Generation and Characterization of Recombinant Pseudorabies Virus Delivering African Swine Fever Virus CD2v and p54

African swine fever (ASF) leads to high mortality in domestic pigs and wild boar, and it is caused by the African swine fever virus (ASFV). Currently, no commercially available vaccine exists for its prevention in China. In this study, we engineered a pseudorabies recombinant virus (PRV) expressing ASFV CD2v and p54 proteins (PRV-∆TK-(CD2v)-∆gE-(p54)) using CRISPR/Cas9 and homologous recombination technology. PRV-∆TK-(CD2v)-∆gE-(p54) effectively delivers CD2v and p54, and it exhibits reduced virulence. Immunization with PRV-∆TK-(CD2v)-∆gE-(p54) neither induces pruritus nor causes systemic infection and inflammation. Furthermore, a double knockout of the TK and gE genes eliminates the depletion of T, B, and monocytes/macrophages in the blood caused by wild-type viral infection, decreases the proliferation of granulocytes to eliminate T-cell immunosuppression from granulocytes, and enhances the ability of the immune system against PRV infection. An overexpression of CD2v and p54 proteins does not alter the characteristics of PRV-∆TK/∆gE. Moreover, PRV-∆TK-(CD2v)-∆gE-(p54) successfully induces antibody production via intramuscular (IM) vaccination and confers effective protection for vaccinated mice upon challenge. Thus, PRV-∆TK-(CD2v)-∆gE-(p54) demonstrates good immunogenicity and safety, providing highly effective protection against PRV and ASFV. It potentially represents a suitable candidate for the development of a bivalent vaccine against both PRV and ASFV infections.


Introduction
African swine fever (ASF) is a highly contagious viral disease that leads to significant economic losses in the global swine industry.The severity of ASF clinical signs and the fatality rates varies depending on the animal species and the specific virus strains involved.Acute ASFV infection is associated with nearly 100% mortality [1,2].ASFV, classified as a DNA virus, belongs to the Asfivirus genus within the Asfarviridae family.It is characterized by a 200 nm diameter icosahedral DNA structure comprising an envelope, capsid, inner capsule membrane, core shell, and inner core [3].The viral genome is a linear doublestranded DNA molecule ranging from 170 kb to 190 kb in size, depending on the virus strain.It encodes between 150 and 200 viral proteins, encompassing 68 structural proteins and over 100 non-structural proteins [4,5].
Vaccination represents an effective strategy for preventing viral infection.Current vaccine options include live-attenuated, inactivated, recombinant virus, protein subunit, RNA, DNA, and virus-like particle (VLP) vaccines [6].Live-attenuated or subunit vaccines to infect cells [34], highlighting its significance in subunit vaccine, nucleic acid, and viral vector vaccine research.
In this study, we engineered a recombinant PRV, PRV-∆TK-(CD2v)-∆gE-(p54), expressing both ASFV CD2v and p54 proteins using CRISPR/Cas9 technology, and assessed its safety, ability to stimulate humoral immune responses, and efficacy in providing protection in mice.These findings offer valuable insights for future vaccine development targeting both ASFV and PRV.
The configuration of this recombinant virus is illustrated in Figure 1a.The EGFP and EBFP were driven by the CMV promoter, and the CD2v and p54 were driven by the EF1α promoter.For protein purification and subsequent verification, Flag-tag was added to both the N-and C-termini of CD2v, and 6xHis-tag was added to both the Nand C-termini of p54.To assess the ability of the recombinant virus to efficiently deliver and mediate transgene expression, we infected Vero cells with the wild-type virus PRV-Fa and the recombinant virus PRV-∆TK-(CD2v)-∆gE-(p54).Western blotting (Figure 1b) demonstrated the successful expressions of both CD2v (left) and p54 (right) in Vero, L929, and ST cells.Consistent with the Western blot results, we also confirmed the successful expression of CD2v and p54 in Vero cells following PRV-∆TK-(CD2v)-∆gE-(p54) infection using immunostaining (Figure 1c).As expected, Vero cells infected with PRV-Fa or PRV-∆TK/∆gE did not exhibit the expression of CD2v and p54 (Figure 1c).
In order to determine the genetic stability of PRV-∆TK-(CD2v)-∆gE-(p54), the virus was passaged for at least 40 generations, and the CD2v and p54 genes were detected using PCR and Sanger sequencing.The results showed that the recombinant virus could stably inherit the CD2v and p54 genes (Figure S1).

The Recombinant Pseudorabies Virus Has Reduced Virulence and Is Safe for Mice
To assess the virulence of the recombinant PRV-∆TK-(CD2v)-∆gE-(p54) virus, onestep growth curves were determined in Vero cells for PRV-Fa, PRV-ΔTK/ΔgE, and PRV-∆TK-(CD2v)-∆gE-(p54).The strategy for infection is shown in Supplementary Figure S2.Following infection, crystal violet staining was applied, and viral plaques were quantified on the third day.The findings demonstrate a significant reduction in viral plaques produced by the recombinant virus (Figure 2a), indicating a weakened cell infection capacity.Moreover, utilizing Karber's method, one-step growth curves were computed and graphed subsequent to Vero cell infection with the corresponding virus.Virus titers were assessed at 12 h, 24 h, 36 h, and 48 h, revealing a substantial reduction in titer for the These findings demonstrate the successful construction of the recombinant virus PRV-∆TK-(CD2v)-∆gE-(p54) and its capacity to efficiently deliver and express CD2v and p54 proteins.

