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

Abstract

Background: DNA-launched infectious system is a useful tool with high rescue efficiency that allows the introduction of mutations in specific positions to investigate the function of an individual viral element. Rescued virus particles could be harvested by directly transfecting the DNA-launched recombinant plasmid to the host cells, which will reduce labor and experimental cost by skipping the in vitro transcription assay.

Methods: A total of four overlapping fragments covering the entire viral genome were amplified and then were assembled into a transformation vector based on pIRES2-EGFP to establish the DNA-launched infectious system of duck hepatitis A virus type 1 (DHAV-1), named pIR-DHAV-1. Reverse transcription polymerase chain reaction (RT-PCR) detection, quantitative real-time polymerase chain reaction (qRT-PCR), western blotting assay and indirect immunofluorescence (IFA) were conducted for rescued virus identification. A total of 4.0 μg of recombinant plasmid of pIR-DHAV-1 and in vitro transcribed product of 4.0 μg of RNA-launched infectious clone named pR-DHAV-1 were transfected into BHK-21 cells to analyze the rescue efficiency. Following that, tissue tropism of rescued virus (rDHAV-1) and parental virus (pDHAV-1) were assayed for virulence testing in 1-day-old ducklings.

Results: Rescued virus particles carry the designed genetic marker which could be harvested by directly transfecting pIR-DHAV-1 to BHK-21 cells. The qRT-PCR and western blotting results indicated that rDHAV-1 shared similar growth characteristics with pDHAV-1. Furthermore, DNA-launched infectious system possessed much higher rescue efficiency assay compared to RNA-launched infectious system. The mutation at position 3042 from T to C has no impact on viral replication and tissue tropism. From 1 h post infection (hpi) to 48 hpi, the viral RNA copies of rDHAV-1 in liver were the highest among the six tested tissues (with an exception of thymus at 6 hpi), while the viral RNA copy numbers in heart and kidney were alternately the lowest.

Conclusion: We have constructed a genetically stable and highly pathogenic DNA-launched infectious clone, from which the rescued virus could be harvested by direct transfection with recombinant plasmids. rDHAV-1 shared similar growth characteristics and tissue tropism with pDHAV-1. The DNA-launched infectious system of DHAV-1 possessed higher rescue efficiency compared to the traditional RNA-launched infectious system.

Keywords: DHAV-1; DNA-launched infectious clone; Rescue efficiency; Ribozyme.

Fig. 1

Strategy for the construction of a full-length DNA-launched infectious clone based on a virulent strain LY0801 of DHAV-1. The fragment IR was derived from the plasmid pIRES2-EGFP (Clontech) using primers pIR-BamHI-XhoIF and pIR-AscI-BamHIR, and then was digested with BamH I and ligated with T4 DNA ligase to yield pIR vector. The LY0801 virus genome with two ribozyme sequences introduced at the both ends was placed downstream of the CMV promoter in pIR vector to construct the DNA-launched infectious clone, pIR-DHAV-1. A total of 4 overlapping fragments amplified from total RNAs extracted from the 5th passage of LY0801 virus were assembled successively into the pIR vector by unique restriction enzyme indicated for each fragment. A copy of hammerhead ribozyme sequence and Asc I restrict enzyme site were engineered at the 5′ end while a copy of hepatitis delta virus ribozyme (HDVRs) sequence and Xho I restrict enzyme site were engineered at the 3′ end of the cDNA copy of DHAV full-length genome, respectively. The base T at position of 3042 was mutated into base C to generate a BamH I restrict enzyme site which was treated as a genetic marker to distinguish the rescued virus from the parental virus

Fig. 2

Recombinant plasmids digested with appropriate enzymes during construction. Four fragments that amplified with the four pairs primers 3endRibo-F/R, Cloning 2-F/R, DHAV-Seq-6F/9R and 5HeadRibo-F/R were assembled into pIR vector by one multi-step strategy, respectively. The yield recombinant plasmids were digested with appropriate enzymes and verified by electrophoresis in 2% agarose gel. (M) DNA Marker DL2000; (1) The recombinant plasmid that consisted of pIR vector and cloning 1 was digested with BamH I and Xho I; (2) The recombinant plasmid that consisted of pIR vector and cloning 1 and cloning 2 was digested with BamH I and EcoR V; (3) The recombinant plasmid that consisted of pIR vector and cloning 1 to cloning 3 was digested with EcoR V; (4) The complete DNA-launched infectious clone was digested with Asc I and BamH I; (5) The complete DNA-launched infectious clone digested with BamH I; (6) The complete DNA-launched infectious clone digested with Xho I

Fig. 3

IFA observation of transfected/infected BHK-21 cells. (a) BHK-21 cells were transfected with the recombinant plasmid DNA of pIR-DHAV-1; (b) BHK-21 cells were infected by the parental virus of LY0801 strain; (c) BHK-21 cells were transfected with the plasmid DNA of pIR. After passaging for three times, IFA was conducted by incubation of anti-DHAV-1 mAb 4F8 (dilution of 1:1000 with PBS) for 1 h at 37 °C, followed by incubation in 37 °C for 1 h with FITC-labeled goat anti-mouse antibody. Red bars represent 10 μm

