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.

Swine pseudorabies virus attenuated vaccine reprograms the kidney cancer tumor microenvironment and synergizes with PD-1 blockade.

The global incidence rate of kidney cancer (KC) has been steadily increasing over the past 30 years. With the aging global population, kidney cancer has become an escalating concern that necessitates vigilant surveillance. Nowadays, surgical intervention remains the optimal therapeutic approach for kidney cancer, while the availability of efficacious treatments for advanced tumors remains limited. Oncolytic viruses, an emerging form of immunotherapy, have demonstrated encouraging anti-neoplastic properties and are progressively garnering public acceptance. However, research on oncolytic viruses in kidney cancer is relatively limited. Furthermore, given the high complexity and heterogeneity of kidney cancer, it is crucial to identify an optimal oncolytic virus agent that is better suited for its treatment. The present study investigates the oncolytic activity of the Pseudorabies virus live attenuated vaccine (PRV-LAV) against KC. The findings clearly demonstrate that PRV-LAV exhibits robust oncolytic activity targeting KC cell lines. Furthermore, the therapeutic efficacy of PRV-LAV was confirmed in both a subcutaneous tumor-bearing nude mouse model and a syngeneic mouse model of KC. Combined RNA-seq analysis and flow cytometry revealed that PRV-LAV treatment substantially enhances the infiltration of a diverse range of lymphocytes, including T cells, B cells, macrophages, and NK cells. Additionally, PRV-LAV treatment enhances T cell activation and exerts antitumor effects. Importantly, the combination of PRV-LAV with anti-PD-1 antibodies, an approved drug for KC treatment, synergistically enhances the efficacy against KC. Overall, the discovery of PRV-LAV as an effective oncolytic virus holds significant importance for improving the treatment efficacy and survival rates of KC patients.

Brincidofovir Effectively Inhibits Proliferation of Pseudorabies Virus by Disrupting Viral Replication.

Pseudorabies is an acute and febrile infectious disease caused by pseudorabies virus (PRV), a member of the family Herpesviridae. Currently, PRV is predominantly endemoepidemic and has caused significant economic losses among domestic pigs. Other animals have been proven to be susceptible to PRV, with a mortality rate of 100%. In addition, 30 human cases of PRV infection have been reported in China since 2017, and all patients have shown severe neurological symptoms and eventually died or developed various neurological sequelae. In these cases, broad-spectrum anti-herpesvirus drugs and integrated treatments were mostly applied. However, the inhibitory effect of the commonly used anti-herpesvirus drugs (e.g., acyclovir, etc.) against PRV were evaluated and found to be limited in this study. It is therefore urgent and important to develop drugs that are clinically effective against PRV infection. Here, we constructed a high-throughput method for screening antiviral drugs based on fluorescence-tagged PRV strains and multi-modal microplate readers that detect fluorescence intensity to account for virus proliferation. A total of 2104 small molecule drugs approved by the U.S. Food and Drug Administration (FDA) were studied and validated by applying this screening model, and 104 drugs providing more than 75% inhibition of fluorescence intensity were selected. Furthermore, 10 drugs that could significantly inhibit PRV proliferation in vitro were strictly identified based on their cytopathic effects, virus titer, and viral gene expression, etc. Based on the determined 50% cytotoxic concentration (CC50) and 50% inhibitory concentration (IC50), the selectivity index (SI) was calculated to be 26.3-3937.2 for these 10 drugs, indicating excellent drugability. The antiviral effects of the 10 drugs were then assessed in a mouse model. It was found that 10 mg/kg brincidofovir administered continuously for 5 days provided 100% protection in mice challenged with lethal doses of the human-origin PRV strain hSD-1/2019. Brincidofovir significantly attenuated symptoms and pathological changes in infected mice. Additionally, time-of-addition experiments confirmed that brincidofovir inhibited the proliferation of PRV mainly by interfering with the viral replication stage. Therefore, this study confirms that brincidofovir can significantly inhibit PRV both in vitro and in vivo and is expected to be an effective drug candidate for the clinical treatment of PRV infections.

Construction of and evaluation of the immune response to two recombinant pseudorabies viruses expressing the B119L and EP364R proteins of African swine fever virus.

