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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Main Authors: Hiroho Ishida, Shin Murakami, Haruhiko Kamiki, Hiromichi Matsugo, Misa Katayama, Wataru Sekine, Kosuke Ohira, Akiko Takenaka-Uema, Taisuke Horimoto
Format: Article
Language:English
Published: MDPI AG 2021-10-01
Series:Viruses
Subjects:
Online Access:https://www.mdpi.com/1999-4915/13/11/2166
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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
collection DOAJ
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.
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spelling doaj.art-5acac4dbed744e5b9926fe325cd4c6562023-11-23T01:55:57ZengMDPI AGViruses1999-49152021-10-011311216610.3390/v13112166Construction of an Influenza D Virus with an Eight-Segmented GenomeHiroho Ishida0Shin Murakami1Haruhiko Kamiki2Hiromichi Matsugo3Misa Katayama4Wataru Sekine5Kosuke Ohira6Akiko Takenaka-Uema7Taisuke Horimoto8Department of Veterinary Microbiology, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo 113-8657, JapanDepartment of Veterinary Microbiology, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo 113-8657, JapanDepartment of Veterinary Microbiology, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo 113-8657, JapanDepartment of Veterinary Microbiology, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo 113-8657, JapanDepartment of Veterinary Microbiology, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo 113-8657, JapanDepartment of Veterinary Microbiology, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo 113-8657, JapanDepartment of Veterinary Microbiology, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo 113-8657, JapanDepartment of Veterinary Microbiology, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo 113-8657, JapanDepartment of Veterinary Microbiology, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo 113-8657, JapanInfluenza 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.https://www.mdpi.com/1999-4915/13/11/2166bovine respiratory disease complexinfluenza D virusmutantrecombinant virusreverse geneticsnonstructural protein
spellingShingle 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
Viruses
bovine respiratory disease complex
influenza D virus
mutant
recombinant virus
reverse genetics
nonstructural protein
title 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_short Construction of an Influenza D Virus with an Eight-Segmented Genome
title_sort construction of an influenza d virus with an eight segmented genome
topic bovine respiratory disease complex
influenza D virus
mutant
recombinant virus
reverse genetics
nonstructural protein
url https://www.mdpi.com/1999-4915/13/11/2166
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