Construction of recombinant pseudorabies virus expressing PCV2 Cap, PCV3 Cap, and IL-4: investigation of their biological characteristics and immunogenicity.

Background: Porcine circovirus type 2 (PCV2) is a globally prevalent and recurrent pathogen that primarily causes slow growth and immunosuppression in pigs. Porcine circovirus type 3 (PCV3), a recently discovered virus, commonly leads to reproductive disorders in pigs and has been extensively disseminated worldwide. Infection with a single PCV subtype alone does not induce severe porcine circovirus-associated diseases (PCVD), whereas concurrent co-infection with PCV2 and PCV3 exacerbates the clinical manifestations. Pseudorabies (PR), a highly contagious disease in pigs, pose a significant threat to the swine industry in China. Methods: In this study, recombinant strains named rPRV-2Cap/3Cap and rPRV-2Cap/3Cap/IL4 was constructed by using a variant strain XJ of pseudorabies virus (PRV) as the parental strain, with the TK/gE/gI genes deleted and simultaneous expression of PCV2 Cap, PCV3 Cap, and IL-4. The two recombinant strains obtained by CRISPR/Cas gE gene editing technology and homologous recombination technology has genetic stability in baby hamster Syrian kidney-21 (BHK-21) cells and is safe to mice. Results: rPRV-2Cap/3Cap and rPRV-2Cap/3Cap/IL4 exhibited good safety and immunogenicity in mice, inducing high levels of antibodies, demonstrated 100% protection against the PRV challenge in mice, reduced viral loads and mitigated pathological changes in the heart, lungs, spleen, and lymph nodes during PCV2 challenge. Moreover, the recombinant viruses with the addition of IL-4 as a molecular adjuvant outperformed the non-addition group in most indicators. Conclusion: rPRV-2Cap/3Cap and rPRV-2Cap/3Cap/IL4 hold promise as recombinant vaccines for the simultaneous prevention of PCV2, PCV3, and PRV, while IL-4, as a vaccine molecular adjuvant, effectively enhances the immune response of the vaccine.

Application of CRISPR/Cas9 for Rapid Genome Editing of Pseudorabies Virus and Bovine Herpesvirus-1.

The CRISPR/Cas9 system is widely used to manipulate viral genomes. Although Alphaherpesvirinae genomes are large and complicated to edit, in recent years several Pseudorabies virus (PRV) mutants have been successfully generated using the CRISPR/Cas9 system. However, the application of CRISPR/Cas9 editing on another member of alpha herpesviruses, bovine herpesvirus-1 (BHV-1), is rarely reported. This paper reports a rapid and straightforward approach to manipulating herpesviruses genome using CRISPR/Cas9. The recombinant plasmids contained the left and right arm of the thymidine kinase (TK) gene of PRV or of the glycoprotein I (gI) and glycoprotein E (gE) of BHV-1. Upon the cleavage of the TK or gIgE gene by Cas9 protein, this was replaced by the enhanced green fluorescence protein (eGFP) by homologous recombination. With this approach, we generated recombinant TK-/eGFP+ PRV and gIgE-/eGFP+ BHV-1 mutants and then proceeded to characterize their biological activities in vitro and in vivo. In conclusion, we showed that alpha herpesvirus, including PRV and BHV-1, can be rapidly edited using the CRISPR/Cas9 approach paving the way to the development of animal herpesvirus vaccines.

Single amino acid mutation of nectin-1 provides remarkable resistance against lethal pseudorabies virus infection in mice.