African swine fever (ASF) is an infectious disease caused by ASF virus (ASFV), which is characterized by high infectivity, rapid onset of disease, and a high mortality rate. Outbreaks of ASFV have caused great economic losses to the global pig industry, and there is a need to develop safe and effective vaccines. In this study, two recombinant pseudorabies virus (PRV) strains, rGXGG-2016-ΔgI/ΔgE-EP364R and rGXGG-2016-ΔgI/ΔgE-B119L, expressing the EP364R and B119L protein, respectively, of ASFV, were constructed by homologous recombination technology. Western blotting and immunofluorescence analysis showed that these foreign proteins were expressed in cells infected with the recombinant strains. The strains showed good genetic stability and proliferative characteristics for 20 passages in BHK-21 cells. Both of these strains were immunogenic in mice, inducing the production of specific antibodies against the expressed ASFV proteins while providing protection against lethal challenge with PRV. Thus, the recombinant strains rGXGG-2016-ΔgI/ΔgE-EP364R and rGXGG-2016-ΔgI/ΔgE-B119L could be used as candidate vaccines for both ASFV and PRV. In addition, our study identifies two potential target genes for the development of safe and efficient ASFV vaccines, provides a reference for the construction of bivalent ASFV and PRV vaccines, and demonstrates the feasibility of developing a live ASFV vector vaccine.

Detection and molecular analysis of Pseudorabies virus from free-ranging Italian wolves (Canis lupus italicus) in Italy - a case report.

BACKGROUND: The only natural hosts of Pseudorabies virus (PRV) are members of the family Suidae (Sus scrofa scrofa). In mammals, the infection is usually fatal and typically causes serious neurologic disease. This study describes four Aujeszky's disease cases in free-ranging Italian wolves (Canis lupus italicus). In Italy, the wolf is a strictly protected species and is in demographic expansion. CASE PRESENTATION: Three wolves (Wolf A, B, and C) were found in a regional park in Northern Italy, and one (Wolf D) was found in Central Italy. Wolf A and D were alive at the time of the finding and exhibited a fatal infection with epileptic seizures and dyspnoea, dying after a few hours. Wolf B presented scratching lesions under the chin and a detachment of the right earlobe, whilst Wolf C was partially eaten. The wolves showed hepatic congestion, diffuse enteritis, moderate pericardial effusion, severe bilateral pneumonia, and diffuse hyperaemia in the brain. The diagnostic examinations included virological analyses and detection of toxic molecules able to cause serious neurological signs. All four wolves tested positive for pseudorabies virus (PrV). The analysed sequences were placed in Italian clade 1, which is divided into two subclades, "a" and "b". The sequences of Wolf A, B, and C were closely related to other Italian sequences in the subclade b, originally obtained from wild boars and hunting dogs. The sequence from Wolf D was located within the same clade and was closely related to the French hunting dog sequences belonging to group 4. CONCLUSION: Results showed the presence of PrV strains currently circulating in wild boars and free-ranging Italian wolves. The genetic characterisation of the PrV UL44 sequences from the four wolves confirmed the close relationship with the sequences from wild boars and hunting dogs. This fact supports a possible epidemiological link with the high PrV presence in wild boars and the possibility of infection in wolves through consumption of infected wild boar carcasses or indirect transmission. To the best of our knowledge, this study is the first detection of Pseudorabies virus in free-ranging Italian wolves in northern and central Italy.

Inhibition of pseudorabies virus replication via upregulated interferon response by targeting 7-dehydrocholesterol reductase.

