A xanthan gum and carbomer-codispersed divalent manganese ion-loaded tannic acid nanoparticle adjuvanted inactivated pseudorabies virus vaccine induces balanced humoral and cellular immune responses.

Adjuvants including aluminum adjuvant (Alum) and oil-water emulsion have been widely used in inactivated pseudorabies virus (PRV) vaccines to improve their performance, however, they are not sufficient to protect from PRV infection because of the weak immune response and poor Th1-type immune response. Divalent manganese ion (Mn2+) has been reported to increase the cellular immune response significantly. In this work, a xanthan gum and carbomer-dispersed Mn2+-loaded tannic acid-polyethylene glycol (TPMnXC) nanoparticle colloid is developed and used as an adjuvant to improve the performance of the inactivated PRV vaccine. The good in vitro and in vivo biocompatibility of the developed TPMnXC colloid has been confirmed by the cell viability assay, erythrocyte hemolysis, blood routine analysis, and histological analysis of mouse organs and injection site. The TPMnXC-adjuvanted inactivated PRV vaccine (TPMnXC@PRV) significantly promotes higher and more balanced immune responses indicating with an increased specific total IgG antibody and IgG2a/IgG1 ratio, efficient splenocytes proliferation, and elevated Th1- and Th2-type cytokine secretion than those of control groups. Wild PRV challenge experiment is performed using mice as a model animal, achieving a protection rate of up to 86.67 %, which is much higher than those observed from the commercial Alum. This work not only demonstrates the high potentiality of TPMnXC in practical applications but also provides a new way to develop the Mn2+-loaded nanoadjuvant for veterinary vaccines.

Additional Insertion of gC Gene Triggers Better Immune Efficacy of TK/gI/gE-Deleted Pseudorabies Virus in Mice.

In recent years, pseudorabies virus (PRV) variants have resulted in an epidemic in swine herds and huge economic losses in China. Therefore, it is essential to develop an efficacious vaccine against the spread of PRV variants. Here, the triple-gene-deletion virus and the triple-gene-deletion plus gC virus were constructed by homologous recombination (HR). And then, their growth capacity, proliferation ability, and immune efficacy were evaluated. The results showed that the growth kinetics of the recombinant viruses were similar to those of the parental strain PRV-AH. Compared with the triple-gene-deletion virus group, the more dominant level of neutralizing antibody (NA) can be induced in the triple-gene-deletion plus gC virus group with the same 106.0 TCID50 dose after 4 and 6 weeks post-initial immunization (PII) (p < 0.0001). In addition, the antibody titers in mice immunized with the triple-gene-deletion plus gC virus were significantly higher than those immunized with triple-gene deletion virus with the same 105.0 TCID50 dose after 6 weeks PII (p < 0.001). More importantly, in the triple-gene-deletion plus gC virus group with 105.0 TCID50, the level of NA was close to that in the triple-gene deletion virus group with 106.0 TCID50 at 6 weeks PII. Meanwhile, the cytokines IL-4 and IFN-γ in sera were tested by enzyme-linked immunosorbent assay (ELISA) in each group. The highest level of IL-4 or IFN-γ was also elicited in the triple-gene deletion plus gC virus group at a dose of 106.0 TCID50. After challenge with PRV-AH, the survival rates of the triple-gene deletion plus gC virus immunized groups were higher than those of other groups. In immunized groups with 105.0 TCID50, the survival rate shows a significant difference between the triple-gene deletion plus gC virus group (75%, 6/8) and the triple-gene deletion virus group (12.5%, 1/8). In general, the immune efficacy of the PRV TK/gI/gE-deleted virus can be increased with additional gC insertion in mice, which has potential for developing an attenuated vaccine candidate for PRV control.

Pseudorabies virus UL38 attenuates the cGAS-STING signaling pathway by recruiting Tollip to promote STING for autophagy degradation.