The Recombinant Pseudorabies Virus Has Reduced Virulence and Is Safe for Mice
To assess the virulence of the recombinant PRV-∆TK-(CD2v)-∆gE-(p54) virus, onestep growth curves were determined in Vero cells for PRV-Fa, PRV-∆TK/∆gE, and PRV-∆TK-(CD2v)-∆gE-(p54).The strategy for infection is shown in Supplementary Figure S2.Following infection, crystal violet staining was applied, and viral plaques were quantified on the third day.The findings demonstrate a significant reduction in viral plaques produced by the recombinant virus (Figure 2a), indicating a weakened cell infection capacity.Moreover, utilizing Karber's method, one-step growth curves were computed and graphed subsequent to Vero cell infection with the corresponding virus.Virus titers were assessed at 12 h, 24 h, 36 h, and 48 h, revealing a substantial reduction in titer for the recombinant virus (Figure 2b).These outcomes strongly suggest that double knockout of gE and TK leads to a diminished proliferation of PRV, thereby affirming the heightened safety profile of the PRV-∆TK-(CD2v)-∆gE-(p54) recombinant virus.
groups of 6-week-old male C57BL/6 mice were infected intramuscularly (IM) with 5 × 10 5 TCID50 PRV-Fa, PRV-ΔTK/ΔgE, or PRV-∆TK-(CD2v)-∆gE-(p54).The control group received a PBS vaccination.On the third day post-infection, severe pruritus was observed in the PRV-Fa group but not in the PRV-ΔTK/ΔgE-or PRV-∆TK-(CD2v)-∆gE-(p54)infected groups, indicating that the insertion of CD2v and p54 did not alter the pathogenicity of PRV-ΔTK/ΔgE.The primary cause of mortality in mice infected with highly virulent strains was systemic inflammation, particularly neuroinflammation.IL-6 serves as a crucial marker of systemic inflammation induced by highly virulent infections [35].Serum samples were collected on the third day post-infection prior to mouse sacrifice, and IL-6 levels in serum were measured via an enzyme-linked immunosorbent assay.Our results revealed a significant increase in IL-6 secretion in the mice infected with PRV-Fa, while IL-6 secretion in the mice infected with PRV-∆TK/∆gE or PRV-∆TK-(CD2v)-∆gE-(p54) was comparable to the control group (Figure 2d).
In order to evaluate organ injury in the infected mice, we sacrificed mice on the third day post-infection before the mice died in the PRV-Fa infection group and collected the brain, heart, lung, liver, kidney, colon, and spleen.H&E staining revealed that PRV-Fa infection prompted infiltration of immune cells into the lung and liver, a phenomenon not observed in the other two infection groups.This strongly suggests that systemic inflammation is a direct consequence of the PRV-Fa infection.Conversely, PRV-∆TK/∆gE or PRV-∆TK-(CD2v)-∆gE-(p54) infection did not induce infiltration of immune cells (Figure 3a,b).Furthermore, PRV-Fa infection leads to a significant reduction in lymphocytes in the white pulp of the spleen, indicating that PRV-Fa infection depletes lymphocytes in mice.In contrast, the mice infected with PRV-∆TK/∆gE or PRV-∆TK-(CD2v)-∆gE-(p54) were unaffected (Figure 3c).Nonetheless, the kidney, colon, and brain showed no discernible effects (Figure 3d-f).Moreover, the viral genome DNA was detected in all tissues in PRV-Fa-infected mice but not in the PRV-∆TK/∆gE or PRV-∆TK-(CD2v)-∆gE-(p54)-infected groups on the third day (Figure S3).These findings demonstrate that the double knockout of TK and gE effectively abolishes systemic inflammation triggered by PRV-Fa infection, and an overexpression of CD2v and p54 proteins did not alter the histopathology of PRV-∆TK/∆gE.
were measured via an enzyme-linked immunosorbent assay.Our results revealed significant increase in IL-6 secretion in the mice infected with PRV-Fa, while IL-6 secretio in the mice infected with PRV-ΔTK/ΔgE or PRV-∆TK-(CD2v)-∆gE-(p54) was comparab to the control group (Figure 2d).
In order to evaluate organ injury in the infected mice, we sacrificed mice on the thir day post-infection before the mice died in the PRV-Fa infection group and collected th brain, heart, lung, liver, kidney, colon, and spleen.H&E staining revealed that PRV-F infection prompted infiltration of immune cells into the lung and liver, a phenomenon no observed in the other two infection groups.This strongly suggests that system inflammation is a direct consequence of the PRV-Fa infection.Conversely, PRV-ΔTK/Δg or PRV-∆TK-(CD2v)-∆gE-(p54) infection did not induce infiltration of immune cel (Figure 3a,b).Furthermore, PRV-Fa infection leads to a significant reduction i lymphocytes in the white pulp of the spleen, indicating that PRV-Fa infection deplete lymphocytes in mice.In contrast, the mice infected with PRV-ΔTK/ΔgE or PRV-∆TK (CD2v)-∆gE-(p54) were unaffected (Figure 3c).Nonetheless, the kidney, colon, and brai showed no discernible effects (Figure 3d-f).Moreover, the viral genome DNA wa detected in all tissues in PRV-Fa-infected mice but not in the PRV-ΔTK/ΔgE or PRV-∆TK (CD2v)-∆gE-(p54)-infected groups on the third day (Figure S3).These finding demonstrate that the double knockout of TK and gE effectively abolishes system inflammation triggered by PRV-Fa infection, and an overexpression of CD2v and p5 proteins did not alter the histopathology of PRV-ΔTK/ΔgE.These results revealed that the recombinant pseudorabies virus PRV-∆TK-(CD2v)-∆gE-(p54) is safe for mice.

