The UL24 gene of PRV has been confirmed to play a pivotal role in regulating innate immune responses in host cells (Liu et al., 2021; Wang et al., 2020). Here, a variant PRV SX-10 virus lacking the gI, gE, TK, and UL24 genes was generated using the CRISPR/Cas9 technology, as shown in Figure 1A. Additionally, a similar procedure was utilized to generate a mutant PRV SX-10 virus lacking only the UL24 gene, named PRV SX-10ΔUL24 (Figure 1B). The purified viruses were confirmed via PCR assays with specific primers to the viral gI/gE, TK, UL24, EGFP, and mCherry (Figure 1C).

The peak virus titers of PRV SX-10ΔgI/gE/TK/UL24, PRV SX-10ΔgI/gE/TK, PRV SX-10ΔUL24, Bartha-K61, and wild-type PRV SX-10 were observed at 48 h post-infection, reaching 10^7.67, 10^7.92, 10^8.39, 10^8.36, and 10^8.47 TCID₅₀/mL, respectively. Moreover, the one-step growth curves were similar among the recombinant PRVs and PRV SX-10 (Figure 2A). Compared to the wild-type PRV SX-10 strain, the plaque sizes formed by the recombinant virus strains and Bartha-K61 were significantly smaller (Figures 2B,C). PRV SX-10ΔgI/gE/TK/UL24 was serially passaged in PK-15 cells for 21 generations (F21), and the relevant genes were verified using PCR analysis (Figure 2D). Transmission electron microscopy analysis revealed that the morphology of viral particles from recombinant virus strains was indistinguishable from that of wild-type PRV SX-10, with no discernible structural differences observed (Figure 2E).

The UL24 of herpesviruses suppress IFN-β and reduce pathogenicity in mice (Jacobson et al., 1998; Liu et al., 2021). Real-time PCR analysis revealed that IFN-β transcripts levels were significantly elevated in cells infected with PRV SX-10ΔUL24 and PRV SX-10ΔgI/gE/TK/UL24 compared to those infected with PRV SX-10 or PRV SX-10ΔgI/gE/TK strains, or treated with DMEM (Figure 3A). Moreover, in the mouse challenge experiment, mice infected with PRV SX-10ΔUL24 exhibited a 20% higher survival rate compared to those challenged with wild-type PRV SX-10 (Figure 3B).

The safety and immunogenicity of the recombinant viruses were evaluated in mice (Figure 4A). Mice inoculated with 100 μL containing 10⁵ TCID₅₀/mL of PRV SX-10ΔgI/gE/TK/UL24, PRV SX-10ΔgI/gE/TK, Bartha-K61, or DMEM (control group) all survived without clinical symptoms through 14 days post-immunization (dpi). However, mice inoculated with PRV SX-10 exhibited typical pseudorabies (PR) symptoms, including severe pruritus, ruffled fur, and hyperkinesia, with all subjects succumbing to the infection within 2–4 days post-inoculation (Figure 4B). At 14 dpi, a subset of mice was euthanized and subjected to necropsy. The viral loads in tissues from mice in the PRV SX-10ΔgI/gE/TK/UL24 groups were significantly lower than those in the PRV SX-10 group (Figure 4C). Compared to the DMEM control group, brain, liver, and lung tissues from mice challenged with PRV SX-10ΔgI/gE/TK/UL24, PRV SX-10ΔgI/gE/TK, or Bartha-K61 strains exhibited no significant histopathological changes. In contrast, mice infected with PRV SX-10 exhibited cerebral vascular congestion and perivascular lymphocytic cuffing in brain tissues, pulmonary congestion and edema in the lungs, and congestion in the liver (Figure 4D). Additionally, the PRV SX-10ΔgI/gE/TK/UL24 vaccinated group exhibited a significantly stronger antibody response compared to groups vaccinated with PRV SX-10ΔgI/gE/TK, especially on 21 dpi (p < 0.0001) (Figure 4E). Concurrently, the PRV SX-10ΔgI/gE/TK/UL24 immunized group and the PRV SX-10ΔgI/gE/TK immunized group exhibited similar neutralization titers at 28 dpi, which were significantly higher than those of the Bartha-K61 immunized group (p < 0.0001) (Figure 4F).

