Abstract
The ruminant-associated influenza D virus (IDV) has a broad host tropism and was shown to have zoonotic potential. To identify and characterize molecular viral determinants influencing the host spectrum of IDV, a reverse genetic system is required. For this, we first performed 5′ and 3′ rapid amplification of cDNA ends (RACE) of all seven genomic segments, followed by assessment of the 5′ and 3′ NCR activity prior to constructing the viral genomic segments of a contemporary Swiss bovine IDV isolate (D/CN286) into the bidirectional pHW2000 vector. The bidirectional plasmids were transfected in HRT-18G cells followed by viral rescue on the same cell type. Analysis of the segment specific 5′ and 3′ non-coding regions (NCR) highlighted that the terminal 3′ end of all segments harbours an uracil instead of a cytosine nucleotide, similar to other influenza viruses. Subsequent analysis on the functionality of the 5′ and 3′ NCR in a minireplicon assay revealed that these sequences were functional and that the variable sequence length of the 5′ and 3′ NCR influences reporter gene expression. Thereafter, we evaluated the replication efficiency of the reverse genetic clone on conventional cell lines of human, swine and bovine origin, as well as by using an in vitro model recapitulating the natural replication site of IDV in bovine and swine. This revealed that the reverse genetic clone D/CN286 replicates efficiently in all cell culture models. Combined, these results demonstrate the successful establishment of a reverse genetic system from a contemporary bovine IDV isolate that can be used for future identification and characterization of viral determinants influencing the broad host tropism of IDV.
Highlights
Far no reverse genetic system has been established for a bovine isolate of influenza D virus (IDV), that can be used to characterize viral determinants which influences the broad host tropism of IDV among even-toed ungulates
In this study we demonstrate the establishment of a functional reverse genetic system for a contemporary bovine isolate of IDV
Using 50 and 30 rapid amplification of cDNA ends (RACE), we demonstrate that the 11 and 12 terminal nucleotides on the 30 and 50 non-coding regions (NCR), respectively are conserved on all viral genomic segments
Summary
The Deltainfluenzavirus genus is the newest member of the Orthomyxoviridae virus family, and harbours the influenza D virus (IDV) species [1,2]. In 2011, IDV was first identified in a 15-week-old pig suffering from flu-like symptoms such as sneezing and dry cough [3]. In addition to cattle and swine, IDV was shown to infect a broad range of other animals, such as feral swine, water buffalo, sheep, horses and camelids [3,8,12,13,14,15,16]. The high seroprevalence among humans with frequent exposure to cattle, as
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