Double Knockout of TK and gE Protects Mice from Exhaustion of Multiple Immune Cells Caused by PRV Challenge
In consideration of the role of CD2v in inhibiting lymphocyte proliferation in response to mitogens during ASFV infection [28], we assessed the leukocyte populations in the peripheral blood of immunized mice.Peripheral blood samples, collected on the third day post-infection prior to sacrifice, were processed with Gey's solution to remove red blood cells and resuspended in PBS supplemented with 2% BSA; then, the cells were stained with a combination of B220-APC, Mac-1-PECy7, and Gr-1-PE for B cells and myeloid cells or stained with a combination of CD3-Alexa Fluor 700, CD4-APC, CD8-BUV805, and CD25-FITC for T cells.
The results show that the percentage of B cells was markedly reduced in PRV-Fainfected mice, while PRV-∆TK/∆gE or PRV-∆TK-(CD2v)-∆gE-(p54) infections not only prevented this decrease but also stimulated the proliferation of these cells (Figure 4a,b).Furthermore, our results unveiled a significant reduction in the percentage of T cells, including CD4 + , CD8 + , and CD4 + CD25 + Treg cells, in PRV-Fa-infected mice, while the mice infected with PRV-∆TK/∆gE or PRV-∆TK-(CD2v)-∆gE-(p54) were unaffected (Figure 4c-h).Similar to the B cells and T cells, the monocytes/macrophages also experienced a decrease with PRV-Fa infection, but they remained unaffected by PRV-∆TK/∆gE infection.Interestingly, PRV-∆TK-(CD2v)-∆gE-(p54) infection significantly elevated the percentage of monocytes/macrophages (Figure 4i,j).In contrast, we observed that PRV infection led to an increased percentage of granulocytes, while the mice infected with PRV-∆TK/∆gE or PRV-∆TK-(CD2v)-∆gE-(p54) presented a decrease to a level even lower than that in the control group.Therefore, wild-type PRV-Fa infection depletes various lymphocytes in peripheral blood.However, the double knockout of TK and gE in PRV did not impair the immunity Therefore, wild-type PRV-Fa infection depletes various lymphocytes in peripheral blood.However, the double knockout of TK and gE in PRV did not impair the immunity of mice.Furthermore, the overexpression of the CD2v and p54 proteins had no discernible impact on the characteristics of PRV-∆TK/∆gE.

The Recombinant Pseudorabies Virus-Induced Antibody Production via IM Vaccination in Mice
To ascertain the efficacy of delivering foreign proteins to the spleen via the recombinant PRV, we conducted immunohistochemical staining on spleen sections harvested on the third day post-infection.These sections were treated with anti-His-tag or anti-Flag-tag antibodies.Encouragingly, both CD2v and p54 were distinctly detected in the spleens of mice infected with PRV-∆TK-(CD2v)-∆gE-(p54).Conversely, the mice infected with PRV-Fa or PRV-∆TK/∆gE failed to exhibit any detectable expression of either CD2v or p54 (Figure 5a).
To evaluate the immunogenicity of PRV-∆TK-(CD2v)-∆gE-(p54), we successfully expressed and purified the p54 protein from HEK-293T cells (Figure 5b,c).This purified protein served as the substrate for ELISA analysis, enabling the precise detection of p54specific antibodies.For the CD2v protein, we used the remaining stock from our previous study [32].
recombinant PRV, we conducted immunohistochemical staining on spleen sections harvested on the third day post-infection.These sections were treated with anti-His-tag or anti-Flag-tag antibodies.Encouragingly, both CD2v and p54 were distinctly detected in the spleens of mice infected with PRV-∆TK-(CD2v)-∆gE-(p54).Conversely, the mice infected with PRV-Fa or PRV-ΔTK/ΔgE failed to exhibit any detectable expression of either CD2v or p54 (Figure 5a).

The Recombinant Pseudorabies Virus Confers Effective Protection for Vaccinated Mice after Challenge
Our results demonstrate the immunogenicity and safety of the recombinant PRV-∆TK-(CD2v)-∆gE-(p54) virus vaccine in mice.The next logical step is to evaluate the potential of the vaccine to confer protection against viral infection in mice.
To achieve this, the strategy we used was a homologous prime-boost vaccination regimen.Three distinct groups of mice were subjected to challenge with PRV-Fa via IM injection into the leg (100 µL, 5 × 10 5 TCID 50 ) 14 days after the vaccination period with PBS, PRV-∆TK/∆gE, and PRV-∆TK-(CD2v)-∆gE-(p54).The control group was mock-challenged.The strategy for the vaccination and challenge is shown in Supplementary Figure S4.As anticipated, severe pruritus was observed in the PBS-vaccinated group on the third day Meanwhile, we assessed the detoxification effects in mice following the PRV-Fa challenge.Viral genomic DNA in the feces of each group was detected via qPCR for a total of 7 days post-challenge.Notably, viral DNA loading in the PBS-vaccinated group was significantly higher compared to the groups immunized with PRV-ΔTK/ΔgE or PRV-∆TK-∆TK-(CD2v)-∆gE-(p54) (Figure 6d).This suggests that PRV-ΔTK/ΔgE and PRV-∆TK-(CD2v)-∆gE-(p54) do not undergo rapid replication in immunized mice.Therefore, the recombinant PRV confers highly effective protection to vaccinated mice following challenge.

Discussion
The most efficacious experimental vaccine candidates for the current ASFV strain pandemic are live-attenuated virus vaccines developed by deleting specific virulenceassociated genes [10,11,[36][37][38].Despite this, concerns persist regarding their side effects and Meanwhile, we assessed the detoxification effects in mice following the PRV-Fa challenge.Viral genomic DNA in the feces of each group was detected via qPCR for a total of 7 days post-challenge.Notably, viral DNA loading in the PBS-vaccinated group was significantly higher compared to the groups immunized with PRV-∆TK/∆gE or PRV-∆TK-∆TK-(CD2v)-∆gE-(p54) (Figure 6d).This suggests that PRV-∆TK/∆gE and PRV-∆TK-(CD2v)-∆gE-(p54) do not undergo rapid replication in immunized mice.Therefore, the recombinant PRV confers highly effective protection to vaccinated mice following challenge.