Fig. 4

Identification of the rescued virus. (a) Western blotting assay. The BHK-21 cells infected with parental virus and rescued viruses were harvested at 48 hpi. Anti-DHAV-1 monoclonal antibody 4F8 (dilution of 1:500) and HRP-conjugated goat anti-mouse antibody (dilution of 1:3000) were used to conduct the western blot assay. The BHK-21 cells transfected with pIR vector were used as negative control. (b) Identification of the genetic marker in the rescued virus. The BamH I restriction enzyme site was introduced into the recombinant plasmid to create a genetic marker to distinguish the rescued virus from the parental virus (without BamH I enzyme site). Two 2290 bp-fragments derived from parental and rescued virus with primers DHAV-3F and DHAV-4R were digested with BamH I and analyzed on a 2.0% agarose gel. (M) DNA Marker DL2000; 1. Fragment amplified with template of parental RNA; 2. Fragment derived from the parental virus digested with BamH I; 3. Fragment derived from the rescued virus digested with BamH I; 4. Fragment amplified with template of rescued virus RNA

Fig. 5

Identification the growth characteristics of rDHAV-1. BHK-21 cells were infected with the parental virus and the rescued virus at 0.1 MOI, respectively. Supernatants and cells were separately collected and used for RNA extraction and qRT-PCR amplification and western blotting. (a) Growth curves of the parental/rescued virus in supernatants; (b) Growth characteristics of the parental/rescued virus in BHK-21 cells. (c) Viral replication levels of the rescued viruses in supernatants and BHK-21 cells. (d) Viral replication levels of the parental viruses in supernatants and BHK-21 cells

Fig. 6

Rescue efficiency of RNA/DNA-launched infectious system. (a) 4.0 μg of the DNA-launched infectious clones or in vitro transcribed product of the same amount of RNA-launched infectious clones were used for BHK-21 transfection. The DNA-launched infectious clone (plasmid pIR-DHAV-1) and the empty vector (plasmid pIR) were transfected directly into BHK-21 cells, while the RNA-launched infectious clone was transcribed in vitro, and then the transcription mixture was transfected into BHK-21 cells. At 48 hpt, IFA observation was conducted after staining with anti-DHAV-1 mAb 4F8 (dilution of 1:1000 with PBS) for 1 h at 37 °C, followed by incubation in 37 °C for 1 h with FITC-labeled goat anti-mouse antibody. Bars represent 10 μm. (b) Western blotting assay. A total of 4.0 μg of recombinant plasmid of DNA-launched infectious clone and in vitro transcribed product from 4.0 μg of RNA-launched infectious clone were transfected into BHK-21 cells to analyze rescue efficiency between DNA-launched system and RNA-launched system. Cell culture was collected at 48 hpt and run on SDS 12%-polyacrylamide gels. Anti-DHAV-1 monoclonal antibody 4F8 (dilution of 1:500) and HRP-conjugated goat anti-mouse antibody (dilution of 1:3000) were used to conduct the western blot assay. BHK-21 cells that transfected with pIR vector were conducted as negative control. (c) BHK-21 cells were infected with transfected product of 4.0 μg of the DNA-launched infectious clones or in vitro transcribed product of the same amount of RNA-launched infectious clones, cell lysates were collected at 48 hpi, and were immediately used for RNA extraction and qRT-PCR measurement. Viral RNA copies were calculated with formula X = 6.7 × 10(40.812-y)/3.285. “X” is a standard of viral copies while “y” is a standard of values derived from one step real-time PCR. Bars represent means and standard of three individual repeats. “***” represents p < 0.001

Fig. 7

(a) Survival curves of 1-day ducklings after inoculation of 0.25 ml of the parental virus (10^4.0 TCID₅₀) or the rescued virus (10^4.0 TCID₅₀). All ten ducklings in control group live in healthy condition. (b) Genetic marker detection of the parental/rescued virus group. Total RNA was extracted from liver tissue of dead ducklings of the rescued virus group and the parental virus group, and then immediately used for transcription using the primers BamH I-detect-F/R. The amplified fragments were then digested with restriction enzyme BamH I and verified by electrophoresis in 2% agarose gel. (M) DNA Marker DL2000; (1) Fragments were amplified with the template RNAs of the parental virus group and then digested with BamH I; (2) Fragments were amplified with the template RNAs of the rescued virus group and then digested with BamH I. Nucleotide sequence alignment (c) and amino acid sequence alignment (d) were also conducted by the Clustal W method (DNA Star LaserGene software, DNAStar Inc. Madison, WI) to investigate genetic marker in both groups

Fig. 8

Dynamic analysis of viral load. Same weight of heart (a), liver (b), kidney (c), spleen (d), thymus (e) and bursa of Fabricius (f) of the 1-day-old ducklings were collected at 1, 6, 12, 18, 24, 48 and 72 hpi, and were immediately used for RNA extraction and qRT-PCR measurement as previously described. Viral RNA copies were calculated with formula X = 6.7 × 10(40.812-y)/3.285. “X” is a standard of viral copies while “y” is a standard of values derived from one step real-time PCR. Bars represent means and standard of three individual repeats