Natural immunity is the first defense line of the host immune system, which plays a significant role in combating foreign pathogenic microorganisms. The IFN-ß (interferon-beta) signaling pathway, being a typical example of innate immunity, plays a vital function. This study aimed to elucidate the function of pseudorabies virus (PRV) UL38 protein (unique long region 38) in suppressing the activation of the IFN-ß signaling pathway. The findings from our study indicate that the PRV UL38 protein effectively hampers the activation of IFN-ß by poly (dA: dT) (poly(deoxyadenylic-deoxythymidylic)) and 2'3'-cGAMP (2'-3'-cyclic GMP-AMP). Furthermore, UL38 exhibits spatial co-localization with STING (stimulator of interferon genes) and effectively hinders STING dimerization. Subsequently, STING was downgraded to suppress the production of IFN-ß and ISGs (interferon stimulated genes). Immunoprecipitation analysis revealed that the interaction between UL38 and STING, which subsequently initiated the degradation of STING via selective autophagy mediated by TOLLIP (toll interacting protein). To summarize, this research elucidates the function of UL38 in counteracting the cGAS (cGAMP synthase)-STING-induced IFN-ß pathway. The PRV UL38 protein may attenuate the activation of IFN-ß as a means of regulating the virus's persistence in the host.

Pseudorabies virus usurps non-muscle myosin heavy chain IIA to dampen viral DNA recognition by cGAS for antagonism of host antiviral innate immunity.

Alphaherpesvirus pseudorabies virus (PRV) causes severe economic losses to the global pig industry and has garnered increasing attention due to its broad host range including humans. PRV has developed a variety of strategies to antagonize host antiviral innate immunity. However, the underlying mechanisms have not been fully elucidated. In our previous work, we demonstrated that non-muscle myosin heavy chain IIA (NMHC-IIA), a multifunctional cytoskeleton protein, attenuates innate immune responses triggered by RNA viruses. In the current study, we reported a previously unrecognized role of NMHC-IIA in counteracting PRV-induced cyclic GMP-AMP synthase (cGAS)-dependent type I interferon (IFN-I) production. Mechanistically, PRV infection led to an elevation of NMHC-IIA, strengthening the interaction between poly (ADP-ribose) polymerase 1 (PARP1) and cGAS. This interaction impeded cGAS recognition of PRV DNA and hindered downstream signaling activation. Conversely, inhibition of NMHC-IIA by Blebbistatin triggered innate immune responses and enhanced resistance to PRV proliferation both in vitro and in vivo. Taken together, our findings unveil that PRV utilizes NMHC-IIA to antagonize host antiviral immune responses via impairing DNA sensing by cGAS. This in-depth understanding of PRV immunosuppression not only provides insights for potential PRV treatment strategies but also highlights NMHC-IIA as a versatile immunosuppressive regulator usurped by both DNA and RNA viruses. Consequently, NMHC-IIA holds promise as a target for the development of broad-spectrum antiviral drugs.IMPORTANCECyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) axis plays a vital role in counteracting alphaherpesvirus infections. Alphaherpesviruses exploit various strategies for antagonizing cGAS-STING-mediated antiviral immune responses. However, limited examples of pseudorabies virus (PRV)-caused immunosuppression have been documented. Our findings reveal a novel role of non-muscle myosin heavy chain IIA (NMHC-IIA) in suppressing PRV-triggered innate immune responses to facilitate viral propagation both in vitro and in vivo. In detail, NMHC-IIA recruits poly (ADP-ribose) polymerase 1 (PARP1) to augment its interaction with cGAS, which impairs cGAS recognition of PRV DNA. Building on our previous demonstration of NMHC-IIA's immunosuppressive role during RNA virus infections, these findings indicate that NMHC-IIA acts as a broad-spectrum suppressor of host antiviral innate immunity in response to both DNA and RNA viruses. Therefore, NMHC-IIA will be a promising target for the development of comprehensive antiviral strategies.

Alginate di-aldehyde-modified metal-organic framework nanocarriers as delivery platform and adjuvant in inactivated pseudorabies vaccination.

