In vitro expression of the goose astrovirus Cap protein delivered with a duck enteritis virus vector

Abstract

Background

Goose astrovirus (GAstV) is an emerging pathogen that is widely distributed throughout China and can cause visceral gout, resulting in serious economic losses for the goose industry. Open reading frame 2 (ORF2) of this virus encodes the precursor capsid protein, which is essential for the assembly and antigenicity of these virions. To construct a bi-valent vaccine for controlling GAstV and duck enteritis virus (DEV) infection, an infectious bacterial artificial chromosome (BAC) clone of the DEV vaccine strain pDEV-EF1 was used to establish a recombinant DEV vector for GAstV ORF2 gene delivery.

Methods

GAstV ORF2 expression frame was inserted into the US7 and US8 intergenic region of DEV genome by Red E/T two-step recombinant technology, then the recombinant virus rDEV-GAstV ORF2 was rescued by transfecting recombinant clone pDEV-GAstV ORF2 into chicken embryonic fibroblasts (CEFs). The expression of ORF2 in CEFs and formation of virus-like particles (VLPs) were analysed by Western blotting, indirect immunofluorescence assay (IFA) and immunogold electron microscopy (IEM), individually. And protein celluar localization was analysed by IFA.

Results

Using this rDEV-GAstV ORF2 vector to infect CEFs was sufficient to elicit GAstV Cap protein expression, as confirmed by Western blotting and IFA. IEM also revealed the formation of VLPs within cells expressing this Cap protein.

Conclusions

DEV is a good viral vector for GAstV ORF2 gene delivery and these results provide a basis for the development of a bivalent vaccine for controlling DEV and GAstV infections.

Introduction

Recurring outbreaks of goose astrovirus (GAstV) infections have been reported among geese in coastal China since 2015, and these infections have quickly spread to inland provinces, leading to major economic losses for the goose industry [1,2,3,4,5,6]. Infected geese suffer from joint and visceral gout, and the mortality rate for infected animals is high.

GAstV is a small, round, nonenveloped virus with a diameter of roughly 30 nm. It is a single-stranded positive-sense RNA virus with an approximate genome length of 7.2 kb, encoding three open reading frames (ORFs; ORF1a, ORF1b, and ORF2) [7]. Of these, ORFs1a and ORF1b encode nonstructural proteins important for viral transcriptional and replicative activity, whereas the capsid polyprotein (Cap) is encoded by ORF2 and is required for viral assembly and the induction of neutralizing antibodies, playing key roles in both viral entry and pathogenicity [8]. Currently, no safe and effective vaccines for GAstV-1 are commercially available.

Duck enteritis virus (DEV), sometimes known as Anatid herpesvirus 1, can cause a form of acute, highly contagious, and potentially deadly viral enteritis that affects many waterfowl species in the order Anseriformes. A live attenuated vaccine is the primary tool used for the prevention of this disease, with DEV exhibiting many advantageous features that render it well suited to be developed into live vaccine vector. Similar to other herpesviruses including HVT (Turkey herpesvirus), MDV (Marek’s disease virus), and pseudorabies virus (PRV), the genome of DEV is large and contains an array of nonessential genes which is amenable to exogenous gene insertion; it exhibits good genomic stability, limited interference from maternal antibodies; and it can persist for long time in vivo.

Several studies have achieved good results when utilizing DEV as a live vaccine vector [9,10,11,12,13,14,15,16,17]. Here, the GAstV-1 ORF2 gene was inserted into the DEV genome, and exogenous GAstV-1 ORF2 expression was confirmed, with virus-like particles (VLPs) being verified in recombinant rDEV-GAstV ORF2-infected cells. Given these promising results, this recombinant virus may offer utility as a candidate vaccine against both DEV and GAstV.

Materials and methods

All animal experiments were conducted following the experimental practices and standards approved by the Animal Welfare and Research Ethics Committee of Zhejiang Academy of Agricultural Science, China, with approval number 23ZALAS12. This experiment was conducted under the ARRIVE guidelines to minimize animal suffering.

Viral strains and cells

The pDEV-EF1/GS1783 bacteria containing the full-length DEV vaccine strain BAC clone and the rDEV-EF1 virus were prepared in our laboratory [18, 19]. Chicken embryonic fibroblasts (CEFs) were generated with 10-day-old SPF chicken eggs (Zhejiang JianLiang Bioengineering Co., Ltd., Hangzhou, China) that were specific pathogen-free using a standard protocol, and these cells were cultured with DMEM (Gibco-BRL, MD, USA) with 8% FBS (Gibco-BRL, MD, USA), penicillin (100 U/mL), and streptomycin (100 µg/mL).

