RRC ID 65677
Author Ishida H, Murakami S, Kamiki H, Matsugo H, Takenaka-Uema A, Horimoto T.
Title Establishment of a Reverse Genetics System for Influenza D Virus.
Journal J Virol
Abstract Influenza D virus (IDV) was initially isolated in the United States in 2011. IDV is distributed worldwide and is one of the causative agents of the bovine respiratory disease complex (BRDC), which causes high morbidity and mortality in feedlot cattle. The molecular mechanisms of IDV pathogenicity are still unknown. Reverse genetics systems are vital tools not only for studying the biology of viruses, but also for use in applications such as recombinant vaccine viruses. Here, we report the establishment of a plasmid-based reverse genetics system for IDV. We first verified that the 3'-terminal nucleotide of each 7-segmented genomic RNA contained uracil (U), contrary to previous reports, and we were then able to successfully generate recombinant IDV by cotransfecting 7 plasmids containing these genomic RNAs along with 4 plasmids expressing polymerase proteins and nucleoprotein into human rectal tumor 18G (HRT-18G) cells. The recombinant virus had a growth deficit compared to the wild-type virus, and we determined the reason for this growth difference by examining the genomic RNA content of the viral particles. We found that the recombinant virus incorporated an unbalanced ratio of viral RNA segments into particles compared to that of the wild-type virus, and thus we adjusted the amount of each plasmid used in transfection to obtain a recombinant virus with the same replicative capacity as the wild-type virus. Our work here in establishing a reverse genetics system for IDV will have a broad range of applications, including uses in studies focused on better understanding IDV replication and pathogenicity, as well as in those contributing to the development of BRDC countermeasures.IMPORTANCE The bovine respiratory disease complex (BRDC) causes high mortality and morbidity in cattle, causing economic losses worldwide. Influenza D virus (IDV) is considered to be a causative agent of the BRDC. Here, we developed a reverse genetics system that allows for the generation of IDV from cloned cDNAs and the introduction of mutations into the IDV genome. This reverse genetics system will become a powerful tool for use in studies related to understanding the molecular mechanisms of viral replication and pathogenicity and will also lead to the development of new countermeasures against the BRDC.
Volume 94(10)
Published 2020-5-4
DOI 10.1128/JVI.01767-19
PII JVI.01767-19
PMID 32102883
PMC PMC7199395
MeSH Animals Bovine Respiratory Disease Complex Cattle Cell Line, Tumor DNA, Complementary Genetic Vectors / genetics Genome, Viral HEK293 Cells Hemagglutination Humans Influenza, Human Orthomyxoviridae Infections / virology Plasmids RNA, Viral Rectal Neoplasms / virology Reverse Genetics / methods* Thogotovirus / genetics* Thogotovirus / growth & development Transfection Virion / genetics Virus Replication
IF 4.501
Human and Animal Cells 293T(RCB2202)