Pseudorabies virus (PRV) is a highly contagious viral disease, which leads to severe financial losses in the breeding industry worldwide. Presently, PRV is mainly controlled using live attenuated and inactivated vaccines. However, these vaccines have an innate tendency to lose their structural conformation upon exposure to environmental and chemical stressors and cannot provide full protection against the emerging prevalent PRV variants. In this work, first, we synthesized aminated ZIF-7/8 nanoparticles (NPs), and then chemical bond-coated alginate dialdehyde (ADA, a type of dioxide alginate saccharide) on their surface via Schiff base reaction to obtain ZIF-7/8-ADA NPs. The as-fabricated ZIF-7/8-ADA NPs exhibited high stability, monodispersity and a high loading ratio of antigen. Furthermore, the ZIF-7/8-ADA NPs showed good biocompatibility in vitro and in vivo. Using ZIF-7/8-ADA NPs as an adjuvant and inactivated PRV as a model antigen, we constructed a PR vaccine through a simple mixture. The immunity studies indicated that ZIF-7/8-ADA induced an enhancement in the Th1/Th2 immune response, which was superior to that of the commercial ISA201, alum adjuvant and ZIF-7/8. Due to the pH-sensitive release of the antigen in lysosomes, the as-prepared PR vaccine subsequently accelerated the antigen presentation and improved the immune responses in vitro and in vivo. The results of PRV challenge using mice as the model demonstrated that ZIF-7/8-ADA achieved the same preventive effect as the commercial ISA201 and was much better than the alum adjuvant, and thus can serve as a promising delivery system and adjuvant to enhance humoral and cellular responses against PRV infection.

Swine NONO is an essential factor to inhibit pseudorabies virus infection.

Pseudorabies virus (PRV) is a member of the genus Varicellovirus, family Herpesviridae and causes Aujeszky's disease to lead to huge economic losses in the global pig industry. The Non-POU domain-containing octamer-binding protein (NONO), as a Drosophila behavior/human splicing (DBHS) protein, plays a key role in multiple biological functions in cells, including transcriptional regulation, RNA splicing, DNA repair and so on. However, whether swine NONO (sNONO) inhibits PRV infection is less understood. In this study, we showed that sNONO was a crucial host factor for antagonizing PRV infection and positive regulated transcription levels of ISGs. After PRV infection, sNONO enhanced the activation of IFN-ß promoter and IFN-ß expression. Furthermore, knockout of sNONO in PAM-KNU cells impaired activation of type I IFN pathway and increased PRV propagation. Taken together, we have first elucidated the anti-PRV function and mechanism of sNONO, which may provide a new strategy for preventing DNA virus infection.

Pseudorabies Virus EP0 Antagonizes the Type I Interferon Response via Inhibiting IRF9 Transcription.

The alphaherpesvirus pseudorabies virus (PRV) is the etiologic agent of swine Aujeszky's disease, which can cause huge economic losses to the pig industry. PRV can overcome a type I interferon (IFN)-induced antiviral state in host cells through its encoded EP0 protein. However, the exact role of EP0 in this process is poorly defined. Here, we report that EP0 transcriptionally represses IFN regulatory factor 9 (IRF9), a critical component in the IFN signaling pathway, thereby reducing the cellular levels of IRF9 and inhibiting IFN-induced gene transcription. This activity of EP0 is mediated by its C-terminal region independently of the RING domain. Moreover, compared with EP0 wild-type PRV, EP0-deficient PRV loses the ability to efficiently decrease cellular IRF9, while reintroducing the C-terminal region of EP0 back into the EP0-deficient virus restores the activity. Together, these results suggest that EP0 can transcriptionally modulate IRF9-mediated antiviral pathways through its C-terminal region, contributing to PRV innate immune evasion. IMPORTANCE Alphaherpesviruses can establish lifelong infections and cause many diseases in humans and animals. Pseudorabies virus (PRV) is a swine alphaherpesvirus that threatens pig production. Using PRV as a model, we found that alphaherpesvirus can utilize its encoded early protein EP0 to inhibit the IFN-induced upregulation of antiviral proteins by reducing the basal expression levels of IRF9 through repressing its transcription. Our findings reveal a mechanism employed by alphaherpesvirus to evade the immune response and indicate that EP0 is an important viral protein in pathogenesis and a potential target for antiviral drug development.