Analysis conservation of amino acid sequence of GAstV cap

Protein search was performed with pBLAST [20] (default parameters against all genomes deposited in NCBI database, at November 2024) using the amino acid sequence of the GAstV Cap (GenBank MH052598 as query.

BGH-GAstV ORF2 vector construction

GAstV ORF2 containing separate Not I and Kpn I sites introduced into the 5’and 3’ termini was optimized according to the duck codon preferences, after which it was synthesized by GeneScript (Nanjing, China) with reference to the GAstV gene sequence (GenBank MH052598). The BGH-GAstV ORF2 plasmid was constructed by inserting GAstV ORF2 into the Not I and Kpn I sites of the pEP-BGH-end vector (Fig. 1 ).

Fig. 1

Schematic diagram of recombinant plasmid pEP-BGH-GAstV ORF2

Recombinant mutated BAC clone preparation for the DEV delivery of the GAstV ORF2 gene

The mutated BAC clones for this study were prepared with a two-step Red-mediated recombination (en passant) approach [21] via the insertion of a P_CMV-GAstV ORF2-BGH-pA expression cassette into the DEV intergenic region between US7 and US8 (Fig. 2). Specifically, pDEV-GAstV ORF2 construction was achieved by amplifying the P_CMV-GAstV ORF2-BGH-pA-Kan expression cassette with flanking DEV US7 and US8 homology arms from the BGH-GAstV ORF2-Kan using pDEV vac-in-as and pDEV vac-in-s primers (Table 1). The purified 4322 bp PCR product (100 ng) was then electroporated into competent pDEV-EF1-containing GS1783 bacteria, and these transformed bacteria were plated on LB agar plates with chloramphenicol (cat, 34 µg/mL) and kanamycin (Kan, 50 µg/mL) followed by incubation for 48 h at 32 ℃. Colonies with dual resistance (pDEV-Kan.GAstV ORF2) were then chosen for the next recombination step for the removal of the Kan resistance gene by homologous recombinant that occurred between rec out arm A and B, selecting cat-resistant Kan-sensitive colonies. Positive colonies (pDEV-GAstV ORF2) were identified based on restriction fragment length polymorphisms (RFLP) with BamH I and sequence of PCR products amplified with the Rec-JD-F and Rec-JD-R primers flanking GAstV ORF2 insert region (Table 1).

Fig. 2

Recombinant pDEV-GAstV ORF2 construction. pDEV-EF1 (shown in the dotted box (A and B)) were constructed and detailed in our prior publication [18]. A. Homologous recombination was used to introduce a mini-F BAC vector (pHA2) that allows for large circular DNA to be maintained in E. coli into the intergenic region between UL15B and UL18 in a DEV vaccine strain. B. Two-step Red-mediated recombination (en passant) was used to substitute the P_CMV promoter controlling GFP expression in pDEV-vac for the pEF1 gene. C-D. The recombinant BAC clone pDEV-kan.GAstV ORF2 was constructed by first inserting P_CMV-GAstV ORF2-BGH-pA-Kan expression cassette into the US7 and US8 intergenic spacer in pDEV-EF1(C) and pDEV-kan.GAstV ORF2 (D) were obtained by deleting Kan gene from pDEV-kan.GAstV ORF2 by 2nd homologous recombinant

Table 1 Primers used in this study

Recombinant virus rescue

An alkaline lysis approach was used to isolate pDEV-GAstV ORF2, after which 4 µg of pDEV-GAstV ORF2 DNA was used to transfect CEFs via calcium phosphate precipitation as reported previously by Chen L [17]. These cells were cultured for 3–6 days in DMEM with 8% FBS until 70–80% cytopathic effect (CPE) had occurred. The collected viruses were designated rDEV-GAstV ORF2.

Viral plaque size analyses

To measure rDEV-GAstV ORF2 and control virus rDEV-EF1 plaque sizes, serial dilutions of the viruses were performed followed by added to CEFS in 12-well plates. At 2 h after inoculation, the inoculum was exchanged for DMEM with 1.5% methylcellulose (Sigma-Aldrich, USA). Following incubation for 2 days at 37 °C, 100 fluorescent plaques per virus were selected at random and imaged under fluorescence microscopy (Nikon, Japan), measuring their sizes with Image J software. The statistical analysis was conducted using GraphPad Prism 5 software, the P value was calculated by the independent sample t-test, and a P value less than 0.05 was regarded as significant.