At 28 dpi, mice in the PRV SX-10ΔgI/gE/TK/UL24, PRV SX-10ΔgI/gE/TK, or Bartha-K61 groups were challenged with 100 μL containing 10⁵ TCID₅₀/mL of PRV SX-10, while the DMEM group served as a control. All mice in the PRV SX-10 group and 70% in the Bartha-K61 group exhibited typical PR symptoms and succumbed to the infection between 3 and 6 days post-challenged (dpc). In contrast, all mice in the PRV SX-10ΔgI/gE/TK/UL24, PRV SX-10ΔgI/gE/TK, and DMEM control groups survived (Figure 5A). Compared to the PRV SX-10ΔgI/gE/TK/UL24 and PRV SX-10ΔgI/gE/TK groups, significant viral genomic presence was detected in the brain, lungs, and liver tissues of mice in the Bartha-K61 and PRV SX-10 groups (Figure 5B). At 14 days dpc, histopathological examinations were conducted on the mice. In the PRV SX-10 and Bartha-K61 groups, mice exhibited markedly enlarged vascular sleeve phenomena in brain tissues, accompanied by neuronophagia. Additionally, these mice developed pulmonary edema, severe hemorrhage, and alveolar destruction in the lungs, as well as hepatocyte degeneration in the liver. In contrast, mice immunized with PRV SX-10ΔgI/gE/TK/UL24 exhibited no discernible histopathological lesions or pathological damage. However, mice in the PRV SX-10ΔgI/gE/TK group exhibited mild inflammatory cell infiltration and congestion in the lungs (Figure 5C).

To further evaluate the safety profile and immunogenic potential of recombinant virus, piglets were immunized with either PRV SX-10ΔgI/gE/TK/UL24, PRV SX-10ΔgI/gE/TK, Bartha-K61, or DMEM as a control (Figure 6A). At 14 dpi, all piglets in the vaccinated groups remained alive and in good health (Figure 6B). Rectal temperatures were monitored, and no significant differences were observed compared to the DMEM control group. Temperatures consistently remained below 40.0°C in all groups (Figure 6C). Piglets inoculated with PRV SX-10ΔgI/gE/TK/UL24, PRV SX-10ΔgI/gE/TK, or Bartha-K61 strains produced PRV-specific gB antibodies, with titers increasing progressively over time. However, PRV SX-10ΔgI/gE/TK/UL24 produced significantly higher antibody levels than Bartha-K61 (Figure 6D). At 28 dpi, piglets inoculated with PRV SX-10ΔgI/gE/TK/UL24 or PRV SX-10ΔgI/gE/TK strains showed comparable levels of neutralizing antibodies (NAbs) against PRV SX-10. These groups exhibited significantly higher NAb titers than the Bartha-K61-vaccinated cohort (p < 0.05) (Figure 6E). Furthermore, at 28 dpi, the group inoculated with PRV SX-10ΔgI/gE/TK/UL24 exhibited significantly higher levels of IFN-γ compared to groups inoculated with PRV SX-10ΔgI/gE/TK, Bartha-K61, or DMEM (p < 0.01). Serum levels of IL-2 and IL-4 were also significantly elevated in the group inoculated with PRV SX-10ΔgI/gE/TK/UL24 (p < 0.05) (Figure 6F).

At 28 dpi, piglets from the PRV SX-10ΔgI/gE/TK/UL24, PRV SX-10ΔgI/gE/TK, and Bartha-K61 test groups were challenged with 10⁶ TCID₅₀/mL of virulent PRV SX-10 strain, except for the DMEM control group. Piglets in the Bartha-K61 and PRV SX-10 groups exhibited typical pseudorabies symptoms, including pruritus and sneezing. Notably, two out of five piglets infected with the PRV SX-10 strain succumbed to the infection within 7–9 days post-inoculation. In contrast, piglets inoculated with PRV SX-10ΔgI/gE/TK/UL24, PRV SX-10ΔgI/gE/TK, Bartha-K61, or DMEM exhibited no discernible clinical symptoms (Figure 7A). For the first 9 days after infection, rectal temperatures in the PRV SX-10 and Bartha-K61 groups surpassed 41.0°C, indicating severe febrile responses (Figure 7B). Viral load quantification indicated low viral presence in the tissues of piglets immunized with the PRV SX-10ΔgI/gE/TK/UL24 and PRV SX-10ΔgI/gE/TK strains, with the former group exhibiting significantly lower titers in brain tissue (p < 0.05) (Figure 7C). Cytokine analysis conducted 3 dpc with PRV SX-10 revealed a significant upregulation of IL-6 in the PRV SX-10 and Bartha-K61 groups compared to groups vaccinated with PRV SX-10ΔgI/gE/TK/UL24, PRV SX-10ΔgI/gE/TK, or treated with DMEM (Figure 7D). Histopathological examination revealed severe tissue damage in piglets from the PRV SX-10 group. Affected tissues included lung alveoli with inflammatory infiltration and hemorrhage, brain tissue showing neuronal degeneration, and liver tissue displaying hepatocyte swelling and vascular dilation. Comparable pathological alterations were observed in the Bartha-K61 group. In stark contrast, piglets in the PRV SX-10ΔgI/gE/TK/UL24, PRV SX-10ΔgI/gE/TK, or DMEM groups exhibited no significant histopathological alterations (Figure 7E).

