Pseudorabies Research Articles

Interference of pseudorabies virus infection on functions of porcine granulosa cells via apoptosis modulated by MAPK signaling pathways

BackgroundPseudorabies virus (PRV) is one of the major viral pathogens leading to reproductive disorders in swine. However, little is known about the effects of PRV infection on porcine reproductive system. Ovarian granulosa cells are somatic cells surrounding oocytes in ovary and required for folliculogenesis. The present study aimed to investigate the interference of PRV on functions of porcine ovarian granulosa cells in vitro.MethodsPrimary granulosa cells were isolated from porcine ovaries. To investigate the PRV infectivity, transmission electron microscopy (TEM) was used to check the presence of viral particles, and the expression of viral gE gene was detected by quantitative real-time PCR (qPCR) in PRV-inoculated cells. After PRV infection, cell viability was detected by MTS assay, Ki67 for proliferative status was determined by immunofluorescence assay (IFA), cell cycle and apoptosis were detected by flow cytometry, and progesterone (P4) and estradiol (E2) were determined by radioimmunoassay. The checkpoint genes of cell cycle and apoptosis-related proteins were studied by qPCR and western blotting.ResultsVirus particles were observed in the nucleus and cytoplasm of PRV-infected granulosa cells by TEM imaging, and the expression of viral gE gene increased in a time-dependent manner post infection. PRV infection inhibited cell viability and blocked cell cycle at S phase in porcine granulosa cells, accompanied by decreases in expression of Ki67 protein and checkpoint genes related to S phase. Radioimmunoassay revealed decreased levels in P4 and E2, and the expressions of key steroidogenic enzymes were also down-regulated post PRV-infection. In addition, PRV induced apoptosis with an increase in Bax expression and activation of caspase 9, and the phosphorylation of JNK, ERK and p38 MAPKs were significantly up-regulated in porcine ovarian granulosa cells post PRV infection.ConclusionsThe data indicate that PRV causes infection on porcine ovarian granulosa cells and interferes the cell functions through apoptosis, and the MAPK signaling pathway is involved in the viral pathogenesis.

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-β) 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.

ATG7/GAPLINC/IRF3 axis plays a critical role in regulating pathogenesis of influenza A virus.

Autophagy-related protein 7 (ATG7) is an essential autophagy effector enzyme. Although it is well known that autophagy plays crucial roles in the infections with various viruses including influenza A virus (IAV), function and underlying mechanism of ATG7 in infection and pathogenesis of IAV remain poorly understood. Here, in vitro studies showed that ATG7 had profound effects on replication of IAV. Depletion of ATG7 markedly attenuated the replication of IAV, whereas overexpression of ATG7 facilitated the viral replication. ATG7 conditional knockout mice were further employed and exhibited significantly resistant to viral infections, as evidenced by a lower degree of tissue injury, slower body weight loss, and better survival, than the wild type animals challenged with either IAV (RNA virus) or pseudorabies virus (DNA virus). Interestingly, we found that ATG7 promoted the replication of IAV in autophagy-dependent and -independent manners, as inhibition of autophagy failed to completely block the upregulation of IAV replication by ATG7. To determine the autophagy-independent mechanism, transcriptome analysis was utilized and demonstrated that ATG7 restrained the production of interferons (IFNs). Loss of ATG7 obviously enhanced the expression of type I and III IFNs in ATG7-depleted cells and mice, whereas overexpression of ATG7 impaired the interferon response to IAV infection. Consistently, our experiments demonstrated that ATG7 significantly suppressed IRF3 activation during the IAV infection. Furthermore, we identified long noncoding RNA (lncRNA) GAPLINC as a critical regulator involved in the promotion of IAV replication by ATG7. Importantly, both inactivation of IRF3 and inhibition of IFN response caused by ATG7 were mediated through control over GAPLINC expression, suggesting that GAPLINC contributes to the suppression of antiviral immunity by ATG7. Together, these results uncover an autophagy-independent mechanism by which ATG7 suppresses host innate immunity and establish a critical role for ATG7/GAPLINC/IRF3 axis in regulating IAV infection and pathogenesis.

MicroRNA-194-5p/Heparin-binding EGF-like growth factor signaling mediates dexamethasone-induced activation of pseudorabies virus in rat pheochromocytoma cells