Discussion
The most efficacious experimental vaccine candidates for the current ASFV strain pandemic are live-attenuated virus vaccines developed by deleting specific virulenceassociated genes [10,11,[36][37][38].Despite this, concerns persist regarding their side effects and potential safety issues, including lymphadenopathy, recurrent fever, chronic viremia, persistent chronic infections, and the possibility of virulence recovery [10][11][12][13].In contrast, the advantages of recombinant virus vaccines are their predictability of response and higher efficacy [14].Among them, PRV is a good vector for developing multivalent vaccines [15].
Many genes that are unnecessary for PRV replication, such as TK, gE, and gI, can be replaced by foreign genes [25].The primary virulent genes in PRV, gE, and TK can be deleted, leading to substantial reductions in invasiveness and virulence while retaining the vaccine's immunogenicity.Therefore, current commercial live-attenuated PRV vaccines are developed based on the deletion of gE and TK [9,39,40].Furthermore, studies indicate that by replacing gE and TK with foreign genes, the virulent PRV can be transformed into a safe and effective recombinant virus vector vaccine [32,41].
CD2v and p54 play pivotal roles in the infection, internalization, transport, and assembly of ASFV [26,27,33,34].Consequently, CD2v and p54 represent focal points in the exploration of subunit vaccines, nucleic acid vaccines, and viral vector vaccines.
Systemic inflammation, particularly neuroinflammation, emerges as the primary cause of mortality in mice following infection with highly virulent PRV strains.As IL-6 stands out as a major marker for inflammation induced by such virulent infections [35], its levels were measured via an enzyme-linked immunosorbent assay.The results reveal a significant increase in IL-6 secretion in mice infected with PRV-Fa, whereas the IL-6 levels in those infected with PRV-∆TK/∆gE or PRV-∆TK-(CD2v)-∆gE-(p54) were comparable to the control group.
Moreover, H&E staining showed that PRV-Fa infection induced the infiltration of immune cells into the lung and liver, as well as a significant decrease in lymphocytes in the white pulp of the spleen.In contrast, PRV-∆TK/∆gE or PRV-∆TK-(CD2v)-∆gE-(p54) infections did not induce immune cell infiltration or lymphocyte reduction.This indicates that PRV-Fa infection triggers systemic inflammation and lymphocyte depletion in mice.Conversely, double knockout of TK and gE eliminates the ability of the PRV to induce inflammation and lymphocyte depletion in mice.Furthermore, the overexpression of CD2v and p54 proteins exerted no discernible impact on the characteristics of PRV-∆TK/∆gE.
A previous study showed that inflammation promotes T cell overactivation and eventual exhaustion [42].This strongly implies that systemic inflammation is the underlying cause of lymphocyte depletion resulting from the PRV-Fa infection.This insight suggests that employing anti-inflammatory agents to mitigate inflammation during PRV infection may alleviate lymphocyte exhaustion, thereby fortifying the immunity of afflicted swine and diminishing mortality.
CD2v is a decisive factor for in vitro ASFV infection of peripheral blood lymphocytes to inhibit lymphocyte proliferation in response to mitogens.In vitro, the ASFV deletion of CD2v and the parental virus exhibited indistinguishable growth characteristics on primary porcine macrophage cell cultures; however, mitogen-dependent lymphocyte proliferation of swine PBMCs in vitro was reduced by 90 to 95% following infection with the revertant virus but remained unaltered following infection with the CD2v gene deletion mutant [28].
In another study, the attenuated ASFV with a single amino acid substitution in CD2v, Q96R, induced moderate levels of replication and 100% protection against virulent ASFV in pigs [31].However, it has also been reported that CD2v immunization alone can provide partial protection in the early stages of ASFV infection [30].In addition, our previous study showed that the recombinant PRV that only carries CD2v provides 100% protective ability, induces CD2v-specific humoral and cellular immune responses in mice, and only slightly interferes with the in vivo proliferation of T cells [32].Based on these studies, one possible explanation is that CD2v requires other components of the ASFV to perform immunosuppressive functions.Individually, CD2v can be used as an effective immunogen to activate the immune system and produce effective protection against ASFV infection.In this study, leucocyte subsets in peripheral blood were detected via flow cytometry.Consistent with a previous study [32], our results show that the percentages of T cells, including CD4 + , CD8 + , and CD25 + Treg cells, were markedly reduced in PRV-Fa-infected mice, while those infected with PRV-∆TK/∆gE or PRV-∆TK-(CD2v)-∆gE-(p54) exhibited no such effects.Notably, our study reveals that PRV-∆TK/∆gE or PRV-∆TK-(CD2v)-∆gE-(p54) does not impede T cell proliferation in vivo, suggesting that the double knockout of TK and gE genes effectively eliminates T cell depletion induced by PRV-Fa infection.Additionally, the overexpression of CD2v and P54 proteins exerts no discernible impact on the characteristics of PRV-∆TK/∆gE.Furthermore, we revealed that PRV-Fa infection can also disrupt B cell proliferation, whereas PRV-∆TK/∆gE or PRV-∆TK-(CD2v)-∆gE-(p54) not only refrains from interfering but also stimulates the proliferation of these cells.Prior studies propose that early-stage influenza infection is characterized by substantial upsurges in local type I IFN production, augmenting B cell receptor-mediated proliferation of B cells [43].Presumably, this aids even low-affinity B cells in orchestrating an antiviral response, thereby initiating the expansion of virus-specific clones [44].Within two days of type I IFN-induced CD69 expression, infection of B cells within mediastinal lymph nodes induces upregulation of CD69 and the co-stimulatory surface molecule CD86 [45,46], thereby regulating B cell egress from lymph nodes into the bloodstream [47].The increased B cell levels in mice infected with PRV-∆TK/∆gE or PRV-∆TK-(CD2v)-∆gE-(p54) indicate that the double knockout of TK and gE genes in PRV entirely eliminates the toxicity of PRV-Fa, effectively stimulates the proliferation of low-affinity circulating B cells in peripheral blood, or enhances the ability of B cell egress from lymph nodes into the blood.Moreover, an overexpression of CD2v and P54 proteins does not impact the characteristics of PRV-∆TK/∆gE.
Much like B cells, the proliferation of monocytes/macrophages is not only affected by PRV-Fa but also stimulated by PRV-∆TK/∆gE or PRV-∆TK-(CD2v)-∆gE-(p54).Monocytes/macrophages constitute a diverse group of cells that serve as the vanguards in innate immunity and, subsequently, as mediators for adaptive immunity to help clear infections [48].These cells are recognized as professional antigen-presenting cells and "professional" phagocytes, equipped with general receptors and several sensors, particularly pattern recognition receptors that initiate and regulate immune responses against invading pathogens [49].Increased monocytes/macrophages in mice infected with PRV-∆TK/∆gE or PRV-∆TK-(CD2v)-∆gE-(p54) signifies that the double knockout of TK and gE genes, coupled with recombination of both CD2v and p54 genes in PRV, robustly activate the first responders in innate immunity.In contrast, we revealed that PRV-Fa infection promotes the proliferation of granulocytes while disrupting this in mice infected with PRV-∆TK/∆gE or PRV-∆TK-(CD2v)-∆gE-(p54).The percentage of granulocytes decreased to a level that was lower than the PBS control group.According to previous research, increasing immature granulocytes is associated with clinical severity and prognosis after surgery, and in the early phase of sepsis, immature granulocytes could directly contribute to T-cell immunosuppression and thus facilitate secondary post-cardiac surgery infection [50,51].The observed decrease in granulocytes among mice infected with PRV-∆TK/∆gE or PRV-∆TK-(CD2v)-∆gE-(p54) signifies that the double knockout of TK and gE genes effectively eliminates T-cell immunosuppression from granulocytes, thereby enhancing the ability of the immune system against viral infection.Moreover, the overexpression of CD2v and P54 proteins has shown no discernible impact on the characteristics of PRV-∆TK/∆gE.
Moreover, according to another study, CD2v interacts with CSF2RA, which is a hematopoietic receptor superfamily member in myeloid cells and a key receptor protein that activates receptor-associated JAK and STAT proteins.CD2v deletion downregulates the JAK2-STAT3 pathway and promotes apoptosis to inhibit ASFV replication [52].Our study shows that the proliferation rate of monocytes/macrophages in mice infected with PRV-∆TK-(CD2v)-∆gE-(p54) is higher than that in mice infected with PRV-∆TK/∆gE, indicating that the recombinant PRV-carrying CD2v can specifically promote the proliferation of monocytes/macrophages in myeloid cells rather than granulocytes.The increase in and activation of B cells and monocytes/macrophages, as well as decreased granulocytes, indicates that the recombinant virus can effectively eliminate T cell immunosuppression and activate the immune system in mice, thereby effectively responding to viral infection.
Antibodies play a crucial role in the immune response by fighting off pathogens as well as helping to create strong immunological memory.Pathogen-specific antibodies are a hallmark of an effective immune response following infection or vaccination [53].To evaluate the immunogenicity of PRV-∆TK-(CD2v)-∆gE-(p54), antigen-specific IGs were detected on days 7 and 14 after vaccination.Remarkably, PRV-∆TK-(CD2v)-∆gE-(p54) elicited significant serum IGs in mice against CD2v and p54 on day 7 following IM vaccination, whereas PRV-Fa and PRV-∆TK/∆gE did not elicit serum IGs against CD2v and p54, demonstrating that the recombinant PRV-∆TK-(CD2v)-∆gE-(p54) virus vaccine successfully induced antibody production via IM vaccination in mice.This result shows that PRV-∆TK-(CD2v)-∆gE-(p54) demonstrates good immunogenicity for inducing antibody production against AFSV CD2v and p54, indicating that vaccination with this recombinant virus is able to suppress clinical signs related to PRV infection, while the production of antibodies against CD2v and p54 may offer protection against ASFV infection.