Both BHK-21 and PK-15 cells were grown in DMEM with added serum and antibiotics. Cells were kept at 37°C in a special incubator with controlled humidity and CO₂ levels. The PRV SX-10ΔgI/gE strain was previously constructed and maintained in our laboratory (Luo et al., 2022). The PRV SX-10 variant strain, isolated from a pig farm in Shaanxi, was used for neutralizing antibody tests and challenge protection experiments. Six-week-old female Kunming mice were obtained from Chengdu Dossy Experimental Animals Co., Ltd. (Chengdu, China). Four-week-old male American Landrace piglets, weighing 7–9 kg, seronegative for PRRSV, PCV2, CSFV, PPV, and PRV, were purchased from Shaanxi Shunxin Pig Breeding Co., Ltd. (Shaanxi, China).

To generate a PRV mutant deficient in gI, gE, TK, and UL24, we constructed a donor plasmid with UL24/TK deletions based on the PRV SX-10ΔgI/gE strain. Homology arms of 1,150 bp and 1,398 bp were amplified using primers UL25 LA-F/R and UL22 RA-F/R, respectively, with the SX-10 viral genome serving as the PCR template. These homology arms were subsequently inserted flanking a CMV promoter-driven EGFP reporter gene, resulting in the plasmid designated pUC57-UL25LA-CMV-EGFP-polyA-UL22RA. A similar method was used to construct a plasmid with only the UL24 deletion, which was named pUC57-UL25LA-CMV-EGFP-polyA-TKRA. To facilitate targeted deletions of the UL24/TK and UL24 genes, we employed the CRISPR tool¹ to design an sgRNA system. The dual sgRNAs were then individually cloned into a modified pX335 vector using BbsI and BsaI restriction sites. Primer and sgRNA sequences used are listed in Table 1.

Gene deletion in the virus was achieved by leveraging the host cell’s homology-directed repair (HDR) mechanism and CRISPR/Cas9 technology. BHK-21 cells were cultured in 12-well plates and co-transfected with either pX335 sgRNA1/2-TK/UL24 (2 μg) or pX335 sgRNA3/4-UL24 (2 μg), along with pUC57-UL25LA-CMV-EGFP-polyA-UL22RA (2 μg) or pUC57-UL25LA-CMV-EGFP-polyA-TKRA, using TurboFect transfection reagent as per the manufacturer’s instructions. At 24 h post-transfection, cells were infected with either PRV SX-10ΔgI/gE or PRV SX-10 at a multiplicity of infection (MOI) of 0.01. Upon observation of extensive cytopathic effect (CPE), cells and supernatants were harvested, subjected to multiple freeze–thaw cycles, and used to re-infect fresh cells. These cells were then overlaid with a mixture of 2× DMEM and 1% low-melting agarose. Using an inverted fluorescence microscope, researchers observed the development of plaques exhibiting green and red fluorescence, or solely green fluorescence. These fluorescent plaques underwent multiple rounds of isolation and purification. To confirm the identity of the recombinant viruses, PCR analysis was conducted using the specific primers outlined in Table 1. Finally, the PRV SX-10ΔgI/gE/TK/UL24 and PRV SX-10ΔUL24 deletion viruses were generated via plaque purification.

