A Recombinant Turkey Herpesvirus Expressing the F Protein of Newcastle Disease Virus Genotype XII Generated by NHEJ-CRISPR/Cas9 and Cre-LoxP Systems Confers Protection against Genotype XII Challenge in Chickens

Abstract

In this study, we developed a new recombinant virus rHVT-F using a Turkey herpesvirus (HVT) vector, expressing the fusion (F) protein of the genotype XII Newcastle disease virus (NDV) circulating in Peru. We evaluated the viral shedding and efficacy against the NDV genotype XII challenge in specific pathogen-free (SPF) chickens. The F protein expression cassette was inserted in the unique long (UL) UL45-UL46 intergenic locus of the HVT genome by utilizing a clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 gene-editing technology via a non-homologous end joining (NHEJ) repair pathway. The rHVT-F virus, which expressed the F protein stably in vitro and in vivo, showed similar growth kinetics to the wild-type HVT (wtHVT) virus. The F protein expression of the rHVT-F virus was detected by an indirect immunofluorescence assay (IFA), Western blotting, and a flow cytometry assay. The presence of an NDV-specific IgY antibody was detected in serum samples by an enzyme-linked immunosorbent assay (ELISA) in SPF chickens vaccinated with the rHVT-F virus. In the challenge experiment, the rHVT-F vaccine fully protects a high, and significantly reduced, virus shedding in oral at 5 days post-challenge (dpc). In conclusion, this new rHVT-F vaccine candidate is capable of fully protecting SPF chickens against the genotype XII challenge.

Keywords: CRISPR/Cas9; NHEJ; Newcastle disease virus; Turkey herpesvirus; challenge protection; fusion gene; genotype XII; shed challenge virus.

Figure 1

The strategy used for the generation of the rHVT-F virus. (A) The genome of the wild-type (wt) Herpesvirus of turkey (HVT) is shown with unique long (UL), unique short (US), terminal and internal repeat long (TRL/IRL), and short (TRS/IRS) regions. The single guide (sg)RNA-UL45-46 introduced double-stranded breaks (DSBs) in the intergenic region UL45–UL46 of the wtHVT genome. The scissor symbol (green) represents the sgRNA-UL45-46 targeting site in the UL45-UL46 of the wtHVT genome. The green fluorescence protein (GFP) expression cassette contains: the elongation factor promoter (pEF), the complete open reading frame (ORF) of the GFP, and poly-A signal sequence (pA); Fusion (F) expression cassette contains: murine cytomegalovirus promoter (mCMV), the ORF of the F protein of Newcastle disease virus (NDV), and pA. The GFP and F expression cassettes from the donor plasmid were flanked with sg-A target sites to introduce the desired cleavage for its release and integration using the non-homologous end joining (NHEJ)—clustered regularly interspaced short palindromic repeat (CRISPR) -associated 9 (CRISPR/Cas9) strategy into the genome of wtHVT. The scissor symbol (blacks) represents the sgRNA-sgA targeting sites in the donor plasmid. The GFP reporter expression cassette was excised from rHVT-GFP-F using the Cre-loxP System and the new recombinant HVT was named rHVT-F. The fragments amplified by polymerase chain reaction (PCR) using the primers 1F/1R and 2F/2R showed the correct insertion of the F expression cassette into plasmid pGEM-sgA-GFP-F-XII. The junction primers NDV-F(XII)-5F/HVT UL46-5R (507 bp, base pairs = bp) showed the insertion of the F expression cassette at the correct locus into the genome rHVT-GPF-F and rHVT-F (after excision of the GFP reporter cassette). The primers HVT UL45F/HVT UL46R (3950 bp) showed the presence of the complete NDV F expression cassette in the genome at the correct locus of rHVT-F. This drawing was created with BioRender.com (Not drawn to scale). (B) Comparison of plaque morphology between rHVT-GFP-F, rHVT-F, and wtHVT. The plaque morphology of infected chicken embryo fibroblast (CEF) cells, induced by the rHVT-GFP-F, rHVT-F, and wtHVT viruses, was observed by fluorescence (top panel) and bright-field (bottom panel) microscopy: 100× magnification, and all scale bars represent 500 µm. (C) The presence of the NDV F expression cassette in the HVT genome was verified by PCR using the primers NDV-F(XII)-5F and HVT UL46-5R, as shown in (A) in both rHVT-GFP-F and rHVT-F, respectively.