Pseudorabies Virus Infection Results in a Broad Inhibition of Host Gene Transcription.

Pseudorabies virus (PRV) is a porcine alphaherpesvirus that belongs to the Herpesviridae family. We showed earlier that infection of porcine epithelial cells with PRV triggers activation of the nuclear factor κB (NF-κB) pathway, a pivotal signaling axis in the early immune response. However, PRV-induced NF-κB activation does not lead to NF-κB-dependent gene expression. Here, using electrophoretic mobility shift assays (EMSAs), we show that PRV does not disrupt the ability of NF-κB to interact with its κB target sites. Assessing basal cellular transcriptional activity in PRV-infected cells by quantitation of prespliced transcripts of constitutively expressed genes uncovered a broad suppression of cellular transcription by PRV, which also affects the inducible expression of NF-κB target genes. Host cell transcription inhibition was rescued when viral genome replication was blocked using phosphonoacetic acid (PAA). Remarkably, we found that host gene expression shutoff in PRV-infected cells correlated with a substantial retention of the NF-κB subunit p65, the TATA box binding protein, and RNA polymerase II-essential factors required for (NF-κB-dependent) gene transcription-in expanding PRV replication centers in the nucleus and thereby away from the host chromatin. This study reveals a potent mechanism used by the alphaherpesvirus PRV to steer the protein production capacity of infected cells to viral proteins by preventing expression of host genes, including inducible genes involved in mounting antiviral responses. IMPORTANCE Herpesviruses are highly successful pathogens that cause lifelong persistent infections of their host. Modulation of the intracellular environment of infected cells is imperative for the success of virus infections. We reported earlier that a DNA damage response in epithelial cells infected with the alphaherpesvirus pseudorabies virus (PRV) results in activation of the hallmark proinflammatory NF-κB signaling axis but, remarkably, that this activation does not lead to NF-κB-induced (proinflammatory) gene expression. Here, we report that PRV-mediated inhibition of host gene expression stretches beyond NF-κB-dependent gene expression and in fact reflects a broad inhibition of host gene transcription, which correlates with a substantial recruitment of essential host transcription factors in viral replication compartments in the nucleus, away from the host chromatin. These data uncover a potent alphaherpesvirus mechanism to interfere with production of host proteins, including proteins involved in antiviral responses.

Pseudorabies virus tegument protein UL13 recruits RNF5 to inhibit STING-mediated antiviral immunity.

Pseudorabies virus (PRV) has evolved various immune evasion mechanisms that target host antiviral immune responses. However, it is unclear whether and how PRV encoded proteins modulate the cGAS-STING axis for immune evasion. Here, we show that PRV tegument protein UL13 inhibits STING-mediated antiviral signaling via regulation of STING stability. Mechanistically, UL13 interacts with the CDN domain of STING and recruits the E3 ligase RING-finger protein 5 (RNF5) to promote K27-/K29-linked ubiquitination and degradation of STING. Consequently, deficiency of RNF5 enhances host antiviral immune responses triggered by PRV infection. In addition, mutant PRV lacking UL13 impaired in antagonism of STING-mediated production of type I IFNs and shows attenuated pathogenicity in mice. Our findings suggest that PRV UL13 functions as an antagonist of IFN signaling via a novel mechanism by targeting STING to persistently evade host antiviral responses.

The construction and immunogenicity analyses of a recombinant pseudorabies virus with porcine circovirus type 3 capsid protein co-expression.