An approach to genetically engineered resistance to pseudorabies virus (PRV) infection was examined by using a mouse model with defined point mutation in primary receptor for alphaherpesviruses, nectin-1, by the CRISPR/Cas9 system. It has become clear that phenylalanine at position 129 of nectin-1 is important for binding to viral glycoprotein D (gD), and mutation of phenylalanine 129 to alanine (F129A) prevents nectin-1 binding to gD and virus entry in vitro. Here, to assess the antiviral potential of the single amino acid mutation of nectin-1, F129A, in vivo, we generated genome-edited mutant mouse lines; F129A and 135 knockout (KO). The latter, 135 KO used as a nectin-1 knockout line for comparison, expresses a carboxy-terminal deleted polypeptide consisting of 135 amino acids without phenylalanine 129. In the challenge with 10 LD50 PRV via intranasal route, perfect protection of disease onset was induced by expression of the mutation of nectin-1, F129A (survival rate: 100% in F129A and 135 KO versus 0% in wild type mice). Neither viral DNA/antigens nor pathological changes were detected in F129A, suggesting that viral entry was prevented at the primary site in natural infection. In the challenge with 50 LD50 PRV, lower but still strong protective effect against disease onset was observed (survival rate: 57% in F129A and 75% in 135 KO versus 0% in wild type mice). The present results indicate that single amino acid mutation of nectin-1 F129A provides significant resistance against lethal pseudorabies.

Fast and sensitive differential diagnosis of pseudorabies virus-infected versus pseudorabies virus-vaccinated swine using CRISPR-Cas12a.

IMPORTANCE: Pseudorabies virus (PRV) causes high mortality and miscarriage rates in the infected swine, and the eradication policy coupled with large-scale vaccination of live attenuated vaccines has been adopted globally against PRV. Differential diagnosis of the vaccinated and infected swine is highly demanded. Our multienzyme isothermal rapid amplification (MIRA)-Cas12a detection method described in this study can diagnose PRV with a superior sensitivity comparable to the quantitative PCR (qPCR) and a competitive detection speed (only half the time as qPCR needs). The portable feature and the simple procedure of MIRA-Cas12a make it easier to deploy for clinical diagnosis, even in resource-limited settings. The MIRA-Cas12a method would provide immediate and accurate diagnostic information for policymakers to respond promptly.

Natto extract inhibits infection caused by the Aujeszky's disease virus in mice.

Aujeszky's disease virus (ADV), also known as Suid alphaherpesvirus 1, which mainly infects swine, causes life-threatening neurological disorders. This disease is a serious global risk factor for economic losses in the swine industry. The development of new anti-ADV drugs is highly anticipated and required. Natto, a traditional Japanese fermented food made from soybeans, is a well-known health food. In our previous study, we confirmed that natto has the potential to inhibit viral infections by severe acute respiratory syndrome coronavirus 2 and bovine alphaherpesvirus 1 through their putative serine protease(s). In this study, we found that an agent(s) in natto functionally impaired ADV infection in cell culture assays. In addition, ADV treated with natto extract lost viral infectivity in the mice. We conducted an HPLC gel-filtration analysis of natto extract and molecular weight markers and confirmed that Fraction No. 10 had ADV-inactivating ability. Furthermore, the antiviral activity of Fraction No. 10 was inhibited by the serine protease inhibitor 4-(2-Aminoethyl) benzene sulfonyl fluoride hydrochloride (AEBSF). These results also suggest that Fraction No. 10, adjacent to the 12.5 kDa peak of the marker in natto extract, may inactivate ADV by proteolysis. Our findings provide new avenues of research for the prevention of Aujeszky's disease.

Characterization of a gE/gI/TK gene-deleted pseudorabies virus variant expressing the Cap protein of porcine circovirus type 2d.