Mice
Six-week-old specific-pathogen-free SPF-C57BL/6 male mice were purchased from Wushi Experimental Animal Trade Co., Ltd.(Fuzhou, China) and housed in the Animal Center of Fujian Normal University.All research and animal care procedures were ethically approved by the Animal Ethical and Welfare Committee of Fujian Normal University (IACUC-20230041).
To generate the CD2v and p54 double-knockout virus, PRV-∆TK/∆gE transfected the KO plasmids targeting PRV gE and TK into 5 × 10 5 HEK 293T cells.Six hours posttransfection, we added 1 × 10 5 TCID 50 PRV-Fa.The PRV-Fa strain (GenBank: ON005001.1.:The PRV-Fa strain is the earliest isolated typical strain.It was isolated in the 1960s, and the genomic size was 141,930 nt.This strain caused pseudorabies to become prevalent in China, resulting in most of the cases of pseudorabies and large economic losses in the pig industry [22]), generously provided by the Fujian Academy of Agricultural Sciences, was propagated in Vero cells.The virus culture was harvested once the cytopathic effect reached 90% or more.The TCID 50 (50% tissue culture infective dose) was determined using a microtiter assay as outlined by Reed and Muench.The sgRNA-induced mutation was verified through PCR and sequencing with specific primers (primers for gE-KO are forward 5 ′ -AAAAGGTGGTGTTTGCATAATT-3 ′ and reverse 5 ′ -TCGGTGGTGATGTAGAACG-3 ′ ; primers for TK-KO are forward 5 ′ -TCGTAGAAGCGGTTGTGG-3 ′ and reverse 5 ′ -CGACCA GGACGAACAGG-3 ′ ).Four rounds of plaque purification were conducted in the Vero cells to obtain pseudorabies gE and the TK double-knockout strain PRV-∆TK/∆gE.
To determine the growth kinetics of the virus, 5 × 10 5 Vero cells in 2 mL of DMEM were seeded into each well of 6-well plates.Then, 1 × 10 3 TCID 50 viruses were added to each well, and samples were collected 12, 24, 36, and 48 h post-infection.The virus titer was calculated using the Karber method.
The growth of plaques in Vero cells was observed by crystal violet staining.Vero cells (5 × 10 5 /each well) in the 6-well plates were infected with PRV-Fa, PRV-∆TK/∆gE, or PRV-∆TK-(CD2v)-∆gE-(p54) of 2 × 10 2 TCID 50 for 36 h, and fixed with 4% paraformaldehyde for 30 min, stained with 2.5% crystal violet.Seven plaques were randomly selected to measure their areas under the microscope.The size of the plaques was determined by ZEN blue 2.3, a picture editing software that comes with the microscope (ZEISS Axio Vert.A1, Carl Zeiss AG, Oberkohen, Germany).