Porcine circovirus-associated diseases (PCVADs) and pseudorabies (PR) are highly contagious and economically significant diseases of swine in China. Porcine circovirus type 3 (PCV3) is an emerging swine pathogen of PCVAD. Currently, no PCV3 vaccine is commercially available, and the epidemic caused by it is still spreading worldwide. In this study, we used the PRV variant strain HNX as the parental virus to construct recombinant PRV with TK/gE gene deletion and capsid (Cap) protein co-expression, named HNX-ΔTK/ΔgE-ORF2. The results revealed that PCV3 Cap protein can be detected in HNX-ΔTK/ΔgE-ORF2-infected PK-15 cells by both western blotting and immunofluorescence assays. Vaccination with HNX-ΔTK/ΔgE-ORF2 did not cause pruritus, ruffled fur, systemic infection, or inflammation (without high expression of interleukin-6 (IL-6) and granulocyte colony-stimulating factor (G-CSF) in plasma). Furthermore, HNX-ΔTK/ΔgE-ORF2 immunization induced an anti-Cap specific antibody, activated a PRV-specific cellular immune response, and provided 100 % protection to mice against the challenge of the virulent HNX strain. Thus, HNX-ΔTK/ΔgE-ORF2 appears to be a promising vaccine candidate against PRV and PCV3 for the control of the PRV variant and PCV3.

Alphaherpesvirus-induced activation of plasmacytoid dendritic cells depends on the viral glycoprotein gD and is inhibited by non-infectious light particles.

Plasmacytoid dendritic cells (pDC) are important innate immune cells during the onset of viral infections as they are specialized in the production of massive amounts of antiviral type I interferon (IFN). Alphaherpesviruses such as herpes simplex virus (HSV) or pseudorabies virus (PRV) are double stranded DNA viruses and potent stimulators of pDC. Detailed information on how PRV activates porcine pDC is lacking. Using PRV and porcine primary pDC, we report here that PRV virions, so-called heavy (H-)particles, trigger IFNα production by pDC, whereas light (L-) particles that lack viral DNA and capsid do not. Activation of pDC requires endosomal acidification and, importantly, depends on the PRV gD envelope glycoprotein and O-glycosylations. Intriguingly, both for PRV and HSV-1, we found that L-particles suppress H-particle-mediated activation of pDC, a process which again depends on viral gD. This is the first report describing that gD plays a critical role in alphaherpesvirus-induced pDC activation and that L-particles directly interfere with alphaherpesvirus-induced IFNα production by pDC.

A monoclonal antibody neutralizes pesudorabies virus by blocking gD binding to the receptor nectin-1.

Pseudorabies virus (PRV) was isolated from some human cases recently and the infected patients manifested respiratory dysfunction and acute neurological symptoms. However, no effective drug or vaccine, preventing the progression of PRV infection, is available. Nectin-1 was the only reported receptor for PRV cell entry both swine and human origin, representing an excellent target to block PRV infection, and especially its transmission from pigs to humans. A PRV-gD specific mAbs (10B6) was isolated from hybridomas and its neutralizing activities in vitro and in vivo were determined. 10B6 exhibited effective neutralizing activities in vitro with IC50 = 2.514 µg/ml and 4.297 µg/ml in the presence and absence of complement. And in vivo, 10B6 provided 100% protection against PRV lethal challenge with a dose of 15 mg/kg. Further, 10B6 could bind to a conserved epitope, 316QPAEPFP322, locating in gD pro-fusion domain, and finally blocks the binding of PRV-gD to nectin-1. Moreover, 10B6 showed an effective inhibition on PRV cell-attachment in a cell type-independent manner and could also block the virus spreading among cells. 10B6 exhibited effectively neutralizing activities to Chinese PRV variant strain in vitro and in vivo by blocking gD binding to nectin-1, implied both prophylactic and therapeutic interventions against PRV infections.

Pseudorabies Virus DNA Polymerase Processivity Factor UL42 Inhibits Type I IFN Response by Preventing ISGF3-ISRE Interaction.