Porcine circovirus type 2 (PCV2) plays a key role in the etiology of PCV2-associated disease (PCVAD), and its predominant strain is PCV2d which is not completely controlled by most commercially available vaccines against PCV2a strains. Pseudorabies (PR) caused by pseudorabies virus (PRV) variants re-emerged in Bartha-K61 vaccine-immunized swine herds in late 2011, which brought considerable losses to the global pig husbandry. Therefore, it is significantly important to develop a safe and effective vaccine against both PCV2d and PRV infection. In the present study, the PCV2d ORF2 gene was amplified by PCR, and cloned into the BamHI site of PRV transfer plasmid pG vector to obtain the recombinant transfer plasmid pG-PCV2dCap-EGFP. Subsequently, it was transfected into ST cells infected with the three gene deleted PRV variant strain NY-gE-/gI-/TK- to generate a recombinant virus rPRV NY-gE-/gI-/TK-/PCV2dCap+/EGFP+, and then the EGFP gene was knocked out to harvest the rPRV NY-gE-/gI-/TK-/PCV2dCap+ using gene-editing technology termed CRISPR/Cas9 system. The recombinant virus rPRV NY-gE-/gI-/TK-/PCV2dCap+ had similar genetic stability and proliferation characteristics to the parental PRV as indicated by PCR and one-step growth curve test, and the expression of Cap was validated by Western blot. In animal experiment, higher PCV2-specific ELISA antibodies and detectable PCV2-specific neutralizing antibodies could be elicited in mice immunized with rPRV NY-gE-/gI-/TK-/PCV2dCap+ compared to commercial PCV2 inactivated vaccine. Moreover, the recombinant virus rPRV NY-gE-/gI-/TK-/PCV2dCap+ significantly reduced the viral loads in the hearts, livers, spleens, lungs, and kidneys in mice following a virulent PCV2d challenge. Mice immunized with rPRV NY-gE-/gI-/TK-/PCV2dCap+ developed comparable PRV-specific humoral immune responses and provided complete protection against a lethal PRV challenge. Together, the rPRV NY-gE-/gI-/TK-/PCV2dCap+ recombinant strain has strong immunogenicity.

Carbopol dispersed PAA-modified UIO-66 with high colloidal stability as a combination nano-adjuvant boosts immune response and protection against pseudorabies virus in mice and pigs.

Although inactivated vaccines have higher safety than live-attenuated vaccines in the control of pseudorabies virus (PRV), their protection efficacy is limited due to insufficient immunogenicity when used alone. High-performance adjuvants that can potentiate immune responses are highly desirable to improve the protection efficacy of inactivated vaccines. In this work, we have developed U@PAA-Car, a Carbopol dispersed zirconium-based metal-organic framework UIO-66 modified by polyacrylic acid (PAA), as a promising adjuvant for inactivated PRV vaccines. The U@PAA-Car has good biocompatibility, high colloidal stability, and antigen (vaccine) loading capacity. It significantly potentiates humoral and cellular immune responses over either U@PAA, Carbopol, or commercial adjuvants such as Alum and biphasic 201 by inducing a higher specific antibody titer, IgG2a/IgG1 ratio, cell cytokine secretion, and splenocyte proliferation. A protection rate of over 90% was observed in challenge tests in the model animal mice and the host animal pigs, which is much higher than that observed with commercial adjuvants. The high performance of the U@PAA-Car is attributed to antigen sustainable release at the injection site and highly efficient antigen internalization and presentation. In conclusion, this work not only demonstrates a great potential of the developed U@PAA-Car nano-adjuvant for the inactivated PRV vaccine but also gives a preliminary explanation of its action mechanism. STATEMENT OF SIGNIFICANCE: We have developed a Carbopol dispersed PAA-modified zirconium-based metal-organic framework UIO-66 (U@PAA-Car) as a promising combination nano-adjuvant for the inactivated PRV vaccine. The U@PAA-Car induced higher specific antibody titers and IgG2a/IgG1 ratio, increased cell cytokines secretion, and better splenocyte proliferation than U@PAA, Carbopol, and the commercial adjuvants Alum and biphasic 201, indicating that it induces a significant potentiation of humoral and cellular immune response. In addition, much higher protection rates were achieved with the U@PAA-Car-adjuvanted PRV vaccine in mice and pigs challenge than those observed from the commercial adjuvant groups. This work not only demonstrates the great potential of the U@PAA-Car nano-adjuvant in an inactivated PRV vaccine but also gives a preliminary explanation of its action mechanism.

Enhanced productivity and stability of PRV in recombinant ST-Tret1 cells.

