Establishment of a Reverse Genetic System from a Bovine Derived Influenza D Virus Isolate

Holwerda, Melle; Laloli, Laura; Wider, Manon; Schönecker, Lutz; Becker, Jens; Meylan, Mireille; Dijkman, Ronald (2021). Establishment of a Reverse Genetic System from a Bovine Derived Influenza D Virus Isolate. Viruses, 13(3) Molecular Diversity Preservation International MDPI 10.3390/v13030502

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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.

Item Type:

Journal Article (Original Article)

Division/Institute:

04 Faculty of Medicine > Service Sector > Institute for Infectious Diseases
05 Veterinary Medicine > Department of Infectious Diseases and Pathobiology (DIP) > Institute of Virology and Immunology
04 Faculty of Medicine > Service Sector > Institute for Infectious Diseases > Research
04 Faculty of Medicine > Pre-clinic Human Medicine > BioMedical Research (DBMR)
05 Veterinary Medicine > Department of Clinical Veterinary Medicine (DKV)
05 Veterinary Medicine > Department of Clinical Veterinary Medicine (DKV) > Clinic for Ruminants
05 Veterinary Medicine > Department of Infectious Diseases and Pathobiology (DIP)
05 Veterinary Medicine > Department of Infectious Diseases and Pathobiology (DIP) > Institute of Veterinary Bacteriology

Graduate School:

Graduate School for Cellular and Biomedical Sciences (GCB)

UniBE Contributor:

Holwerda, Melle; Laloli, Laura; Wider, Manon Flore; Schönecker, Lutz; Becker, Jens Michael; Meylan, Mireille and Dijkman, Ronald

Subjects:

500 Science > 570 Life sciences; biology
600 Technology > 610 Medicine & health
600 Technology > 630 Agriculture
500 Science > 590 Animals (Zoology)

ISSN:

1999-4915

Publisher:

Molecular Diversity Preservation International MDPI

Language:

English

Submitter:

Nathalie Viviane Zollinger

Date Deposited:

08 Apr 2021 11:52

Last Modified:

08 Apr 2021 11:52

Publisher DOI:

10.3390/v13030502

PubMed ID:

33803792

BORIS DOI:

10.48350/154365

URI:

https://boris.unibe.ch/id/eprint/154365

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