Alphaherpesviruses are large dsDNA viruses with an ability to establish persistent infection in hosts, which rely partly on their ability to evade host innate immune responses, notably the type I IFN response. However, the relevant molecular mechanisms are not well understood. In this study, we report the UL42 proteins of alphaherpesvirus pseudorabies virus (PRV) and HSV type 1 (HSV1) as a potent antagonist of the IFN-I-induced JAK-STAT signaling pathway. We found that ectopic expression of UL42 in porcine macrophage CRL and human HeLa cells significantly suppresses IFN-α-mediated activation of the IFN-stimulated response element (ISRE), leading to a decreased transcription and expression of IFN-stimulated genes (ISGs). Mechanistically, UL42 directly interacts with ISRE and interferes with ISG factor 3 (ISGF3) from binding to ISRE for efficient gene transcription, and four conserved DNA-binding sites of UL42 are required for this interaction. The substitution of these DNA-binding sites with alanines results in reduced ISRE-binding ability of UL42 and impairs for PRV to evade the IFN response. Knockdown of UL42 in PRV remarkably attenuates the antagonism of virus to IFN in porcine kidney PK15 cells. Our results indicate that the UL42 protein of alphaherpesviruses possesses the ability to suppress IFN-I signaling by preventing the association of ISGF3 and ISRE, thereby contributing to immune evasion. This finding reveals UL42 as a potential antiviral target.

Inhibition of histone deacetylase 1 suppresses pseudorabies virus infection through cGAS-STING antiviral innate immunity.

Pseudorabies virus (PRV) is an enveloped double-stranded DNA virus that is the etiological agent of Aujeszky's disease in pigs. Vaccination is currently available to prevent PRV infection, but there is still an urgent need for new strategies to control this infectious disease. Histone deacetylases (HDACs) are epigenetic regulators that regulate the histone tail, chromatin conformation, protein-DNA interaction and even transcription. Viral transcription and protein activities are intimately linked to regulation by histone acetyltransferases and HDACs that remodel chromatin and regulate gene expression. We reported here that genetic and pharmacological inhibition of HDAC1 significantly influenced PRV replication. Moreover, we demonstrated that inhibition of HDAC1 induced a DNA damage response and antiviral innate immunity. Mechanistically, the HDAC1 inhibition-induced DNA damage response resulted in the release of double-strand DNA into the cytosol to activate cyclic GMP-AMP synthase and the downstream STING/TBK1/IRF3 innate immune signaling pathway. Our results demonstrate that an HDAC1 inhibitor may be used as a new strategy to prevent Aujeszky's disease in pigs.

Inhibition of PARP1 Dampens Pseudorabies Virus Infection through DNA Damage-Induced Antiviral Innate Immunity.

Pseudorabies virus (PRV) is the causative pathogen of Aujeszky's disease in pigs. Although vaccination is currently applied to prevent the morbidity of PRV infection, new applications are urgently needed to control this infectious disease. Poly(ADP-ribose) polymerase 1 (PARP1) functions in DNA damage repair. We report here that pharmacological and genetic inhibition of PARP1 significantly influenced PRV replication. Moreover, we demonstrate that inhibition of PARP1 induced DNA damage response and antiviral innate immunity. Mechanistically, PARP1 inhibition-induced DNA damage response resulted in the release of double-stranded DNA (dsDNA) into the cytosol, where dsDNA interacted with cyclic GMP-AMP (cGAMP) synthase (cGAS). cGAS subsequently catalyzed cGAMP production to activate the STING/TBK1/IRF3 innate immune signaling pathway. Furthermore, challenge of mice with PARP1 inhibitor stimulated antiviral innate immunity and protected mice from PRV infection in vivo. Our results demonstrate that PARP1 inhibitors may be used as a new strategy to prevent Aujeszky's disease in pigs. IMPORTANCE Aujeszky's disease is a notifiable infectious disease of pigs and causes economic losses worldwide in the pig industry. The causative pathogen is PRV, which is a member of the subfamily Alphaherpesvirinae of the family Herpesviridae. PRV has a wide range of hosts, such as ruminants, carnivores, and rodents. More seriously, recent reports suggest that PRV can cause human endophthalmitis and encephalitis, which indicates that PRV may be a potential zoonotic pathogen. Although vaccination is currently the major strategy used to control the disease, new applications are also urgently needed for the pig industry and public health. We report here that inhibition of PARP1 induces DNA damage-induced antiviral innate immunity through the cGAS-STING signaling pathway. Therefore, PARP1 is a therapeutic target for PRV infection as well as alphaherpesvirus infection.