Multi-step growth kinetics

Multi-step growth kinetics of rDEV-GAstV ORF2 were determined and compared with the parental virus rDEV-EF1. Briefly, the CEFs were infected with approximately 0.02 MOI of cell-free viruses of rDEV-GAstV ORF2 and rDEV-EF1. The cells and culture supernatant were harvested at different times (0, 12, 24, 36, 48, 60, and 72 h) after virus infection. The cells were collected by trypsin digestion following two washes with phosphate-buffered saline (PBS, pH 7.0) and were then suspended with 1 mL of DMEM-2% FBS and an equal volume of supernatant. The collected cells and supernatants were stored at -70 °C until all samples had been collected. Before titration on CEFs, the cells were treated with a Tissuelyser-24 at 65 Hz for 60 s and centrifuged at 8000 g for 5 min, and then 100 µL of lysis supernatant and culture supernatant were taken for titering via the TCID₅₀ test according to standard virological methods. The multi-step growth curves were computed from three independent experiments.

Western blot analysis of cap protein expression in recombinant virus- infected CEFs

Cap protein expression was confirmed through Western blot analysis with an anti-GAstV Cap monoclonal antibody (mAb) prepared in our laboratory. Briefly, equal CEFs in T75 cell culture flask were separately infected with rDEV-GAstV ORF2 (MOI = 0.02), or control rDEV-EF1 (MOI = 0.02), and when 80% cells ocurred fluorescent plaque, cells and culture supernatants were separately collected. Cells were resuspended in 100 µL 1× SDS-PAGE Sample Loading Buffer (Beyotime) and heated at 100 °C for 10 min, then the lysis cell sample were taken to centrifugation at 13,000 g for 5 min, and the lysate supernatant were collected and protein concentration were measured using Bradford methods. 2 µL 5× SDS-PAGE Sample Loading Buffer (Beyotime) were added to 10 µL culture supernatants, and heat denaturation at 100 °C for 10 min. At last, 8 µL cell lysate supernatant and 20 µL culture supernatants were taken to 10% SDS-PAGE separation and transfer onto nitrocellulose membranes (Millipore, MA, USA). The samples were prepared in duplicate. After blocking with 10% skim milk in 0.5% Tween20 in PBS (PBST) at 4 ℃, one blot was treated for 1 h with anti-GAstV Cap mAb (1:500) and another inculated with mouse anti-DEV US3 polyclonal antibody (1:500) at 37 ℃, followed by staining with HRP-conjugated goat anti-mouse IgG (1:1000) (Santacruz, CA, USA). Western Blot Hyper HRP Substrate (Takara, Japan) was used for protein band visualization, and the blots were imaged with a Molecular Imager^® ChemiDoc™ XRS⁺ Imaging System (Bio-Rad Laboratories, CA, USA).

Protein localization observed by confocal microscopy

rDEV-GAstV ORF2 at a MOI of 0.02 was inoculated to CEFs seeded on the slides in a 24-well plate, and at 24 h.p.i, the cells were taken to indirect immunofluorescence assay (IFA). The cells were washed twice with PBS (pH 7.2) and then fixed with methanol: acetone (1:1) for 1 h, then wahed with PBS. After 1 h blog in 1% BSA/PBST solution, primary antibody anti-GAstV Cap mAb (1: 100) was added and incubated for 1 h at 37 ℃, then incubation for 1 h at 37 ℃ with Cy3-labeled goat anti-mouse IgG (Beyotime). At last, the cells were stained with DAPI solution (Beyotime), and visualized by fluorescence microscope (Nikon ) and laser scanning confocal microscopy (Leica TCS-SP5) with a 100×oil immersion objective.

Immunoelectron microscopy

Cellular cryosections were prepared for immunogold labeling by fixing rDEV-GAstV ORF2-infected cells at 18 h post-infection using 4% paraformaldehyde in PHEM buffer (60 mM PIPES [piperazine-N, N=-bis (2-ethanesulfonic acid)], 25 mM HEPES, 10 mM EGTA, 2 mM MgSO₄; pH 6.9) for 4 h at room temperature, followed by embedding in 10% (w/v) gelatin and overnight croprotection in a 2.3 M sucrose solution with 20% polyvinylpyrrolidone. Liquid nitrogen was used to rapidly freeze these samples, followed by cryosectioning at 120 °C using a Leica EM FCS cryoultramicrotome. Immunogold labeling was achieved by incubating thawed cryosections (thickness: 90 nm) for 5 min in 20 mM glycine for the quenching of free aldehyde groups, followed by blocking for 5 min with 10% FBS. Samples were next incubated with primary anti-GAstV Cap (1:500) mAb for 30 min at room temperature, after which protein A-conjugated 10-nm gold particles (EM Laboratory, Utrecht University, Utrecht, The Netherlands) were added. Signal amplification was achieved with rabbit anti-mouse IgG (Dako) and subsequent incubation with protein A-conjugated 10-nm gold particles in some cases. Prior to imaging, sections were stained using 1.8% methylcellulose and 0.4% uranyl acetate, followed by examination using a JEOL JEM-1400Flash or JEOL JEM-1010 electron microscope at 80 kV with respective Gatan One View CMOS 4 K or TVIPS F416 cameras.