Productivity and stability of Pseudorabies virus (PRV) are critical for the manufacture and storage of live attenuated pseudorabies vaccine. Trehalose is commonly used as a cryoprotectant to stabilize organisms during freezing and lyophilization. Trehalose transporter 1 (Tret1), derived from Polypedilum vanderplanki, can deliver trehalose with a reversible transporting direction. In this study, we demonstrated that productivity and stability of PRV proliferated in recombinant ST cells with stable expression of Tret1 were enhanced. As a result, a five-fold increase of intracellular trehalose amount was observed, and the significant increase of progeny viral titer was achieved in recombinant cells with the addition of 20 mM trehalose. Particularly, after storage for 8 weeks at 20 °C, the loss of viral titer was 0.8 and 1.7 lgTCID50/mL lower than the control group with or without the addition of trehalose. Additionally, the freeze-thaw resistance at -20 °C and -70 °C of PRV was significantly enhanced. Furthermore, according to standard international protocols, a series of tests, including karyotype analysis, tumorigenicity, and the ability of proliferation PRV, were conducted. Our results demonstrated that the recombinant ST cell with Tret1 is a promising cell substrate and has a high potential for producing more stable PRV for the live attenuated vaccine.

Genomic Characterization and gE/gI-Deleted Strain Construction of Novel PRV Variants Isolated in Central China.

Pseudorabies virus (PRV) variants have caused substantial economic losses in the swine industry in China since 2011. To surveil the genetic variation in PRV field strains, here, two novel variant strains of PRV were isolated from Shanxi Province in central China and were designated SX1910 and SX1911. To identify the genetic characteristics of the two isolates, their complete genomes were sequenced, and phylogenetic analysis and sequence alignment revealed that field PRV variants have undergone genetic variations; notably, the protein-coding sequences UL5, UL36, US1 and IE180 exhibited extensive variation and contained one or more hypervariable regions. Furthermore, we also found that the glycoproteins gB and gD of the two isolates had some novel amino acid (aa) mutations. Importantly, most of these mutations were located on the surface of the protein molecule, according to protein structure model analysis. We constructed a mutant virus of SX1911 with deletion of the gE and gI genes via CRISPR/Cas9. When tested in mice, SX1911-ΔgE/gI-vaccinated mice were protected within a comparable range to Bartha-K61-vaccinated mice. Additionally, a higher dose of inactivated Bartha-K61 protected the mice from lethal SX1911 challenge, while a lower neutralization titer, higher viral load and more severe microscopic lesions were displayed in Bartha-K61-vaccinated mice. These findings highlight the need for continuous monitoring of PRV and novel vaccine development or vaccination program design for PRV control in China.

Immunogenic response of recombinant pseudorabies virus carrying B646L and B602L genes of African swine fever virus in mice.

African swine fever (ASF) is an acute infectious disease that poses a high lethality risk to domestic pigs and wild boars, causing substantial economic losses to the global pig industry. The prevention and control of ASF remain challenging, necessitating the urgent development of a safe and effective vaccine. This study focused on the essential structural protein p72 of ASFV (encoded by the B646L gene) and its chaperone protein pB602L (encoded by the B602L gene) as the target antigenic proteins. Based on CRISPR/Cas9 gene-editing technology, we constructed a live attenuated recombinant pseudorabies virus vector expressing the p72 and pB602L proteins (designated as rPRVXJ-EGFP/B602L/B646L), and assessed its immunization effect in mice. The recombinant virus rPRVXJ-EGFP/B602L/B646L successfully proliferated and demonstrated stable expression of the p72 and pB602L proteins in BHK-21 cells. Moreover, it exhibited excellent safety when used in mice and induced specific humoral and cellular immune responses targeting p72 and pB602L. In addition, it provided complete protection (100%) against the virulent PRV strain (PRV-XJ). These results indicate that the recombinant virus rPRVXJ-EGFP/B602L/B646L possesses robust immunogenicity and safety in mice. In conclusion, PRV represents a promising viral vector for expressing ASFV gene, and our study serves as an essential reference for the development of viral vector vaccines against ASFV.