Pathogenicity and immunogenicity of a gI/gE/TK/UL13-gene-deleted variant pseudorabies virus strain in swine.

Pseudorabies is a highly infectious disease with severe clinical symptoms, causing acute death in infected pigs and leading to substantial economic losses among swine producers. In this study, a vaccine candidate strain in which the protein kinase UL13 gene was deleted was constructed with the CRISPR/Cas9 system based on the recombinant pseudorabies virus (PRV) ZJ01-ΔgI/gE/TK. Pigs immunized with ZJ01-ΔgI/gE/TK or ZJ01-ΔgI/gE/TK/UL13 produced high levels of anti-gB antibodies and virus-neutralizing antibodies. ZJ01-ΔgI/gE/TK/UL13 provided greater protective efficacy against challenge with PRV variant strain ZJ01 than did Bartha-K61 or ZJ01-ΔgI/gE/TK. The pigs vaccinated with ZJ01-ΔgI/gE/TK/UL13 excreted significantly less virus than those vaccinated with Bartha-K61 or ZJ01-ΔgI/gE/TK. The viral loads in the lungs of pigs treated with ZJ01-ΔgI/gE/TK/UL13 were lower than those in pigs treated with ZJ01-ΔgI/gE/TK after challenge with PRV variant strain ZJ01. These data indicated that ZJ01-ΔgI/gE/TK/UL13 had greater protective efficacy and safety than the commercial ZJ01-ΔgI/gE/TK and Bartha-K61 vaccines, and could be developed as a promising vaccine candidate for the prevention and control of this disease.

Recombinant pseudorabies virus with gI/gE deletion generated by overlapping polymerase chain reaction and homologous recombination technology induces protection against the PRV variant PRV-GD2013.

BACKGROUND: Since 2011, numerous highly virulent and antigenic variant viral strains have been reported in pigs that were vaccinated against the swine pseudorabies virus. These infections have led to substantial economic losses in the Chinese swine industry. RESULTS: This study, constructed a novel recombinant vaccine strain with gI/gE deletion (PRV-GD2013-ΔgI/gE) by overlapping PCR and homologous recombination technology. The growth curves and plaque morphology of the recombinant virus were similar to those of the parental strain. However, PRV-GD2013-ΔgI/gE infection was significantly attenuated in mice compared with that of PRV-GD2013. Two-week-old piglets had normal rectal temperatures and displayed no clinical symptoms after being inoculated with 105 TCID50 PRV-GD2013-ΔgI/gE, indicating that the recombinant virus was avirulent in piglets. Piglets were immunized with different doses of PRV-GD2013-ΔgI/gE, or a single dose of Bartha-K61 or DMEM, and infected with PRV-GD2013 at 14 days post-vaccination. Piglets given high doses of PRV-GD2013-ΔgI/gE showed no obvious clinical symptoms, and their antibody levels were higher than those of other groups, indicating that the piglets were completely protected from PRV-GD2013. CONCLUSIONS: The PRV-GD2013-ΔgI/gE vaccine strain could be effective for immunizing Chinese swine herds against the pseudorabies virus (PRV) strain.

Seroprevalence investigation and genetic analysis of pseudorabies virus within pig populations in Henan province of China during 2018-2019.

In late 2011, the outbreak of pseudorabies (PR) occurred in Bartha-K61-vaccinated pig farms and spread rapidly to many provinces of China, causing substantial economic losses to the swine industry. A total of 4708 pig serum samples from Henan province during 2018-2019 were collected to screen for the presence of pseudorabies virus (PRV) gE-specific antibodies, and phylogenetic analysis based on the gE gene of PRV was performed. Of the 4708 serum samples tested, 30.14% (1419/4708) were seropositive for PRV antibodies, based on PRV gE-coated enzyme-linked immunosorbent assay (ELISA), with slaughterhouses having the highest seroprevalence. The seropositive rates of PRV also varied with the region and the season. Phylogenetic analysis showed that three PRV isolates from this study were clustered in an independent branch together with the Chinese variant PRV strains (after 2012), and had a closer genetic relationship with the Chinese variant PRV strains, but differed genetically from the 4 early Chinese PRV strains and 4 European-American strains. This study suggests that three PRV isolates may belong to PRV variants, and the development of a novel vaccine against PRV variants is particularly urgent.