Pseudorabies virus (PRV) is an alpha-herpesvirus capable of infecting a range of animal species, particularly its natural host, pigs, resulting in substantial economic losses for the swine industry. Recent research has shed light on the significant role of cholesterol metabolism in the replication of various viruses. However, the specific role of cholesterol metabolism in PRV infection remains unknown. Here, we demonstrated that the expression of 7-dehydrocholesterol reductase (DHCR7) is upregulated following PRV infection, as evidenced by the proteomic analysis. Subsequently, we showed that DHCR7 plays a crucial role in promoting PRV replication by converting 7-dehydrocholesterol (7-DHC) into cholesterol, leading to increased cellular cholesterol levels. Importantly, DHCR7 inhibits the phosphorylation of interferon regulatory factor 3 (IRF3), resulting in reduced levels of interferon-beta (IFN-ß) and interferon-stimulated genes (ISGs). Finally, we revealed that the DHCR7 inhibitor, trans-1,4-bis(2-chlorobenzylaminomethyl) cyclohexane dihydrochloride (AY9944), significantly suppresses PRV replication both in vitro and in vivo. Taken together, the study has established a connection between cholesterol metabolism and PRV replication, offering novel insights that may guide future approaches to the prevention and treatment of PRV infections.

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.

Intradermal vaccination with Porcilis® Begonia can clinically protect against fatal PRV challenge with the highly virulent ZJ01 field strain.

Since pseudorabies (PR) re-emerged and rapidly spread in China at the end of 2011, researchers have focused on effective vaccine strategies to prevent and control pseudorabies virus (PRV) infection in pig herds. Due to the extensive application of an attenuated vaccine based on the Bartha-K61 strain isolated in Hungary in 1961 and the variation of the PRV strain, it has been suggested that traditional vaccines based on the Bartha-K61 strain offer only partial protection against variant strains. It was therefore evaluated whether the Porcilis® Begonia vaccine, which is based on the NIA-3 strain with deletions in the gE and TK genes, is efficacious against experimental infection with the virulent, contemporary Chinese PRV strain ZJ01. In this study, piglets were vaccinated with Porcilis® Begonia through either the intradermal (ID) route or the intramuscular (IM) route and subsequently challenged intranasally with strain ZJ01 at 4 weeks post-vaccination. An unvaccinated challenge group and an unvaccinated/nonchallenged group were also included in the study. All animals were monitored for 14 days after challenge. Vaccinated and negative control pigs stayed healthy during the study, while the unvaccinated control animals developed lesions associated with PRV ZJ01 challenge, and 44% of these pigs died before the end of the experiment. This study demonstrated that ID or IM vaccination of pigs with a vaccine based on the NIA-3 strain Porcilis® Begonia clinically protects against fatal PRV challenge with the ZJ01 strain.

Unveiling Hypothalamic Molecular Signatures via Retrograde Viral Tracing and Single-Cell Transcriptomics.

Despite the importance of hypothalamic neurocircuits in regulating homeostatic and survival-related behaviors, our understanding of the intrinsic molecular identities of neural components involved in these complex multi-synaptic interactions remains limited. In this study, we constructed a Cre recombinase-dependent pseudorabies virus (PRVs) capable of crossing synapses, coupled with transcriptome analysis of single upstream neurons post-infection. By utilizing this retrograde nuclear Connect-seq (nuConnect-seq) approach, we generated a single nuclei RNA-seq (snRNA-seq) dataset of 1,533 cells derived from the hypothalamus of CRH-IRES-Cre (CRH-Cre) mice. To ensure the technical validity of our nuConnect-seq dataset, we employed a label transfer technique against an integrated reference dataset of postnatal mouse hypothalamus comprising 152,524 QC-passed cells. The uniqueness of our approach lies in the integration of diverse datasets for validation, providing a more nuanced diversity of hypothalamic cell types. The presented validated dataset may deepen our understanding of hypothalamic neurocircuits and underscore the essential role of comprehensive integrated transcriptomic data for technical validity.

Transcriptomic analysis reveals impact of gE/gI/TK deletions on host response to PRV infection.

BACKGROUND: Pseudorabies virus (PRV) causes substantial losses in the swine industry worldwide. Attenuated PRV strains with deletions of immunomodulatory genes glycoprotein E (gE), glycoprotein I (gI) and thymidine kinase (TK) are candidate vaccines. However, the effects of gE/gI/TK deletions on PRV-host interactions are not well understood. METHODS: To characterize the impact of gE/gI/TK deletions on host cells, we analyzed and compared the transcriptomes of PK15 cells infected with wild-type PRV (SD2017), PRV with gE/gI/TK deletions (SD2017gE/gI/TK) using RNA-sequencing. RESULTS: The attenuated SD2017gE/gI/TK strain showed increased expression of inflammatory cytokines and pathways related to immunity compared to wild-type PRV. Cell cycle regulation and metabolic pathways were also perturbed. CONCLUSIONS: Deletion of immunomodulatory genes altered PRV interactions with host cells and immune responses. This study provides insights into PRV vaccine design.