Five PRV strains (PRV SX-10ΔgI/gE/TK/UL24, PRV SX-10ΔgI/gE/TK, Bartha-K61, PRV SX-10, and PRV SX-10ΔUL24) were evaluated for their one-step growth kinetics and plaque morphology in PK-15 cells. For growth kinetics, PK-15 cells cultured in 12-well plates were infected with each virus at a MOI of 0.01. Cell supernatants were harvested at 12, 24, 36, 48, 60, and 72 h post-infection, subjected to three freeze–thaw cycles, and stored at −80°C. Virus titers were determined by TCID₅₀ assay and used to construct growth curves for each strain. For plaque assays, PK-15 cells in 12-well plates were exposed to viruses at a MOI of 0.01 for 90 min, then overlaid with DMEM containing 2% FBS and 1% low-melting agarose. At 96 h post-infection, plaques were visualized by crystal violet staining, and their sizes were quantified using ImageJ 2.7 software.² To assess the genetic stability of the different PRV strains, recombinant viruses were serially passaged 21 times in BHK cells. Viral DNA was extracted every five passages, and the TK, gEgI, EGFP, and mCherry genes were amplified using gene-specific primers TK-F/R, gEgI-F/R, EGFP-F/R and mCherry-F/R, respectively (Table 1).

PK-15 cells were cultured in 6-well plates. When the cell density reached 80%, each well was inoculated with PRV SX-10ΔgI/gE/TK/UL24, PRV SX-10ΔgI/gE/TK, PRV SX-10ΔUL24, and PRV SX-10, respectively, at a MOI of 0.1. The plates were incubated at 37°C in a CO₂ incubator for 90 min. Following incubation, the supernatant was removed and replaced with DMEM supplemented with 2% FBS. The cultures were then maintained for an additional 24 h. Post-incubation, cells were harvested and sequentially fixed with 0.5 and 0.3% glutaraldehyde solutions. The fixed samples were sent to Chengdu Rilai Biotechnology Co., Ltd. for further processing. Viral ultrastructure was visualized using a JEM-1400plus transmission electron microscope.

The four PRV strains—SX-10ΔgI/gE/TK/UL24, SX-10ΔgI/gE/TK, SX-10ΔUL24, and SX-10—were used to infect PK-15 cells cultured in 12-well plates at a MOI of 1.0. DMEM was used as a negative control. At 12 h post-infection, the supernatant was discarded, and 800 μL of TRNzol Universal reagent (Tiangen Biotech, Beijing, China) was added to each well to lyse the cells and extract total RNA. Subsequently, the extracted total RNA was reverse transcribed into cDNA using a reverse transcription kit (Genestar, Beijing, China) according to the manufacturer’s instructions. RT-qPCR was performed on the obtained cDNA using SYBR reagent (DiNing Biotechnology, Beijing, China) to measure the expression levels of β-actin and IFN-β; the corresponding primers are shown in Table 1. The RT-qPCR was performed using the Tianlong 988 Real-Time PCR System (Tianlong Science & Technology, Shaanxi, China) with the following cycling conditions: initial denaturation at 95°C for 2 min, followed by 40 cycles of 95°C for 15 s, 60°C for 30 s, and 72°C for 30 s. A melting curve analysis was included to verify the specificity of the reaction.

Twenty-five mice were randomly allocated into three groups. Mice were intramuscularly administered 100 μL of either PRV SX-10ΔUL24 (n = 10) or PRV SX-10 (n = 10) with a virus doses of 10⁵ TCID₅₀/mL, except for the DMEM control group (n = 5). The inoculation method and dosage were based on previous studies conducted by our research group (Luo et al., 2022). Clinical signs (including pruritus or scratching) and mortality were monitored and recorded daily to assess the pathogenicity of the PRV SX-10ΔUL24 strain.

Six-week-old female KM mice were randomly allocated into six groups (n = 10 per group). Mice were intramuscularly administered 100 μL containing 10⁵ TCID₅₀/mL of PRV SX-10ΔgI/gE/TK/UL24, PRV SX-10ΔgI/gE/TK, Bartha-K61, or PRV SX-10, while the DMEM group served as a control. Clinical signs and survival rates were monitored daily. The clinical signs of PRV infection in mice were primarily observed through the following symptoms: pruritus, scratching and biting of the infected area, tachypnea, circling behavior in some cases, ocular inflammation in others, and in certain instances, sudden death without prior symptoms. At 14 dpi, mice received a second immunization. Serum samples were collected from the retro-orbital sinus of each mouse at designated time points (7, 14, 21, and 28 dpi) for the assessment of PRV-gB-specific antibody levels and neutralizing antibody titers (NAbs). It is noteworthy that the sample collection procedure should not interfere with the normal physiological functions of the mice.

At 28 dpi, all immunized groups, except for the PRV SX-10 and DMEM groups, were challenged intramuscularly (I.M.) with 100 μL 10⁵ TCID₅₀/mL of PRV SX-10. At 14 dpc, all mice were euthanized and necropsied. Brain, lung, and liver samples were collected for viral load determination and histopathological analysis.