Epidemiological Investigation, Risk Factors, Spatial-Temporal Cluster, and Epidemic Trend Analysis of Pseudorabies Virus Seroprevalence in China (2017 to 2021).

Pseudorabies virus (PRV) is a double-stranded linear DNA virus capable of infecting various animals, including humans. We collected blood samples from 14 provinces in China between December 2017 and May 2021 to estimate PRV seroprevalence. The PRV gE antibody was detected using the enzyme-linked immunosorbent assay (ELISA). Logistic regression analysis identified potential risk factors associated with PRV gE serological status at the farm level. Spatial-temporal clusters of high PRV gE seroprevalence were explored using SaTScan 9.6 software. Time-series data of PRV gE seroprevalence were modeled using the autoregressive moving average (ARMA) method. A Monte Carlo sampling simulation based on the established model was performed to analyze epidemic trends of PRV gE seroprevalence using @RISK software (version 7.0). A total of 40,024 samples were collected from 545 pig farms across China. The PRV gE antibody positivity rates were 25.04% (95% confidence interval [CI], 24.61% to 25.46%) at the animal level and 55.96% (95% CI, 51.68% to 60.18%) at the pig farm level. Variables such as farm geographical division, farm topography, African swine fever (ASF) outbreak, and porcine reproductive and respiratory syndrome virus (PRRSV) control in pig farms were identified as risk factors for farm-level PRV infection. Five significant high-PRV gE seroprevalence clusters were detected in China for the first time, with a time range of 1 December 2017 to 31 July 2019. The monthly average change value of PRV gE seroprevalence was -0.826%. The probability of a monthly PRV gE seroprevalence decrease was 0.868, while an increase was 0.132. IMPORTANCE PRV is a critical pathogen threatening the global swine industry. Our research fills knowledge gaps regarding PRV prevalence, infection risk factors, spatial-temporal clustering of high PRV gE seroprevalence, and the epidemic trend of PRV gE seroprevalence in China in recent years. These findings are valuable for the clinical prevention and control of PRV infection and suggest that PRV infection is likely to be successfully controlled in China.

High expression of the classical swine fever virus (CSFV) envelope protein E2 by a single amino acid mutation and its embedded in the pseudorabies virus (PRV) vector for immunization.

Pseudorabies (PR) and classical swine fever (CSF) are economically important infectious diseases in pigs. Most pig farms in China are vaccinated against these two diseases. Gene-deleted pseudorabies virus (PRV) can be used to develop promising and economical multivalent live attenuated viral vector vaccines. It has been reported that recombinant PRV can express a truncated E2 protein (1-338 aa), but it has not been reported that recombinant PRV can express a full-length E2 protein. We constructed nine groups of E2 proteins with different expression forms and found that the E2 protein could be expressed in vitro only when the transmembrane region of E2 was removed and the signal peptide was added. Analysis of the transmembrane region of E2 revealed that the high hydrophobicity of the E2 transmembrane region was the main reason for its inability to express. By mutating an amino acid to reduce the hydrophobicity of the transmembrane region, it was found that the full-length mutant of E2 (E2FL-muta3 or E2FL-muta4) could be expressed. The expressed full-length mutant E2 could also localize to the cell membrane. Mice immunized with a PRV vector vaccine expressing E2FL-muta3 or E2FL-muta4 developed specific cellular immunity to the E2 protein and stimulated higher levels of E2 antibody than mice immunized with a PRV vector expressing truncated E2. After immunizing the rabbits, the lethal challenge by PRV-ZJ2013 and the febrile response elicited by CSFV were simultaneously prevented. These results suggest that rPRV-dTK/gE-E2FL-muta4 is a promising bivalent vaccine against CSFV and PRV infections.

A bivalent ß-carboline derivative inhibits macropinocytosis-dependent entry of pseudorabies virus by targeting the kinase DYRK1A.