Results

Analysis conservation of amino acid sequence of GAstV cap

GAstV Cap sequence was aligned with pBLAST. Results showed that amino acid sequences of GAstV Cap from GAstV-1 disease samples collected from 12 provinces of China during 2016–2023 were high conservation and their homology was between 98.72 and 100%.

BAC mutant identification

The successful identification of mutated BAC clones was performed through a RFLP approach. The DNA of the mutated BAC clones and intermediates was digested using BamH I, followed by electrophoretic examination (Fig. 3a). RFLP patterns revealed clear concordance between the obtained BamH I pattern matched and the patterns predicted in silico based on the reference DEV vaccine strain genome [GenBank: KF487736] [22]. The sizes and sequences of the PCR products amplified from this recombinant BAC DNA with the Rec-JD-F/Rec-JD-R primers were also consistent with predictions (Fig. 3b). These results confirmed the successful insertion of the exogenous genes into the DEV genome at the expected site.

Fig. 3

Bam HI digestion and PCR-based identification of recombinant BAC clones. a. Bam HI digestion-based analysis of pDEV-GAstV ORF2 mutants. 1: pDEV-EF1; 2: pDEV-Kan.GAstV ORF2; 3: pDEV-GAstV ORF2 mutants. M: 15,000 bp marker; 1: rDEV-EF1; 2: pDEV-Kan. GAstV ORF2; 3: pDEV-GAstV ORF2. b. PCR-based recombinant mutated BAC clone identification. 1: pDEV-EF1 (641 bp); 2: pDEV-Kan.GAstV ORF2 (4234 bp); 3 pDEV-GAstV ORF2 (3818 bp); M: 250 bp marker (4500, 3000, 2250, 1500, 1000, 750, 500, 250)

Recombinant virus rescue

The prepared mutated BAC DNA constructs were used to transfect CEFs via calcium phosphate precipitation. At 48 h post-transfection, fluorescent plaques were visible, which demonstrated that recombinant virus were successfully rescued, collecting cells when over 80% of cells were fluorescent, this virus stock was named rDEV-GAstV ORF2 (Fig. 4).

Fig. 4

Rescued of recombinant virus. Fluorescence plaque of the rescued rDEV-GAstV ORF2 (100×)

Plaque size measurements

Plaque sizes for the rDEV-GAstV ORF2 virus were next examined, revealing them to be significantly (16.44%) smaller than those generated by the parental rDEV-EF1 virus on day 2 post-infection. There was significant difference between rDEV-GAstV ORF2 and rDEV-EF1 (P = 0.03) (Fig. 5).

Fig. 5

Plaque size of rDEV-GAstV ORF2 and control virus rDEV-EF1 on CEFs. Image J was used to compute means and standard deviations when measuring the sizes of 100 plaques, setting the mean plaque size for rDEV-EF1 to 100%. Error bars represent the standard deviation. The P value was calculated by the independent sample t-test, and a P value less than 0.05 was regarded as significant

Multi-step growth kinetics

As shown in Fig. 6, the reconstituted viruses rDEV-GAstV ORF2 (both intracellular and extracellular) exhibited growth characteristics that were virtually identical to those of parental rDEV-EF1. In addition, the virus titers steadily increased from 12 to 60 h post-infection.

Fig. 6

Multi-step growth curves of rDEV-GAstV ORF2 amd rDEV-EF1 on CEFs. Comparison of the in vitro growth of viruses reconstructed with parental DEV. The virus titers of infectedcells (A) and supernatants (B) were determined at different times (0, 12, 24, 36, 48, 60, and 72 h) after inoculation of approximately 0.02 MOI of cell-free viruses of rDEV-GAstV ORF2 and rDEV-EF1. The multi-step growth curves were computed from three independent experiments

Analysis of the GAstV cap protein in recombinant virus-infected CEFs

Cap protein expression in CEFs and culture infected with the recombinant virus was assessed via Western blotting performed with anti-GAstV Cap mAb as primary antibody and HRP-labelled goat anti-mouse IgG as secondary antibody. This approach confirmed the presence of a specific band at ~ 82 kDa in both rDEV-GAstV ORF2-infected cell and supernatant samples that was absent in rDEV-EF1-infected cells (Fig. 7), consistent with predictions. The result of IFA also verified expression of GAstV ORF2 in rDEV-GAstV ORF2-infected cells (Fig. 8). These demonstrated that GAstV Cap was successfully expressed in CEFs.