Four-week-old weaned piglets, seronegative for PRRSV, PCV2, CSFV, PPV, and PRV, were randomly allocated into five groups (n = 5 per group) and housed separately. Piglets in immunization groups 1–3 were intramuscularly inoculated with 2 mL (10⁶ TCID₅₀/mL) of PRV SX-10ΔgI/gE/TK/UL24, PRV SX-10ΔgI/gE/TK, or Bartha-K61, respectively. The positive control group (group 4) received 2 mL (10⁶ TCID₅₀/mL) of PRV SX-10, while the negative control group (group 5) received 2 mL of DMEM. The inoculation method and dosage for PRV infection in piglets were determined based on previous studies (Lv et al., 2021; Sun et al., 2022; Zhao et al., 2023). At 14 dpi, groups 1–3 received a booster immunization. Serum samples were collected from the anterior vena cava of piglets at specified time points (7, 14, 21, and 28 dpi or 3 dpc) for the assessment of PRV-gB-specific antibodies, neutralizing antibody titers, and cytokine levels.

At 28 dpi, groups 1 through 4 were challenged with 2 mL of PRV SX-10 at 10⁶ TCID₅₀/mL. Piglets were monitored daily for 14 dpi, clinical signs and rectal temperatures were recorded. The primary clinical signs of PRV infection in piglets include pyrexia, ataxia, tachypnea, diarrhea, and pronounced cutaneous erythema. At 14 dpc, piglets were euthanized and necropsied. Brain, lung, and liver samples were collected to assess viral load and histopathology.

Serum samples from mice and piglets were collected at predetermined time points for analysis. PRV-specific gB antibodies in pigs were detected using commercially available ELISA kits (JNT, China) according to the manufacturer’s instructions. For mice, PRV-specific gB antibodies were detected using ELISA kits (Finder Biotech, China), with the secondary antibody in the kit replaced by HRP-conjugated goat anti-mouse IgG (Wuhan) at a 1:1,000 dilution. In addition, serum collected from experimental pigs at 28 dpi or 3 dpc was used to quantify IFN-γ, IL-2, IL-4, or IL-6 levels using ELISA kits (Cusabio, Wuhan, China), following the manufacturer’s protocol.

Neutralizing antibodies (NAbs) against PRV SX-10 strains were assessed using a modified version of previously described methods (Yu et al., 2016; He et al., 2019; Gerdts et al., 1999). The serum processing involved multiple steps: initial centrifugation (2,800 rpm, 30 min), followed by heat inactivation (56°C, 30 min). The treated serum underwent twofold serial dilutions before being combined with PRV SX-10 strain (100 TCID₅₀/mL). This mixture was then incubated at 37°C for 1 h. Subsequently, 100 μL of this mixture was added to confluent PK-15 cells in 96-well plates and incubated for 1 h. The mixture was then replaced with DMEM containing 2% FBS, and the culture was incubated at 37°C under 5% CO₂ for 4 days. Neutralizing antibody titers were calculated using the Reed-Muench method.

Mice and piglets were euthanized at designated time points, and fresh samples were promptly collected and immediately cryopreserved at −80°C for subsequent viral load quantification. One g of tissue sample was weighed and homogenized in a grinding tube containing 1 mL of physiological saline using an ultrasonic homogenizer. Following homogenization, 100 μL of the supernatant was collected and subjected to DNA extraction using a DNA extraction kit (Tiangen Biotech, Beijing, China). Subsequently, real-time quantitative PCR (qPCR) was employed to determine the copy number of the PRV gH gene in brain, liver, and lung tissues. The primers used for amplification are listed in Table 1. A standard curve was established using serial dilutions (10⁻⁴ to 10⁻⁸ copies/μL) of the pMD19T-gH positive control plasmid as a template. The qPCR cycling conditions were set as follows: 2 min at 50°C, 2 min at 95°C, followed by 40 cycles of 15 s at 95°C, 30 s at 60°C, and 10 s at 25°C.

Statistical analyses were conducted using one-way ANOVA in GraphPad Prism version 9.0 (GraphPad Software Inc., United States). Significant differences between groups are denoted by asterisks: *p < 0.05, **p < 0.01, and ***p < 0.001. Within each strain, different letters indicate significant differences at a threshold of p < 0.05. All data are presented as means ± standard error of the mean (SEM), derived from at least three independent experiments.