Pseudorabies virus (PRV) has become a "new life-threatening zoonosis" since the human-originated PRV strain was first isolated in 2020. To identify novel anti-PRV agents, we screened a total of 107 ß-carboline derivatives and found 20 compounds displaying antiviral activity against PRV. Among them, 14 compounds showed better antiviral activity than acyclovir. We found that compound 45 exhibited the strongest anti-PRV activity with an IC50 value of less than 40 nM. Our in vivo studies showed that treatment with 45 significantly reduced the viral loads and protected mice challenged with PRV. To clarify the mode of action of 45, we conducted a time of addition assay, an adsorption assay, and an entry assay. Our results indicated that 45 neither had a virucidal effect nor affected viral adsorption while significantly inhibiting PRV entry. Using the FITC-dextran uptake assay, we determined that 45 inhibits macropinocytosis. The actin-dependent plasma membrane protrusion, which is important for macropinocytosis, was also suppressed by 45. Furthermore, the kinase DYRK1A (dual-specificity tyrosine phosphorylation-regulated kinase 1A) was predicted to be a potential target for 45. The binding of 45 to DYRK1A was confirmed by drug affinity responsive target stability and cellular thermal shift assay. Further analysis revealed that knockdown of DYRK1A by siRNA suppressed PRV macropinocytosis and the tumor necrosis factor alpha-TNF-induced formation of protrusions. These results suggested that 45 could restrain PRV macropinocytosis by targeting DYRK1A. Together, these findings reveal a unique mechanism through which ß-carboline derivatives restrain PRV infection, pointing to their potential value in the development of anti-PRV agents.

Recombinant Pseudorabies Virus Usage in Vaccine Development against Swine Infectious Disease.

Pseudorabies virus (PRV) is the pathogen of pseudorabies (PR), which belongs to the alpha herpesvirus subfamily with a double stranded DNA genome encoding approximately 70 proteins. PRV has many non-essential regions for replication, has a strong capacity to accommodate foreign genes, and more areas for genetic modification. PRV is an ideal vaccine vector, and multivalent live virus-vectored vaccines can be developed using the gene-deleted PRV. The immune system continues to be stimulated by the gene-deleted PRVs and maintain a long immunity lasting more than 4 months. Here, we provide a brief overview of the biology of PRV, recombinant PRV construction methodology, the technology platform for efficiently constructing recombinant PRV, and the applications of recombinant PRV in vaccine development. This review summarizes the latest information on PRV usage in vaccine development against swine infectious diseases, and it offers novel perspectives for advancing preventive medicine through vaccinology.

Recombinant pseudorabies virus (PRV) expressing stabilized E2 of classical swine fever virus (CSFV) protects against both PRV and CSFV.

Pseudorabies (PR) and classical swine fever (CSF) are economically important infectious diseases of pigs. Most pig farms in China are immunized against these two diseases. Here, we describe a stabilized E2 protein as an immunogen inserted into the PRV genome as a bivalent live virus-vectored vaccine. The E2 protein has 48 variant sites, there are 2-5 candidate amino acids per variant site, and the relative energy contribution of each amino acid to E2 energy was calculated. Combined substitutions of amino acids at the neighbor variant site (neighbor substitution) were performed to obtain the E2 protein sequence with the lowest energy (stabilized E2). Multiple amino acid substitutions at 48 variant sites were performed, and the results were consistent with neighbor substitutions. The stabilized E2 sequence was obtained, and its energy decreased by 22 Rosetta Energy Units (REUs) compared with the original sequence. After the recombinant PRV expressing stabilized E2 of CSFV was constructed, the secretion efficiency of stabilized E2 was increased by 2.97 times, and the thermal stability was increased by 10.5 times. Immunization of mice resulted in a 2-fold increase in antibody production, and a balanced antibody level against subtype 1.1 and subtype 2.1d E2 was achieved. In rabbits immunized, the lethal challenge of PRV-ZJ and the fever response induced by CSFV could be prevented simultaneously. These findings suggest that rPRV-muta/287aaE2 is a promising bivalent vaccine against CSFV and PRV infections.

Establishment of an indirect ELISA method for antibody detection of porcine pseudorabies by recombinant gB, gC, and gD proteins.

