T00:00:00Zhttps://www.mdpi.com/1999-4915/13/11/2166https://doaj.org/toc/1999-4915Influenza D virus (IDV) may cause the bovine respiratory disease complex, which is the most common and costly disease affecting the cattle industry. Previously, we revealed that eight segments could be actively packaged in its single virion, suggesting that IDV with the seven-segmented genome shows an agnostic genome packaging mechanism. Herein, we engineered an eight-segmented recombinant IDV in which the <i>NS1</i> or <i>NS2</i> genes were separated from NS segment into independent segments (NS1 or NS2 segments, respectively), leading to monocistronic translation of each NS protein. We constructed two plasmids: one for the viral RNA (vRNA)-synthesis of the NS1 segment with a silent mutation at the splicing acceptor site, which controls NS2 transcription in the NS segment; and another for the RNA synthesis of the NS2 segment, with deletion of the intron in the NS segment. These plasmids and six other vRNA-synthesis plasmids were used to fabricate an infectious eight-segmented IDV via reverse genetics. This system enables analysis of the functions of NS1 or NS2. We tested the requirement of the N-terminal overlapping region (NOR) in these proteins for viral infectivity. We rescued a virus with NOR-deleted NS2 protein, which displayed a growth rate equivalent to that of the eight-segmented virus with intact NS2. Thus, the NOR may not influence viral growth. In contrast, a virus with NOR-deleted NS1 protein could not be rescued. These results indicate that the eight-segmented rescue system of IDV may provide an alternative method to analyze viral proteins at the molecular level.Hiroho IshidaShin MurakamiHaruhiko KamikiHiromichi MatsugoMisa KatayamaWataru SekineKosuke OhiraAkiko Takenaka-UemaTaisuke HorimotoMDPI AGarticlebovine respiratory disease complexinfluenza D virusmutantrecombinant virusreverse geneticsnonstructural proteinMicrobiologyQR1-502ENViruses, Vol 13, Iss 2166, p 2166 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	bovine respiratory disease complex influenza D virus mutant recombinant virus reverse genetics nonstructural protein Microbiology QR1-502
spellingShingle	bovine respiratory disease complex influenza D virus mutant recombinant virus reverse genetics nonstructural protein Microbiology QR1-502 Hiroho Ishida Shin Murakami Haruhiko Kamiki Hiromichi Matsugo Misa Katayama Wataru Sekine Kosuke Ohira Akiko Takenaka-Uema Taisuke Horimoto Construction of an Influenza D Virus with an Eight-Segmented Genome
description	Influenza D virus (IDV) may cause the bovine respiratory disease complex, which is the most common and costly disease affecting the cattle industry. Previously, we revealed that eight segments could be actively packaged in its single virion, suggesting that IDV with the seven-segmented genome shows an agnostic genome packaging mechanism. Herein, we engineered an eight-segmented recombinant IDV in which the <i>NS1</i> or <i>NS2</i> genes were separated from NS segment into independent segments (NS1 or NS2 segments, respectively), leading to monocistronic translation of each NS protein. We constructed two plasmids: one for the viral RNA (vRNA)-synthesis of the NS1 segment with a silent mutation at the splicing acceptor site, which controls NS2 transcription in the NS segment; and another for the RNA synthesis of the NS2 segment, with deletion of the intron in the NS segment. These plasmids and six other vRNA-synthesis plasmids were used to fabricate an infectious eight-segmented IDV via reverse genetics. This system enables analysis of the functions of NS1 or NS2. We tested the requirement of the N-terminal overlapping region (NOR) in these proteins for viral infectivity. We rescued a virus with NOR-deleted NS2 protein, which displayed a growth rate equivalent to that of the eight-segmented virus with intact NS2. Thus, the NOR may not influence viral growth. In contrast, a virus with NOR-deleted NS1 protein could not be rescued. These results indicate that the eight-segmented rescue system of IDV may provide an alternative method to analyze viral proteins at the molecular level.
format	article
author	Hiroho Ishida Shin Murakami Haruhiko Kamiki Hiromichi Matsugo Misa Katayama Wataru Sekine Kosuke Ohira Akiko Takenaka-Uema Taisuke Horimoto
author_facet	Hiroho Ishida Shin Murakami Haruhiko Kamiki Hiromichi Matsugo Misa Katayama Wataru Sekine Kosuke Ohira Akiko Takenaka-Uema Taisuke Horimoto
author_sort	Hiroho Ishida
title	Construction of an Influenza D Virus with an Eight-Segmented Genome
title_short	Construction of an Influenza D Virus with an Eight-Segmented Genome
title_full	Construction of an Influenza D Virus with an Eight-Segmented Genome
title_fullStr	Construction of an Influenza D Virus with an Eight-Segmented Genome
title_full_unstemmed	Construction of an Influenza D Virus with an Eight-Segmented Genome
title_sort	construction of an influenza d virus with an eight-segmented genome
publisher	MDPI AG
publishDate	2021
url	https://doaj.org/article/5acac4dbed744e5b9926fe325cd4c656
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Construction of an Influenza D Virus with an Eight-Segmented Genome

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