Establishment of a Reverse Genetics System for Influenza D Virus

Influenza D virus (IDV) was initially isolated in the USA in 2011. IDV is distributed worldwide and is one of the causative agents of bovine respiratory disease complex (BRDC), which exhibits high morbidity and mortality in feedlot cattle. 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 in contrary to the previous report, and were then able to successfully generate recombinant IDV by co-transfecting 7 plasmids containing these genomic RNAs along with 4 plasmids expressing polymerase proteins and NP into 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 recombinant virus incorporated an unbalanced ratio of viral RNA segments into particles as compared to the wild-type virus, and thus we adjusted the amount of each plasmid used in transfection to obtain recombinant virus with the same replicative capacity as 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 those contributing to the development of BRDC countermeasures.\n\nIMPORTANCEBovine respiratory disease complex (BRDC) exhibits high mortality and morbidity in cattle, causing economic losses worldwide. Influenza D virus (IDV) is considered to be a causative agent of 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 BRDC.