Pseudorabies virus (PRV), as a neuroherpes virus, leads to heavy economic losses in the pig industry worldwide. This study was designed to establish recombinant PRV glycoprotein B (gB), C, and D proteins as PRV diagnostic antigens. The gB/C, gC/D, and gB/C/D fusion sequences were synthesized and inserted into pET-28a+ vector to generate the recombinant plasmids. The identified positive recombinant plasmids were transformed into BL21 Escherichia coli. The results of the polymerase chain reaction and enzyme digestion showed that the gB/C, gC/D, and gB/C/D fusion proteins were successfully expressed. An indirect sandwich ELISA was developed with the gB/C, gC/D, and gB/C/D as coating antigens. The results of indirect enzyme-linked immunosorbent assay (ELISA) analysis of 184 PRV-positive porcine sera showed that the positive coincidence rates of three recombinant proteins ELISAs relative to IDEXX kit were 98.25%, 95.32%, and 98.83%, and the negative coincidence rates were 85.71%, 75% and 100%, respectively. The inter and intra batch repeatability tests showed that the coefficient of variations of our kits were all less than 5%. Especially, the gB/C/D-ELISA has the highest specificity and sensitivity among the ELISA methods developed in this study. We established a series expression system of gB/C, gC/D, and gB/C/D antigen epitope genes and Recombinant protein-based indirect ELISA, providing new ideas for PV diagnosis and vaccine development.

Proteomic Comparison of Three Wild-Type Pseudorabies Virus Strains and the Attenuated Bartha Strain Reveals Reduced Incorporation of Several Tegument Proteins in Bartha Virions.

Pseudorabies virus (PRV) is a member of the alphaherpesvirus subfamily and the causative agent of Aujeszky's disease in pigs. Driven by the large economic losses associated with PRV infection, several vaccines and vaccine programs have been developed. To this day, the attenuated Bartha strain, generated by serial passaging, represents the golden standard for PRV vaccination. However, a proteomic comparison of the Bartha virion to wild-type (WT) PRV virions is lacking. Here, we present a comprehensive mass spectrometry-based proteome comparison of the attenuated Bartha strain and three commonly used WT PRV strains: Becker, Kaplan, and NIA3. We report the detection of 40 structural and 14 presumed nonstructural proteins through a combination of data-dependent and data-independent acquisition. Interstrain comparisons revealed that packaging of the capsid and most envelope proteins is largely comparable in-between all four strains, except for the envelope protein pUL56, which is less abundant in Bartha virions. However, distinct differences were noted for several tegument proteins. Most strikingly, we noted a severely reduced incorporation of the tegument proteins IE180, VP11/12, pUS3, VP22, pUL41, pUS1, and pUL40 in Bartha virions. Moreover, and likely as a consequence, we also observed that Bartha virions are on average smaller and more icosahedral compared to WT virions. Finally, we detected at least 28 host proteins that were previously described in PRV virions and noticed considerable strain-specific differences with regard to host proteins, arguing that the potential role of packaged host proteins in PRV replication and spread should be further explored. IMPORTANCE The pseudorabies virus (PRV) vaccine strain Bartha-an attenuated strain created by serial passaging-represents an exceptional success story in alphaherpesvirus vaccination. Here, we used mass spectrometry to analyze the Bartha virion composition in comparison to three established WT PRV strains. Many viral tegument proteins that are considered nonessential for viral morphogenesis were drastically less abundant in Bartha virions compared to WT virions. Interestingly, many of the proteins that are less incorporated in Bartha participate in immune evasion strategies of alphaherpesviruses. In addition, we observed a reduced size and more icosahedral morphology of the Bartha virions compared to WT PRV. Given that the Bartha vaccine strain elicits potent immune responses, our findings here suggest that differences in protein packaging may contribute to its immunogenicity. Further exploration of these observations could aid the development of efficacious vaccines against other alphaherpesvirus vaccines such as HSV-1/2 or EHV-1.