Suppression of NF-κB signaling by Pseudorabies virus DNA polymerase processivity factor UL42 via recruiting SOCS1 to promote the ubiquitination degradation of p65.

The NF-κB pathway is a critical signaling involved in the regulation of the inflammatory and innate immune responses. Previous studies have shown that Pseudorabies Virus (PRV), a porcine alpha herpesvirus, could lead to the phosphorylation and nucleus translocation of p65 while inhibiting the expression of NF-κB-dependent inflammatory cytokines, which indicated that there may be unknown mechanisms downstream of p65 that downregulate the activation of NF-κB signaling. Here, we found that PRV DNA polymerase factor UL42 inhibited TNFα-, LPS-, IKKα-, IKKß-, and p65-mediated transactivation of NF-κB signaling, which demonstrated UL42 worked either at or downstream of p65. In addition, it was found that the DNA-binding activity of UL42 was required for inhibition of NF-κB signaling. Importantly, it was revealed that UL42 could induce the ubiquitination degradation of p65 by upregulating the suppressor of cytokine signaling 1 (SOCS1). Additionally, it was found that UL42 could promote the K6/K29-linked ubiquitination of p65. Finally, knockdown of SOCS1 attenuated the replication of PRV and led to a significant increase of the inflammatory cytokines. Taken together, our findings uncovered a novel mechanism that PRV-UL42 could upregulated SOCS1 to promote the ubiquitination degradation of p65 to prevent excessive inflammatory response during PRV infection.

A Triple Gene-Deleted Pseudorabies Virus-Vectored Subunit PCV2b and CSFV Vaccine Protect Pigs against a Virulent CSFV Challenge.

Classical swine fever (CSF) remains one of the most economically significant viral diseases affecting domestic pigs and wild boars worldwide. To develop a safe and effective vaccine against CSF, we have constructed a triple gene-deleted pseudorabies virus (PRVtmv)-vectored bivalent subunit vaccine against porcine circovirus type 2b (PCV2b) and CSFV (PRVtmv+). In this study, we determined the protective efficacy of the PRVtmv+ against virulent CSFV challenge in pigs. The results revealed that the sham-vaccinated control group pigs developed severe CSFV-specific clinical signs characterized by pyrexia and diarrhea, and became moribund on or before the seventh day post challenge (dpc). However, the PRVtmv+-vaccinated pigs survived until the day of euthanasia at 21 dpc. A few vaccinated pigs showed transient diarrhea but recovered within a day or two. One pig had a low-grade fever for a day but recovered. The sham-vaccinated control group pigs had a high level of viremia, severe lymphocytopenia, and thrombocytopenia. In contrast, the vaccinated pigs had a low-moderate degree of lymphocytopenia and thrombocytopenia on four dpc, but recovered by seven dpc. Based on the gross pathology, none of the vaccinated pigs had any CSFV-specific lesions. Therefore, our results demonstrated that the PRVtmv+ vaccinated pigs are protected against virulent CSFV challenge.

Molecular epidemiological and genetic characterization of pseudorabies virus in Guangxi, China.