Viral Copy Analyses in Mice
The viral copy number was assessed in the fecal samples.Fecal matter from the mice was collected every 24 h post-virus challenge.One gram of feces was mixed with 5 mL of phosphate buffered saline (PBS) and soaked for 2 h, and the supernatant was obtained via centrifugation at 3000× g for 10 min.This supernatant was used for extracting the genomic DNA for qPCR to determine viral nucleic acid copies.Genomic DNA was extracted and purified using the TIANamp Genomic DNA Kit (TIANGEN BIOTECH, Beijing, China, DP304-03).The qPCR primers were as follows: forward (5 ′ -AACGTCACCTTCGA-GGTGTA-3 ′ ) and reverse (5 ′ -AGTCTGAACTCGTGCTTG-3 ′ ).The PRV UL42 gene served as the standard control and was inserted into the pCDH plasmid to create pCDH-UL42.The UL42 gene was also amplified from the genomic DNA of the PRV-Fa strain using the forward primer 5 ′ -ATGTCGCTGTTCGACGAC-3 ′ and the reverse primer 5 ′ -TTAGAATAAATCTCCGTAGGCG-3 ′ .

Hematoxylin-Eosin Staining (H&E Staining) and Immunohistochemistry
Upon the onset of scratching in the control PRV-Fa-infected mouse group (approximately 72 h post-infection), all mice across the groups were humanely euthanized using CO 2 inhalation.Tissues from the heart, brain, lung, liver, spleen, and kidney were collected.These tissues were fixed with 4% paraformaldehyde for 12 h.Subsequently, the samples underwent a series of ethanol treatments (30%, 50%, 70%, 90%, 95%, and anhydrous ethanol) for 30 min each.This was followed by a transparent treatment involving a mixture of ethanol and xylene (1:1), with xylene ultimately replacing the ethanol in the tissues.The tissues were further treated with a mixture of paraffin and xylene (1:1) overnight before being embedded in the paraffin.Finally, the tissues were sliced into 7 µm sections.
The images were captured by scanning with a ZEISS LSM700 microscope (software: ZEN 2.3) (Carl Zeiss AG, Oberkohen, Germany).

Data Statistical Analysis and Image Processing
The images from the Western blotting, H&E staining, crystal violet staining, and immunofluorescence experiments were processed using Adobe Illustrator CS6 software.All of the experiments were independently repeated at least three times.The data differences between groups were analyzed using unpaired t-tests or two-way ANOVA (Graphpad Prism 9.5 Software, San Diego, CA, USA).The data are presented as the mean ± SD for the same treatment.

Conclusions
In summary, we successfully engineered a recombinant PRV capable of expressing the ASFV CD2v and p54 proteins (PRV-∆TK-(CD2v)-∆gE-(p54)) using CRISPR/Cas9 technology.This recombinant virus exhibits reduced virulence, ensuring safety for mice.It confers effective protection for vaccinated mice after the wild-type PRV-Fa challenge; meanwhile, intramuscular vaccination-induced CD2v-and p54-specific antibodies may offer protection against ASFV infection.Therefore, it holds promise as a potential candidate for the development of a bivalent vaccine.However, further investigations are warranted to confirm its effectiveness in swine populations.

Figure 1 .
Figure 1.Construction and verification of recombinant virus PRV-∆TK-(CD2v)-∆gE-(p54).(a) Construct of recombinant virus PRV-∆TK-(CD2v)-∆gE-(p54); the scissors logo indicates the Cas9 protein cleavage site as well as the location of homologous template integration into the genome.(b) The expressions of the CD2v (left panel) and p54 (right panel) proteins in different cell lines after infection by the PRV-∆TK-(CD2v)-∆gE-(p54) recombinant virus were determined using Western blo ing; 48 h post-infection, the cells were harvested and lysed, resolved through SDS-PAGE, and transferred to PVDF membranes; the membrane was blo ed with anti-Flag-tag (CD2v) and anti-His-tag (p54) antibodies, respectively; the red arrow indicates the band of the target proteins; the blue arrow indicates the band of GAPDH.M, marker.(c) The CD2v (left panel) and p54 (right panel) protein expressions in Vero cells were determined under a fluorescence microscope at 24 h post-infection.Scale bar, 200 µm.

Figure 1 .
Figure 1.Construction and verification of recombinant virus PRV-∆TK-(CD2v)-∆gE-(p54).(a) Construct of recombinant virus PRV-∆TK-(CD2v)-∆gE-(p54); the scissors logo indicates the Cas9 protein cleavage site as well as the location of homologous template integration into the genome.(b) The expressions of the CD2v (left panel) and p54 (right panel) proteins in different cell lines after infection by the PRV-∆TK-(CD2v)-∆gE-(p54) recombinant virus were determined using Western blotting; 48 h post-infection, the cells were harvested and lysed, resolved through SDS-PAGE, and transferred to PVDF membranes; the membrane was blotted with anti-Flag-tag (CD2v) and anti-His-tag (p54) antibodies, respectively; the red arrow indicates the band of the target proteins; the blue arrow indicates the band of GAPDH.M, marker.(c) The CD2v (left panel) and p54 (right panel) protein expressions in Vero cells were determined under a fluorescence microscope at 24 h post-infection.Scale bar, 200 µm.