Figure 2

Evaluation of the cell surface expression of the F protein and its expression over time. The cells were infected with the rHVT-GFP-F virus at a multiplicity of infection (MOI) of 0.01 and harvested at 48 and 72 h post-infection (hpi) to evaluate the cell surface expression and overall expression levels of the F protein. The CEF cells were labeled with anti-NDV chicken serum as the primary antibody and Alexa Fluor^® 647-labeled anti-chicken IgY as a secondary antibody. (A) Percentage of infected cells expressing the GFP (Q1) or GFP and F protein (Q2). (B) The F protein expression was evaluated by determining the median fluorescence intensity (MFI) and gated in the GFP+ population. The MFI of cells infected with the rHVT-GFP-F at 48 hpi is shown in red, and at 72 hpi is in light blue. Black and blue correspond to the MFI of uninfected cells (Mock) from 48 to 72 hpi, respectively. The cells were assessed by flow cytometry (Beckman Coulter Gallios, CA, USA) in a 3 laser/10 color configuration, and the data were analyzed using FlowJo software v7.6.5 (TreeStar, Ashland, OR, USA). For each sample, 30,000 events were acquired. Data are shown as mean ± standard deviation (SD) of three independent experiments, but only a representative image is shown.

Figure 3

Characterization of the rHVT-F. Expression of the NDV F protein in CEF cells and their incorporation into rHVT-F virus particles. CEF cells were infected with the recombinant virus at an MOI of 0.01. Then, the CEF cells were harvested at 72 hpi and processed to prepare cell lysates. Additionally, viruses from the supernatants of CEF cells infected with the recombinant virus were concentrated using tangential filtration on Sartoflow Advanced and subjected to ultracentrifugation on sucrose gradients to obtain partially purified virus particles. These samples were evaluated by Western blot analysis using a specific rabbit polyclonal antibody, against the NDV F protein, as primary antibody and a mouse anti-rabbit IgG antibody, conjugated to horseradish peroxidase (HRP), as a secondary antibody. (A) From the left to the right are molecular weight (MW) markers (lane 1), cell lysates from wtHVT-infected CEF cells (lane 2), and rHVT-F-infected CEF cells (lane 3). The Beta-actin protein (~42 kDa) was used as a loading control in cell lysate. (B) From the left to the right are MW markers (lane 1), partially purified virus particles from the supernatant: of wtHVT-infected CEF cells (lane 2), and rHVT-F-infected CEF cells (lane 3). The black arrow indicates a ~52 kDa band corresponding to the cleaved subunit (F1) of the NDV F protein. (C) In vitro replication kinetics of the rHVT-F virus. CEF cells were infected with the rHVT-F virus and wtHVT at 100 plaque-forming units (PFUs). The infected cells were harvested at 24, 48, 72, 96, and 120 hpi and titrated by the plaque assay. Then, the viral titers were analyzed on two-way ANOVA with multiple comparison tests. The results were not statistically significant (p > 0.05).

