[Source: Journal of Virology, full page: (LINK). Abstract, edited.]
Development and characterization of a reverse genetics system for influenza D virus
Jieshi Yu, Runxia Liu, Bin Zhou, Tsui-wen Chou, Elodie Ghedin, Zizhang Sheng, Rongyuan Gao, Shao-lun Zhai, Dan Wang, Feng Li
Influenza D virus (IDV) of the Orthomyxoviridae family has a wide host range and a broad geographical distribution. Recent IDV outbreaks in swine, along with serological and genetic evidence of IDV infection in humans have raised concerns regarding the zoonotic potential of this virus. To better study IDV at the molecular level, a reverse genetics system (RGS) is urgently needed, but to date no RGS had been described for IDV. In this study, we rescued the recombinant influenza D/swine/Oklahoma/1314/2011 (D/OK) virus by using a bidirectional seven plasmid-based system, and further characterized rescued viruses in terms of growth kinetics, replication stability, and receptor-binding capacity. Our results collectively demonstrated that RGS-derived viruses resembled the parental viruses for these properties, thereby supporting the utility of this RGS to study IDV infection biology. In addition, we developed an IDV mini-genome replication assay and identified the E697K mutation in PB1 and the L462F mutation in PB2 that directly affected the activity of the IDV ribonucleoprotein complex (RNP), resulting in either attenuated or replication-incompetent viruses. Finally, by using the mini-genome replication assay, we demonstrated that a single nucleotide polymorphism at position 5 of the 3′ conserved noncoding region in IDV and ICV resulted in the inefficient cross-recognition of the heterotypic promoter by the viral RNP complex. In conclusion, we successfully developed a mini-genome replication assay and a robust reverse genetics system that can be used to further study replication, tropism, and pathogenesis of IDV.
Influenza D virus (IDV) is a new type of influenza virus that uses cattle as the primary reservoir and infects multiple agricultural animals. Increased outbreaks in pigs, and serological and genetic evidence of human infection have raised concerns about potential IDV adaptation in humans. Here, we have developed a plasmid-based IDV reverse genetics system that can generate infectious viruses similar in replication kinetics to wild-type viruses following transfection of cultured cells. Further characterization demonstrated that viruses rescued from the described RGS resembled the parental viruses in biological and receptor binding properties. We also developed and validated an IDV minireplicon reporter system that specifically measures viral RNA polymerase activity. In summary, the reverse genetics system and minireplicon reporter assay as described in this study should be of value in identifying viral determinants of cross-species transmission and pathogenicity of novel influenza D viruses.
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Keywords: Influenza D.