A novel method to rescue and culture duck Astrovirus type 1 in vitro

Abstract

Background Reverse 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. Methods In 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. Results The 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. Conclusions An 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.

Identification of a Type-Specific Epitope in the ORF2 Protein of Duck Astrovirus Type 1

“…The epitope motif identified in this study was conserved among all the DAstV-1 strains, but was obviously different from the strains of DAstV-2, DAstV-3, and DAstV-4 ( Figure 6). To date, only DAstV-1 can be cultured and passaged in cells [28], while other serotypes of DAstV can only be passaged by ducklings [15][16][17]. Reactivity analysis showed that the mAb 3D2 could only react with the epitope 454 STTESA 459 in DAstV-1 ORF2, while not with 454 GDWQSN 459 in DAstV-2 ORF2, 454 DSWKSH 459 in DAstV-3 ORF2, and 454 TNWNGN 459 in DAstV-4 ORF2 (Figure 7).…”

“…DAstV-1 virulent strain D51 (GenBank accession no. MH712856) was isolated from the liver of sick cherry valley ducks in the Shandong province of China in 2012 [28]. The ORF2 gene of DAstV-1 D51 strain was cloned into the prokaryotic expression vectors pET-32a (+) (Novagen, Darmstadt, Germany) and pGEX-6p-1 (GE Healthcare, Amersham, UK) to generate recombinant histidines tagged ORF2 (His-ORF2) and glutathione S-transferase tagged ORF2 (GST-ORF2).…”

“…The baby hamster kidney (BHK-21) cells and duck embryo fibroblasts (DEF) cells were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS) at 37 • C in a 5% CO 2 atmosphere. A DNA-launched infectious clone of DAstV-1 D51 strain, named pABX-D51, was constructed and stored in our lab [28]. The complete ORF2 genes of DAstV-2 SL1 strain (AHX26592), DAstV-3 CPH strain (AID55207), and DAstV-4 YP2 strain (AIS22433), and all the primers used in this study were synthesized by Sangon Biotech Co., Ltd. (Shanghai, China).…”

Beyond the Gastrointestinal Tract: The Emerging and Diverse Tissue Tropisms of Astroviruses

“…One organ system that raises further questions is the detection of astroviruses from tissues of the cardiovascular system. In a small number of studies, astrovirus RNA has been detected in infected human and animal hearts, including pigs, ducks, chickens, and geese [ 19 , 66 , 142 , 143 , 144 ]. In addition, histological findings of myocarditis or epicarditis were identified from two pigs infected with porcine astrovirus [ 66 ].…”

“…Similarly, it is unknown whether astroviruses can infect cells of the immune or hematological systems. Astroviruses have been detected by PCR from splenic tissue, and, in some cases, viral protein has been detected by immunofluorescence in specimens from the spleen [ 51 , 53 , 82 , 143 ]. In addition, the detection of astroviruses in stool has been associated with an increased risk of immune thrombocytopenia in children [ 145 ].…”