Figure 4

Stability of the rHVT-F virus. (A) The F protein expression was detected by indirect immunofluorescence assay (IFA) in the rHVT-F virus-infected CEF cells at 20th passage by staining with anti-NDV chicken serum, followed by Goat anti-chicken IgY H&L Alexa Fluor^® 405 (blue fluorescence). HVT was detected with a mouse antibody against Marek’s disease virus (MDV) (B149M), followed by goat anti-mouse IgG H&L Alexa Fluor^® 594 (red fluorescence) and visualized by fluorescence and bright-field microscopy: 50× magnification, all scale bars represent 500 µm. (B) The genetic stability of rHVT-F virus was detected from infected CEF cells, so viral deoxyribonucleic acid (DNA) was extracted and analyzed by PCR with primers HVT UL45F and HVT UL46R at passage 1, 5, 10, 15, and 20. The primer sequences and direction are shown in Figure 1A. The molecular sizes are indicated to the left of the gel in bp.

Figure 5

Humoral immune response and protection conferred by rHVT-F vaccine against genotype XII NDV challenge. Two groups of specific pathogen-free (SPF) chickens: Group #1 = vaccinated with rHVT-F virus (n = 14) and group #2 = non-vaccinated control to challenge wtHVT (n = 7) were immunized once at 1-day-old, and the challenge performed 49 days post-vaccination (dpv) with 10⁵ median lethal doses (LD₅₀)/chicken via the oculo-nasal route. (A) NDV-specific IgY antibodies in vaccinated chickens at 20, 34, and 49 dpv. Serum samples that were negative for enzyme-linked immunosorbent assay (ELISA) titer are shown below the limit of detection (Cutoff = 993). SD: Standard Deviation. (B) The survival rate of vaccinated chickens showed no mortality in 14 days following the challenge (dpc). (C) Titers of challenge virus shedding, from oral and cloacal swabs from chickens, after the challenge is quantitated by a plaque assay. ND = not detected; NS = no survivors; dpc = days post-challenge. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001.

Figure 6

Detection of the replication and stability of rHVT-F virus in vivo. The isolation of rHVT-F and wtHVT from lymphocytes was confirmed by IFA, PCR, and Western blot analysis at 28, 35, and 74 dpv. A pool of peripheral blood lymphocytes (PBLs) from chickens (n = 5) was used to isolate each virus, but only a representative image from each virus is shown. (A) The IFA was made in CEF cells infected with the isolated viruses by staining with anti-NDV chicken serum, followed by goat anti-chicken IgY H&L Alexa Fluor^® 405 (blue fluorescence). HVT was detected with a mouse antibody against MDV (B149M), followed by goat anti-mouse IgG H&L Alexa Fluor^® 594 (red fluorescence) and visualized by fluorescence and bright-field microscopy: 50× magnification, all scale bars represent 500 µm). (B) The genetic stability of isolated viruses was detected from infected CEF cells, so viral DNA was extracted and analyzed by PCR with primers HVT UL45F and HVT UL46R. From the left to the right are MW marker (lane 1), extracted DNA from CEF cells infected with the isolated viruses: rHVT-F virus at 28 (lane 2), 35 (lane 3), 74 dpv (lane 4), as well as wtHVT virus at 28 (lane 5), 35 (lane 6), 74 dpv (lane 7). The molecular sizes are indicated to the left of the gel in the bp. The black arrow indicates the MW expected of the band. (C) Expression of the NDV F protein was evaluated by Western blot analysis in CEF cells infected with isolated viruses using a specific rabbit polyclonal antibody against the NDV F protein as a primary antibody and a mouse anti-rabbit IgG antibody conjugated to HRP as a secondary antibody. From the left to the right are MW marker (lane 1), cell lysates from CEF cells infected with the isolated viruses: rHVT-F virus at 28 (lane 2), 35 (lane 3), 74 dpv (lane 4) and wtHVT virus at 28 (lane 5), 35 (lane 6), 74 dpv (lane 7). The black arrow indicates a ~59 kDa band corresponding to the inactive precursor (F0), a band ~52 kDa corresponding to cleaved subunit (F1), and a band ~120 kDa corresponding to the F1 dimer (dF1) of the NDV F protein. The Beta-actin protein (~42 kDa) was used as a loading control in lysate cells.