Figure 3 .
Figure 3.Recombinant PRV protects mice from tissue injury and the reduction in lymphocytes in the white pulp of the spleen caused by PRV infection.To assess the safeguarding potential of the recombinant PRV, mice were infected with wild-type (PRV-Fa), PRV-∆TK-∆gE and PRV-∆TK-(CD2v)-∆gE-(p54) viruses, respectively, by intramuscular injection into the leg (100 µL, 5 × 10 5 TCID 50 ); PBS was used as a control.Mice were sacrificed on day 3 after infection before they died in the PRV-Fa infection group.H&E staining was used for assessment.(a,b) PRV-Fa infection induced infiltration of immune cells in the lung and liver (red arrow) but not in the other two infection groups, indicating that systemic inflammation was caused by PRV-Fa infection.(c) PRV-Fa infection leads to a significant reduction in lymphocytes in the white pulp of the spleen, indicating that PRV-Fa infection depletes lymphocytes in mice.In contrast, the mice infected with PRV-∆TK/∆gE or PRV-∆TK-(CD2v)-∆gE-(p54) were unaffected.(d-f) Kidney, colon, and brain tissues showed no discernible effects.H&E, hematoxylin and eosin; scale bar, 100 µm (lung and kidney) or 200 µm.

Figure 4 .
Figure 4. Recombinant PRV protects mice from exhaustion of multiple immune cells caused by PRV infection.(a,b) The percentage of B cells was markedly reduced in PRV-Fa-infected mice, while PRV-ΔTK/ΔgE or PRV-∆TK-(CD2v)-∆gE-(p54) infections not only prevented this decrease but also stimulated the proliferation of these cells.(c-h) The percentages of T cells, including CD3 + , CD4 + , CD8 + , and CD4 + CD25 + Treg cells, in PRV-Fa-infected mice were significantly reduced, while the mice infected with PRV-ΔTK/ΔgE or PRV-∆TK-(CD2v)-∆gE-(p54) were unaffected.(i,j) The percentage of monocytes/macrophages was decreased by PRV-Fa infection but not affected by the infection of PRV-ΔTK/ΔgE, while the infection of PRV-∆TK-(CD2v)-∆gE-(p54) significantly increased the percentage of monocytes/macrophages; in contrast, PRV infection led to an increased percentage of granulocytes, while in the mice infected with PRV-ΔTK/ΔgE or PRV-∆TK-(CD2v)-∆gE-(p54) granulocyte percentage was decreased to a level even lower than the control group.The percentages represent the indicated cells within the gated live cell population.The data presented are derived from five mice in each group.Error bars indicate ± SD.Statistical analysis was performed with the 2-tailed unpaired Student t-test.* p < 0.05; ** p < 0.01; **** p < 0.0001; ns, not significant.

Figure 4 .
Figure 4. Recombinant PRV protects mice from exhaustion of multiple immune cells caused by PRV infection.(a,b) The percentage of B cells was markedly reduced in PRV-Fa-infected mice, while PRV-∆TK/∆gE or PRV-∆TK-(CD2v)-∆gE-(p54) infections not only prevented this decrease but also stimulated the proliferation of these cells.(c-h) The percentages of T cells, including CD3 + , CD4 + , CD8 + , and CD4 + CD25 + Treg cells, in PRV-Fa-infected mice were significantly reduced, while the mice infected with PRV-∆TK/∆gE or PRV-∆TK-(CD2v)-∆gE-(p54) were unaffected.(i,j) The percentage of monocytes/macrophages was decreased by PRV-Fa infection but not affected by the infection of PRV-∆TK/∆gE, while the infection of PRV-∆TK-(CD2v)-∆gE-(p54) significantly increased the percentage of monocytes/macrophages; in contrast, PRV infection led to an increased percentage of granulocytes, while in the mice infected with PRV-∆TK/∆gE or PRV-∆TK-(CD2v)-∆gE-(p54) granulocyte percentage was decreased to a level even lower than the control group.The percentages represent the indicated cells within the gated live cell population.The data presented are derived from five mice in each group.Error bars indicate ± SD.Statistical analysis was performed with the 2-tailed unpaired Student t-test.* p < 0.05; ** p < 0.01; **** p < 0.0001; ns, not significant.

Figure 5 .
Figure 5. Production of anti-p54 and anti-CD2v-antibodies in mice.(a) Mice were infected with PRV-Fa, PRV-∆TK/∆gE, and PRV-∆TK-(CD2v)-∆gE-(p54) by IM injection into the leg (100 µL, 5 × 10 5 TCID50); PBS was used as a control.Post-infection, mice were sacrificed on day 3, and spleens were fixed in 4% paraformaldehyde.Then, paraffin-embedded sections were prepared and subjected to immunohistochemical staining for Flag-tag (top) and His-tag (bo om), representing the CD2v and p54 proteins, respectively.Scale bar, 100 µm.Insets (scale bar, 20 µm) represent an enlarged local area.The red arrow indicates cells presenting and expressing CD2v or p54 protein.(b,c) The p54-6×His-tag protein (~45 kD) was purified using a Ni-NTA beads column.Coomassie blue-stained SDS-PAGE (b) and Western blot (c) of samples from a typical purification of P54 protein from overexpressed 293T cells.Lanes are labeled as follows: M, molecular-weight standards; 293T, untransfected 293T cells; T, total protein; FT, flowthrough (nonbound) from the Ni-NTA beads column; W, protein from the Ni-NTA beads column that was eluted in the wash fractions; P, purified protein after elution from the Ni-NTA beads column; B, Ni-NTA beads after protein elution.The red arrow indicates the target protein.(d) PRV-∆TK/∆gE and PRV-∆TK-(CD2v)-∆gE-(p54) were immunized into C57BL/6 mice via IM injection into the leg (100 µL, 5 × 10 5 TCID50).Serum samples were collected on day 7/14 post-immunization for p54-and CD2v-specific IgG detection via ELISA.The results demonstrate that mice immunized with PRV-R produced both p54-and CD2v-specific IgG.The data shown were obtained from five mice in each group.Error bars represent ± SD.