Pseudorabies Virus Associations in Wild Animals: Review of Potential Reservoirs for Cross-Host Transmission.

Pseudorabies virus (PRV) has received widespread attention for its potential health effects on humans, wildlife, domestic animals, and livestock. In this review, we focus on PRV dynamics in wildlife, given the importance of wild-origin PRV transmission to domestic and farm animals. Wild boars, pigs, and raccoons can serve as reservoirs of PRV, with viral transmission to domestic livestock occurring via several routes, such as wild herd exposure, contaminated meat consumption, and insect vector transmission. Many endangered feline and canine species can be infected with PRV, with acute disease and death within 48 h. The first confirmed human case of PRV infection in mainland China was reported in 2017. Thus, PRV exhibits potentially dangerous cross-host transmission, which is likely associated with inappropriate vaccination, poor awareness, and insufficient biosecurity. Currently, no vaccine provides full protection against PRV in all animals. Here, we summarize the epidemiology and pathogenesis of PRV infection in wild, domestic, and farmed animals, which may facilitate the design of novel therapeutics and strategies for controlling PRV infection and improving wildlife protection in China.

A novel Pseudorabies virus vaccine developed using HDR-CRISPR/Cas9 induces strong humoral and cellular immune response in mice.

Outbreaks of Pseudorabies (PR) by numerous highly virulent and antigenic variant Pseudorabies virus (PRV) strains have been causing severe economic losses to the pig industry in China since 2011. However, current commercial vaccines are often unable to induce thorough protective immunity. In this study, a TK/gI/gE deleted recombinant PRV expressing GM-CSF was developed by using the HDR-CRISPR/Cas9 system. Here, a four-sgRNA along with the Cas9D10A targeting system was utilized for TK/gI/gE gene deletion and GM-CSF insertion. Our study showed that the four-sgRNA targeting system appeared to have higher knock-in efficiency for PRVs editing. The replication of the recombinant PRVs were slightly lower than that of the parental strain, but they appeared to have similar properties in terms of growth curves and plaque morphology. The mice vaccinated with the recombinant PRV expressing GM-CSF via intramuscular injection showed no obvious clinical symptoms, milder pathological lesions, and were completely protected against wild-type PRV challenge. When compared to the triple gene-deleted PRV, the gB antibodies and neutralizing antibody titers were improved and the immunized mice appeared to have lower viral load and higher mRNA levels of IL-2, IL-4, IL-6, and IFN-γ in spleens. Our study offers a novel approach for recombinant PRV construction, and the triple gene-deleted PRV expressing GM-CSF could serve as a promising vaccine candidate for PR control.

Comprehensive evaluation of the safety and immunogenicity of a gene-deleted variant pseudorabies virus attenuated vaccine.

Pseudorabies virus (PRV) variant infections have caused a substantial economic impact on swine production in the absence of new powerful candidate vaccines. In this study, we developed and evaluated a gene-deleted variant pseudorabies virus (PRV)-attenuated vaccine, PRV GX-ΔTK/IES, in which the genes TK, gI, gE, US9 and US2 were deleted. During a study of innocuousness, all mice inoculated with PRV GX-ΔTK/IES survived, neither clinical signs nor pathological changes were observed, and viral genomes could not be detected in the blood and tissues. All piglets inoculated with high titres of PRV GX-ΔTK/IES remained clinically healthy, and neither fever nor clinical signs were observed. Viral detection results were negative in nasal swab samples, blood and tissue samples. Moreover, none of the cohabitated piglets seroconverted during a trial on horizontal transmission. The immunogenicity was assessed through a vaccination and challenge experiment in piglets. Piglets vaccinated with PRV GX-ΔTK/IES and the commercial vaccine were completely protected from subsequent PRV infection, and the level of immunity and protection induced by PRV GX-ΔTK/IES was better than that provided by the live commercial vaccine. Thus, PRV GX-ΔTK/IES is completely safe for both nontarget and target animals and can be regarded as a novel live gene-deleted PRV vaccine candidate.