Pseudorabies virus (PRV) is an important pathogen that can cause harm to the pig population. Since 2011, there have been a number of large-scale outbreaks of pseudorabies on Chinese farms where animals had been vaccinated with the Bartha-K61 vaccine. In order to understand the epidemiological trend and genetic variations of PRV in Guangxi province, China, 819 tissue samples were collected from swine farms where PRV infection was suspected from 2013 to 2019, and these were tested for infectious wild strains of PRV. The results showed a positive rate of PRV in Guangxi province of 28.21% (231/819). Thirty-six wild-type PRV strains were successfully isolated from PRV-positive tissue samples, and a genetic evolutionary analysis was performed based on the gB, gC, gD, gE, and TK genes. Thirty of the PRV strains were found to be closely related to the Chinese variant strains HeN1-China-2012 and HLJ8-China-2013. In addition, five PRV strains were genetically related to Chinese classical strains, and one isolate was a recombinant of the PRV variant and the vaccine strain Bartha-K61. Amino acid sequence analysis showed that all 36 PRV strains had characteristic variant sites in the amino acid sequences of the gB, gC, gD, and gE proteins. Pathogenicity analysis showed that, compared to classical PRV strains, the PRV variant strains were more pathogenic in mice and had a lower LD50. Taken together, our results show that wild-type PRV infections are common on pig farms in Guangxi province of China and that the dominant prevalent strains were those of the PRV variants. The PRV variant strains also had increased pathogenicity in mice. Our data will provide a useful reference for understanding the prevalence and genetic evolution of PRV in China.

Viral Attachment Blocking Chimera Composed of DNA Origami and Nanobody Inhibits Pseudorabies Virus Infection In Vitro.

Antivirals are indispensable tools that can be targeted at viral domains directly or at cellular domains indirectly to obstruct viral infections and reduce pathogenicity. Despite their transformative use in healthcare, antivirals have been clinically approved to treat only 10 of the more than 200 known pathogenic human viruses. Additionally, many virus functions are intimately coupled with host cellular processes, which presents challenges in antiviral development due to the limited number of clear targets per virus, necessitating extensive insight into these molecular processes. Compounding this challenge, many viral pathogens have evolved to evade effective antivirals. We hypothesize that a viral attachment blocking chimera (VirABloC) composed of a viral binder and a bulky scaffold that sterically blocks interactions between a viral particle and a host cell may be suitable for the development of antivirals that are agnostic to the extravirion epitope that is being bound. We test this hypothesis by modifying a nanobody that specifically recognizes a nonessential epitope presented on the extravirion surface of pseudorabies virus strain 486 with a 3-dimensional wireframe DNA origami structure ∼100 nm in diameter. The nanobody switches from having no inhibitory properties to 4.2 ± 0.9 nM IC50 when conjugated with the DNA origami scaffold. Mechanistic studies support that inhibition is mediated by the noncovalent attachment of the DNA origami scaffold to the virus particle, which obstructs the attachment of the viruses onto host cells. These results support the potential of VirABloC as a generalizable approach to developing antivirals.

Ginkgolic acid inhibits the replication of pseudorabies virus in vitro and in vivo by suppressing the transcription of viral late genes.

Pseudorabies virus (PRV) belongs to the species of alphaherpesvirus that can cause substantial economic losses to the world swine industry. Therefore, research on anti-PRV compounds is of great value. In this study, it was found that ginkgolic acid could efficiently inhibit the replication of PRV, and the IC50 and CC50 were 3.407 µM and 102.3 µM, respectively. Moreover, it was discovered that ginkgolic acid had no effect on the adsorption, entry, and release stages of the PRV replication cycle. Importantly, it was found that ginkgolic acid could significantly suppress the transcription of PRV late genes, while the transcription of viral immediate early and early genes was not affected. Finally, in vivo experiments showed that ginkgolic acid could significantly reduce the viral load of PRV in multiple tissues and increase 30% survival rate of mice upon the challenge of PRV. Taken together, a novel PRV replication inhibitor, ginkgolic acid, which worked through suppressing the transcription of the late genes, was found in this study. This study provides a potential therapy method for the infection of PRV.

The mutations on the envelope glycoprotein D contribute to the enhanced neurotropism of the pseudorabies virus variant.

