Construction and characterization of an improved DNA-launched infectious clone of duck hepatitis a virus type 1

Chen, Junhao; Zhang, Ruihua; Lin, Shaoli; Li, Pengfei; Lan, Jingjing; Xie, Zhixun; Wang, Yu; Jiang, Shijin

doi:10.1186/s12985-017-0883-5

Cited by 9 publications

(6 citation statements)

References 28 publications

(31 reference statements)

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“…Secondly, we speculated that the organs of healthy ducklings with low levels of ILF2 might be preferentially infected with DHAV-1 since ILF2 inhibits DHAV-1 replication. Previous studies have shown that DHAV-1 preferentially infects liver tissue since the viral genome could only be detected in liver as early as 1 hpi [ 37 ]. Therefore, the highly low initial transcription levels of ILF2 in liver tissue (Figure 7 A) might contribute to the hepatotropism of DHAV-1.…”

Section: Discussionmentioning

confidence: 99%

Interleukin-2 enhancer binding factor 2 negatively regulates the replication of duck hepatitis A virus type 1 by disrupting the RNA-dependent RNA polymerase activity of 3D polymerase

An,

Yu,

et al. 2024

Vet Res

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The interaction between viral components and cellular proteins plays a crucial role in viral replication. In a previous study, we showed that the 3′—untranslated region (3′—UTR) is an essential element for the replication of duck hepatitis A virus type 1 (DHAV-1). However, the underlying mechanism is still unclear. To gain a deeper understanding of this mechanism, we used an RNA pull-down and a matrix-assisted laser desorption/ionization time-of-flight mass spectrometry assay to identify new host factors that interact with the 3′—UTR. We selected interleukin-2 enhancer binding factor 2 (ILF2) for further analysis. We showed that ILF2 interacts specifically with both the 3′—UTR and the 3D polymerase (3Dpol) of DHAV-1 through in vitro RNA pull-down and co-immunoprecipitation assays, respectively. We showed that ILF2 negatively regulates viral replication in duck embryo fibroblasts (DEFs), and that its overexpression in DEFs markedly suppresses DHAV-1 replication. Conversely, ILF2 silencing resulted in a significant increase in viral replication. In addition, the RNA-dependent RNA polymerase (RdRP) activity of 3Dpol facilitated viral replication by enhancing viral RNA translation efficiency, whereas ILF2 disrupted the role of RdRP in viral RNA translation efficiency to suppress DHAV-1 replication. At last, DHAV-1 replication markedly suppressed the expression of ILF2 in DEFs, duck embryo hepatocytes, and different tissues of 1 day-old ducklings. A negative correlation was observed between ILF2 expression and the viral load in primary cells and different organs of young ducklings, suggesting that ILF2 may affect the viral load both in vitro and in vivo.

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Section: Discussionmentioning

confidence: 99%

Interleukin-2 enhancer binding factor 2 negatively regulates the replication of duck hepatitis A virus type 1 by disrupting the RNA-dependent RNA polymerase activity of 3D polymerase

An,

Yu,

et al. 2024

Vet Res

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“…For RNA viruses, the HamRz and HdvRz as self-cleaving RNA ribozyme elements had been introduced into DNA-launched infectious cDNA clones to successfully improve the rescue efficiency [25,26]. In this study, the two self-cleaving elements were added at both termini of the viral genomic cDNA to generate a DNA-launched infectious clone of DAstV-1.…”

Section: Discussionmentioning

confidence: 99%

“…Cells were examined using IFA every five generations as previous described [26]. At 60 hpt, the transfected BHK-21 cells and the infected DEF cells were washed three times with phosphate-buffered saline (PBS, 8.1 mM Na 2 HPO4, 1.5 mM KH 2 PO 4 , 140 mM NaC1, 3.0 mM KC1, pH 7.2) and then fixed by a mixture of acetone and formaldehyde (1:1) for 20 min at room temperature.…”

Section: Detection Of the Rescued Virus By Immunofluorescence Assay (mentioning

confidence: 99%

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

Zhang

Lan

et al. 2019

Virol J

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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.

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“…The RNA-launched infectious clone of DHAV-1 named pR-DHAV-1 (Figure 1C) was previously established based on the strain LY0801 (accession no. FJ436047) [27].…”

Section: Methodsmentioning

confidence: 99%

IGF2BP1 Significantly Enhances Translation Efficiency of Duck Hepatitis A Virus Type 1 without Affecting Viral Replication

et al. 2019

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As a disease characterized by severe liver necrosis and hemorrhage, duck viral hepatitis (DVH) is mainly caused by duck hepatitis A virus (DHAV). The positive-strand RNA genome of DHAV type 1 (DHAV-1) contains an internal ribosome entry site (IRES) element within the 5′ untranslated region (UTR), structured sequence elements within the 3′ UTR, and a poly(A) tail at the 3′ terminus. In this study, we first examined that insulin-like growth factor-2 mRNA-binding protein-1 (IGF2BP1) specifically interacted with the DHAV-1 3′ UTR by RNA pull-down assay. The interaction between IGF2BP1 and DHAV-1 3′ UTR strongly enhanced IRES-mediated translation efficiency but failed to regulate DHAV-1 replication in a duck embryo epithelial (DEE) cell line. The viral propagation of DHAV-1 strongly enhanced IGF2BP1 expression level, and viral protein accumulation was identified as the key point to this increment. Collectively, our data demonstrated the positive role of IGF2BP1 in DHAV-1 viral proteins translation and provided data support for the replication mechanism of DHAV-1.

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Construction and characterization of an improved DNA-launched infectious clone of duck hepatitis a virus type 1

Cited by 9 publications

References 28 publications

Interleukin-2 enhancer binding factor 2 negatively regulates the replication of duck hepatitis A virus type 1 by disrupting the RNA-dependent RNA polymerase activity of 3D polymerase

Interleukin-2 enhancer binding factor 2 negatively regulates the replication of duck hepatitis A virus type 1 by disrupting the RNA-dependent RNA polymerase activity of 3D polymerase

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

IGF2BP1 Significantly Enhances Translation Efficiency of Duck Hepatitis A Virus Type 1 without Affecting Viral Replication

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