Figure 5 .
Figure 5. Production of anti-p54 and anti-CD2v-antibodies in mice.(a) Mice were infected with PRV-Fa, PRV-∆TK/∆gE, and PRV-∆TK-(CD2v)-∆gE-(p54) by IM injection into the leg (100 µL, 5 × 10 5 TCID 50 ); PBS was used as a control.Post-infection, mice were sacrificed on day 3, and spleens were fixed in 4% paraformaldehyde.Then, paraffin-embedded sections were prepared and subjected to immunohistochemical staining for Flag-tag (top) and His-tag (bottom), representing the CD2v and p54 proteins, respectively.Scale bar, 100 µm.Insets (scale bar, 20 µm) represent an enlarged local area.The red arrow indicates cells presenting and expressing CD2v or p54 protein.(b,c) The p54-6×His-tag protein (~45 kD) was purified using a Ni-NTA beads column.Coomassie blue-stained SDS-PAGE (b) and Western blot (c) of samples from a typical purification of P54 protein from overexpressed 293T cells.Lanes are labeled as follows: M, molecular-weight standards; 293T, untransfected 293T cells; T, total protein; FT, flowthrough (nonbound) from the Ni-NTA beads column; W, protein from the Ni-NTA beads column that was eluted in the wash fractions; P, purified protein after elution from the Ni-NTA beads column; B, Ni-NTA beads after protein elution.The red arrow indicates the target protein.(d) PRV-∆TK/∆gE and PRV-∆TK-(CD2v)-∆gE-(p54) were immunized into C57BL/6 mice via IM injection into the leg (100 µL, 5 × 10 5 TCID 50 ).Serum samples were collected on day 7/14 post-immunization for p54-and CD2v-specific IgG detection via ELISA.The results demonstrate that mice immunized with PRV-R produced both p54-and CD2v-specific IgG.The data shown were obtained from five mice in each group.Error bars represent ± SD.Statistical analysis was performed with the 2-tailed unpaired Student t-test.* p < 0.05; ** p < 0.01; ns, not significant.

Figure 6 .
Figure 6.Recombinant PRV provides protection for mice from rapid death caused by the wild-type PRV.(a) The behavioral phenotype of mice in each group (n = 5) after challenge was observed early from day 3; PRV-Fa caused pruritus in mice vaccinated with PBS and quick death; conversely, mice in the groups vaccinated with PRV-∆TK/∆gE and PRV-∆TK-(CD2v)-∆gE-(p54) were protected from these symptoms.In the group of mice vaccinated with PBS (with a blue arrow), mice curled up and bit the itchy thigh where the virus was injected.The figure at the bo om of the fourth column represents 1 mouse from each group, from left to right: A, mock; B, mouse vaccinated with PRV-∆TK/∆gE and challenged with PRV-Fa; C, mouse vaccinated with PRV-∆TK-(CD2v)-∆gE-(p54) and challenged with PRV-Fa; and D, mouse vaccinated with PBS and challenged with PRV-Fa.The image on the upper right side shows a mouse biting its thigh, and the image on the lower right side shows the depilated thigh of the mouse that bit itself due to pruritus.(b) The rectal temperature of mice in each group (n = 5) was recorded over the course of 7 days post-challenge; the rectal temperature of the mice in each group was comparable in the first two days, while it decreased in the group of mice vaccinated with PBS on the third day due to the mice being close to death.(c) The survival rate of mice in each group (n = 5) was recorded for a total of 14 days following the challenge with the corresponding virus; mice were mostly dead by the later part of day 3 in the groups vaccinated with PBS.(d) Viral DNA in the feces of mice in each group (n = 5, feces of each group mixed together every day) was detected using qPCR after challenge; feces samples were collected for a total of 7 days; viral DNA loading in the PBS-vaccinated group was significantly higher compared to the groups immunized with PRV-ΔTK/ΔgE or PRV-∆TK-∆TK-(CD2v)-∆gE-(p54).

Figure 6 .
Figure 6.Recombinant PRV provides protection for mice from rapid death caused by the wild-type PRV.(a) The behavioral phenotype of mice in each group (n = 5) after challenge was observed early from day 3; PRV-Fa caused pruritus in mice vaccinated with PBS and quick death; conversely, mice in the groups vaccinated with PRV-∆TK/∆gE and PRV-∆TK-(CD2v)-∆gE-(p54) were protected from these symptoms.In the group of mice vaccinated with PBS (with a blue arrow), mice curled up and bit the itchy thigh where the virus was injected.The figure at the bottom of the fourth column represents 1 mouse from each group, from left to right: A, mock; B, mouse vaccinated with PRV-∆TK/∆gE and challenged with PRV-Fa; C, mouse vaccinated with PRV-∆TK-(CD2v)-∆gE-(p54) and challenged with PRV-Fa; and D, mouse vaccinated with PBS and challenged with PRV-Fa.The image on the upper right side shows a mouse biting its thigh, and the image on the lower right side shows the depilated thigh of the mouse that bit itself due to pruritus.(b) The rectal temperature of mice in each group (n = 5) was recorded over the course of 7 days post-challenge; the rectal temperature of the mice in each group was comparable in the first two days, while it decreased in the group of mice vaccinated with PBS on the third day due to the mice being close to death.(c) The survival rate of mice in each group (n = 5) was recorded for a total of 14 days following the challenge with the corresponding virus; mice were mostly dead by the later part of day 3 in the groups vaccinated with PBS.(d) Viral DNA in the feces of mice in each group (n = 5, feces of each group mixed together every day) was detected using qPCR after challenge; feces samples were collected for a total of 7 days; viral DNA loading in the PBS-vaccinated group was significantly higher compared to the groups immunized with PRV-∆TK/∆gE or PRV-∆TK-∆TK-(CD2v)-∆gE-(p54).