Fig. 7

Analyses of GAstV Cap protein expression in virus-infected CEFs and supernatant samples by Western blot analysis. Detection of GAstV ORF2 protein (A) and inner protein DEV US3 (C) by Western blot analysis. Corresonding picture of SDS-PAGE (C). M: Prestained Protein Ladder (10-180 kDa); 1: rDEV-EF1-infected culture supernatants; 2: rDEV-GAstV ORF2-infected culture supernatants; 3: rDEV-EF1-infected CEFs; 4: rDEV-GAstV ORF2-infected CEFs

Fig. 8

Analysis of GAstV Cap protein expression and localizaiton in CEFs by IFA. rDEV-GAstV ORF2 or control virus rDEV-EF1-infected CEFs were fixed and taken to IFA performed with anti-GAstV Cap mAb as primary antibody and Cy3-labelled goat anti-mouse IgG as secondary antibody. And then staining with DAPI solution. Red florescence represent Cap protien

Protein localization observed by confocal microscopy

GAstV Cap localization in CEFs were detected by IFA. Results showed that GAstV Cap were major localized in cytoplasm (Fig. 8).

Immunoelectron microscopy analyses of protein expression

Immuno-TEM analyses revealed the ability of GAstV Cap to form VLPs in CEFs infected with the recombinant virus (Fig. 9). These VLPs were arranged in a lattice-like pattern surrounded by the proteins encoded by ORF2. Asterisks denote VLPs while arrows show colloidal gold dots corresponding to GAstV Cap.

Fig. 9

Immunogold labeling electron microscopy analysis of Cap protein in rDEV-GAstV ORF2-infected CEFs. Nucleoprotein (N) immunogold labeling was achieved with an antibody against the Cap protein. GAstV Cap proteins are denoted by arrows pointing to colloidal gold spots. Scale bar: 200 nm; VLPs are marked with asterisks; Scale bar: 500 nm

Discussion

GAstV-1 exhibits a high degree of pathogenicity in goslings 5–15 days of age. No safe or effective commercial vaccines or treatments for GAstV have been devised to date, and only a specific egg yolk-derived immunoglobulin Y (IgY) can reportedly protect goslings against GAstV infection [23]. The design of effective vaccines that can protect against GAstV-1 is thus urgently required. Potential approaches include inactivated viral vaccines, weakened viral vaccines, subunit vaccines, mRNA vaccines, and viral vector vaccines. Efforts to produce weakened or inactivated GAstV vaccines have been hampered by the absence of any effective in vitro system for GAstV culture. Subunit vaccine development is also challenging as extended periods of time are required for antibody production after the immunization of goslings, but hosts are susceptible to disease from 5 to 15 days of age, hampering efforts to assess the protective efficacy of this vaccine in goslings after challenge with virulent GAstV strains. Analyses of protective efficacy are thus limited to immunized breeding geese based on activity mediated by maternal antibodies. In addition, mRNA vaccines are not cost-effective options for waterfowl vaccine preparation. Viral vectors are a relatively novel vaccine platform that relies on recombinant viruses for the delivery of selected immunogens into hosts to efficiently elicit robust protection [24].

The commercial attenuated DEV VAC strain is associated with good safety and immunogenicity profiles, and is capable of eliciting protection rapidly within three days of initial vaccination such that it is an ideal viral vector candidate. Both preclinical and clinical studies have assessed the protective benefits and safety of DEV-vectored vaccines in chickens, ducks and broilers [15, 25, 26]. The degree to which this candidate vaccine vector is suitable for geese, as another DEV host species, however, warrants further evaluation.

Here, an infectious full-length DEV BAC clone generated in our laboratory [21] was used to express the GAstV-1 ORF2 gene, leading to the formation of VLPs within the cells infected with this recombinant virus. There are many advantages to VLPs including their good safety and stability together with their ability to more effectively mimic parental viral features, thereby facilitating helper T cell activation to achieve superior immunogenicity even at lower dose levels [27]. The potential ability of Cap to form VLPs is thus crucial for the induction of high levels of neutralizing antibodies directed against GAstV. Further studies in the future will thus aim to fully evaluate the possibility of rDEV-GAstV ORF2 as a bivalent vaccine.

Conclusion

A recombinant virus made DEV as viral vector was constructed, and DEV is testified to be a good viral vector for GAstV ORF2 gene delivery and these results provide a basis for the development of a bivalent vaccine for controlling DEV and GAstV-1 infections.