The pseudorabies virus (PRV) TJ strain, a variant of PRV, induces more severe neurological symptoms and higher mortality in piglets and mice than the PRV SC strain isolated in 1980. However, the mechanism underlying responsible for the discrepancy in virulence between these strains remains unclear. Our study investigated the differences in neurotropism between PRV TJ and PRV SC using both in vitro and in vivo models. We discovered that PRV TJ enters neural cells more efficiently than PRV SC. Furthermore, we found that PRV TJ has indistinguishable genomic DNA replication capability and axonal retrograde transport dynamics compared to the PRV SC. To gain deeper insights into the mechanisms underlying these differences, we constructed gene-interchanged chimeric virus constructs and assessed the affinity between envelope glycoprotein B, C, and D (gD) and corresponding receptors. Our findings confirmed that mutations in these envelope proteins, particularly gD, significantly contributed to the heightened attachment and penetration capabilities of PRV TJ. Our study revealed the critical importance of the gDΔR278/P279 and gDV338A in facilitating viral invasion. Furthermore, our observations indicated that mutations in envelope proteins have a more significant impact on viral invasion than on virulence in the mouse model. Our findings provide valuable insights into the roles of natural mutations on the PRV envelope glycoproteins in cell tropism, which sheds light on the relationship between cell tropism and clinical symptoms and offers clues about viral evolution.

Genome-wide analysis of long noncoding RNA profiles in pseudorabies-virus-infected PK15 cells.

Recently, an increasing number of studies have shown that long noncoding RNAs (lncRNAs) are involved in host metabolism after infection with pseudorabies virus (PRV). In our study, via RNA sequencing analysis, a total of 418 mRNAs, 137 annotated lncRNAs, and 312 new lncRNAs were found to be differentially expressed. These lncRNAs were closely associated with metabolic regulation and immunity-related signalling pathways, including the T-cell receptor signalling pathway, chemokine signalling pathway, mitogen-activated protein kinase (MAPK) signalling pathway, TNF signalling pathway, Ras signalling pathway, calcium signalling pathway, and phosphatidylinositol signalling system. Real-time PCR indicated that several mRNAs and lncRNAs involved in the regulation of the immune effector process, T-cell receptor signalling pathway, TNF signalling pathway, MAPK signalling pathway, and chemokine signalling pathways were significantly expressed. These mRNAs and lncRNAs might play a role in PRV infection.

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.

Directed stepwise tracing of polysynaptic neuronal circuits with replication-deficient pseudorabies virus.

Brain functions are accomplished by polysynaptic circuits formed by neurons wired together through multiple orders of synaptic connections. Polysynaptic connectivity has been difficult to examine due to a lack of methods of continuously tracing the pathways in a controlled manner. Here, we demonstrate directed, stepwise retrograde polysynaptic tracing by inducible reconstitution of replication-deficient trans-neuronal pseudorabies virus (PRVΔIE) in the brain. Furthermore, PRVΔIE replication can be temporally restricted to minimize its neurotoxicity. With this tool, we delineate a wiring diagram between the hippocampus and striatum-two major brain systems for learning, memory, and navigation-that consists of projections from specific hippocampal domains to specific striatal areas via distinct intermediate brain regions. Therefore, this inducible PRVΔIE system provides a tool for dissecting polysynaptic circuits underlying complex brain functions.

Transneuronal Circuit Analysis with Pseudorabies Viruses.

Our ability to understand the function of the nervous system is dependent upon defining the connections of its constituent neurons. Development of methods to define connections within neural networks has always been a growth industry in the neurosciences. Transneuronal spread of neurotropic viruses currently represents the best means of defining synaptic connections within neural networks. The method exploits the ability of viruses to invade neurons, replicate, and spread through the intimate synaptic connections that enable communication among neurons. Since the method was first introduced in the 1970s, it has benefited from an increased understanding of the virus life cycle, the function of viral genomes, and the ability to manipulate the viral genome in support of directional spread of virus and the expression of transgenes. In this article, we review these advances in viral tracing technology and the ways in which they may be applied for functional dissection of neural networks. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Retrograde infection of CNS circuits by peripheral injection of virus Basic Protocol 2: Transneuronal analysis by intracerebral injection Alternate Protocol 1: Transneuronal analysis with multiple recombinant strains Alternate Protocol 2: Conditional replication and spread of PRV Alternate Protocol 3: Conditional reporters of PRV infection and spread Alternate Protocol 4: Reporters of neural activity in polysynaptic circuits Support Protocol 1: Growing and titering a PRV viral stock Support Protocol 2: Immunohistochemical processing and detection Support Protocol 3: Dual-immunofluorescence localization.