Establishment of inflammatory model induced by Pseudorabies virus infection in mice.

BACKGROUND: Pseudorabies virus (PRV) infection leads to high mortality in swine. Despite extensive efforts, effective treatments against PRV infection are limited. Furthermore, the inflammatory response induced by PRV strain GXLB-2013 is unclear. OBJECTIVES: Our study aimed to investigate the inflammatory response induced by PRV strain GXLB-2013, establish an inflammation model to elucidate the pathogenesis of PRV infection further, and develop effective drugs against PRV infection. METHODS: Kunming mice were infected intramuscularly with medium, LPS, and different doses of PRV-GXLB-2013. Viral spread and histopathological damage to brain, spleen, and lung were determined at 7 days post-infection (dpi). Immune organ indices, levels of reactive oxygen species (ROS), nitric oxide (NO), and inflammatory cytokines, as well as levels of activity of COX-2 and iNOS were determined at 4, 7, and 14 dpi. RESULTS: At 105-106 TCID50 PRV produced obviously neurological symptoms and 100% mortality in mice. Viral antigens were detectable in kidney, heart, lung, liver, spleen, and brain. In addition, inflammatory injuries were apparent in brain, spleen, and lung of PRV-infected mice. Moreover, PRV induced increases in immune organ indices, ROS and NO levels, activity of COX-2 and iNOS, and the content of key pro-inflammatory cytokines, including interleukin (IL)-1ß, IL-6, tumor necrosis factor-α, interferon-γ and MCP-1. Among the tested doses, 10² TCID50 of PRV produced a significant inflammatory mediator increase. CONCLUSIONS: An inflammatory model induced by PRV infection was established in mice, and 10² TCID50 PRV was considered as the best concentration for the establishment of the model.

Diagnosis and gI antibody dynamics of pseudorabies virus in an intensive pig farm in Hei Longjiang Province.

BACKGROUND: Pseudorabies (PR), caused by the pseudorabies virus (PRV), is an endemic disease in some regions of China. Although there are many reports on epidemiological investigations into pseudorabies, information on PRV gI antibody dynamics in one pig farm is sparse. OBJECTIVES: To diagnose PR and analyze the course of PR eradication in one pig farm. METHODS: Ten brains and 1,513 serum samples from different groups of pigs in a pig farm were collected to detect PRV gE gene and PRV gI antibody presence using real-time polymerase chain reaction and enzyme-linked immunosorbent assay, respectively. RESULTS: The July 2015 results indicated that almost all brain samples were PRV gE gene positive, but PRV gI antibody results in the serum samples of the same piglets were all negative. In the boar herd, from October 2015 to July 2018 three positive individuals were culled in October 2015, and the negative status of the remaining boars was maintained in the following tests. In the sow herd, the PRV gI antibody positive rate was always more than 70% from October 2015 to October 2017; however, it decreased to 27% in January 2018 but increased to 40% and 52% in April and July 2018, respectively. The PRV gI antibody positive rate in 100-day pigs markedly decreased in October 2016 and was maintained at less than 30% in the following tests. For 150-day pigs, the PRV gI antibody positive rate decreased notably to 10% in April 2017 and maintained a negative status from July 2017. The positive trend of PRV gI antibody with an increase in pig age remarkably decreased in three tests in 2018. CONCLUSIONS: The results indicate that serological testing is not sensitive in the early stage of a PRV infection and that gilt introduction is a risk factor for a PRV-negative pig farm. The data on PRV gI antibody dynamics can provide reference information for pig farms wanting to eradicate PR.