Abstract
BackgroundReverse genetics systems enable the manipulation of viral genomes and therefore serve as robust reverse genetic tools to study RNA viruses. A DNA-launched rescue system initiates the transcription of viral genomic cDNA from eukaryotic promoter in transfected cells, generating homogenous RNA transcripts in vitro and thus enhancing virus rescue efficiency. As one of the hazardous pathogens to ducklings, the current knowledge of the pathogenesis of duck astrovirus type 1 (DAstV-1) is limited. The construction of a DNA-launched rescue system can help to accelerate the study of the virus pathogenesis. However, there is no report of such a system for DAstV-1.MethodsIn this study, a DNA-launched infectious clone of DAstV-1 was constructed from a cDNA plasmid, which contains a viral cDNA sequence flanked by hammerhead ribozyme (HamRz) and a hepatitis delta virus ribozyme (HdvRz) sequence at both terminals of the viral genome. A silent nucleotide mutation creating a Bgl II site in the ORF2 gene was made to distinguish the rescued virus (rDAstV-1) from the parental virus (pDAstV-1). Immunofluorescence assay (IFA) and western blot were conducted for rescued virus identification in duck embryo fibroblast (DEF) cells pre-treated with trypsin. The growth characteristics of rDAstV-1 and pDAstV-1 in DEF cells and the tissue tropism in 2-day-old ducklings of rDAstV-1 and pDAstV-1 were determined.ResultsThe infectious DAstV-1 was successfully rescued from baby hamster kidney (BHK-21) cells and could propagate in DEF cells pre-treated with 1 μg/ml trypsin. Upon infection of DEF cells pre-treated with trypsin, DAstV-1 mRNA copies were identified after serial passaging, and the result showed that rDAstV-1 and pDAstV-1 shared similar replication kinetics. Animal experiment showed that the rDAstV-1 had an extensive tissue tropism, and the virus was capable of invading both the central and the peripheral immune organs in infected ducklings.ConclusionsAn improved DNA-launched reverse genetics system for DAstV-1 was firstly constructed. Infectious virus recovered from BHK-21 cells could propagate in DEF cells pre-treated with trypsin. This is the first report of the successful in vitro cultivation of DAstV-1. We believe this valuable experimental system will contribute to the further study of DAstV-1 genome function and pathogenesis.
Highlights
Reverse genetics systems enable the manipulation of viral genomes and serve as robust reverse genetic tools to study RNA viruses
The rescued duck astrovirus type 1 (DAstV-1) could be propagated in duck embryonic fibroblasts (DEF) cells pre-treated with trypsin
Comparison of growth characteristics between the pDAstV-1 and rDAstV-1 The pDAstV-1 of D51 could be propagated in duck embryo fibroblast (DEF) cells pre-treated with trypsin as described above
Summary
Reverse genetics systems enable the manipulation of viral genomes and serve as robust reverse genetic tools to study RNA viruses. Members of the Astroviridae family are non-enveloped, positive and single-stranded RNA viruses, typically 28 to 30 nm in diameter. This family is subdivided into two genera with Mamastrovirus and Avastrovirus, known to infect mammalian and avian species, respectively [1]. As a member of the genus Avastrovirus of the family Astroviridae, duck astrovirus (DAstV) was first reported in England [7]. Astrovirus infection usually causes mild and self-limiting gastroenteritis in most animal species but leads to severe symptoms in poultry, closely related to enteric diseases [10,11,12,13], even associated with fatal hepatitis in ducklings [14,15,16,17]
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