Pseudorabies virus (PRV) is a neurotropic virus, which infects a wide range of mammals. The activity of PRV is gradually suppressed in hosts that have tolerated the primary infection. Increased glucocorticoid levels resulting from stressful stimuli overcome repression of PRV activity. However, the host cell mechanism involved in the activation processes under stressful conditions remains unclear. In this study, infection of rat PC-12 pheochromocytoma cells with neuronal properties using PRV at a multiplicity of infection (MOI) = 1 for 24 h made the activity of PRV be the relatively repressed state, and then incubation with 0.5 μM of the corticosteroid dexamethasone (DEX) for 4 h overcomes the relative repression of PRV activity. RNA-seq deep sequencing and bioinformatics analyses revealed different microRNA and mRNA profiles of PC-12 cells with/without PRV and/or DEX treatment. qRT-PCR and western blot analyses confirmed the negative regulatory relationship of miRNA-194–5p and its target heparin-binding EGF-like growth factor (Hbegf); a dual-luciferase reporter assay revealed that Hbegf is directly targeted by miRNA-194–5p. Further, miRNA-194–5p mock transfection contributed to PRV activation, Hbegf was downregulated in DEX-treated PRV infection cells, and Hbegf overexpression contributed to returning activated PRV to the repression state. Moreover, miRNA-194–5p overexpression resulted in reduced levels of HBEGF, c-JUN, and p-EGFR, whereas Hbegf overexpression suppressed the reduction caused by miRNA-194–5p overexpression. Overall, this study is the first to report that changes in the miR-194–5p-HBEGF/EGFR pathway in neurons are involved in DEX-induced activation of PRV, laying a foundation for the clinical prevention of stress-induced PRV activation.

Screening and Stability Evaluation of Freeze-Dried Protective Agents for a Live Recombinant Pseudorabies Virus Vaccine.

Infection of pigs with the pseudorabies virus (PRV) causes significant economic losses in the pig industry. Immunization with live vaccines is a crucial aspect in the prevention of pseudorabies in swine. The TK/gE/gI/11k/28k deleted pseudorabies vaccine is a promising alternative for the eradication of epidemic pseudorabies mutant strains. This study optimized the lyophilization of a heat-resistant PRV vaccine to enhance the quality of a live vaccine against the recombinant PRV rHN1201TK-/gE-/gI-/11k-/28k-. The A4 freeze-dried protective formulation against PRV was developed by comparing the reduction in virus titer after lyophilization and after seven days of storage at 37 °C. The formulation contains 1% gelatin, 5% trehalose, 0.5% poly-vinylpyrimidine (PVP), 0.5% thiourea, and 1% sorbitol. The A4 freeze-dried vaccine demonstrated superior protection and thermal stability. It experienced a freeze-dried loss of 0.31 Lg post-freeze-drying and a heat loss of 0.42 Lg after being stored at a temperature of 37 °C for 7 consecutive days. The A4 freeze-dried vaccine was characterized through XRD, FTIR, and SEM analyses, which showed that it possessed an amorphous structure with a consistent porous interior. The trehalose component of the vaccine formed stable hydrogen bonds with the virus. Long-term and accelerated stability studies were also conducted. The A4 vaccine maintained viral titer losses of less than 1.0 Lg when exposed to 25 °C for 90 days, 37 °C for 28 days, and 45 °C for 7 days. The A4 vaccine had a titer loss of 0.3 Lg after storage at 2-8 °C for 24 months, and a predicted shelf life of 6.61 years at 2-8 °C using the Arrhenius equation. The A4 freeze-dried vaccine elicited no side effects when used to immunize piglets and produced specific antibodies. This study provides theoretical references and technical support to improve the thermal stability of recombinant PRV rHN1201TK-/gE-/gI-/11k-/28k- vaccines.

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.

Retracted: Antiviral Activity of Plantago asiatica Polysaccharide against Pseudorabies Virus In Vitro.

[This retracts the article DOI: 10.1155/2022/3570475.].

The immunity protection of intestine induced by pseudorabies virus del gI/gE/TK in piglets.

Compared to the classical strain of Pseudorabies virus (PRV), the PRV variant exhibits stronger transmissibility and pathogenicity, causing immense disasters for the global pig industry. Based on this variant, our laboratory has preliminarily constructed a modified pseudorabies virus with deletions in the gE/gI/TK genes. In this study, the protective efficacy of PRV XJ del gI/gE/TK against piglet intestinal damage was evaluated. The results demonstrated that piglets immunized with PRV XJ del gI/gE/TK exhibited alleviated intestinal damage caused by the PRV XJ variant strain. This included reduced viral load, suppressed inflammation, and maintenance of intestinal structure and function. Additionally, PRV XJ del gI/gE/TK also strongly activated the innate immune response in the intestines, increasing the expression of antiviral factor mRNA and the secretion of SIgA to counteract the attack of the PRV XJ variant strain. Our study indicates that PRV XJ del gI/gE/TK can inhibit intestinal damage caused by PRV XJ variant strain and activate the innate immune response in the intestines.

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

BackgroundThe 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 presentationThree 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.ConclusionResults 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.

Infectivity and Potential Zoonotic Characteristics of Porcine Pseudorabies Virus in Human Cells

Pseudorabies virus (PRV) is widely spread, characterized by high contagiousness, high viral load, and strong infectivity, and poses severe threats to the global pig farming industry. Apart from pigs, PRV can also infect several other mammals, including mice, cattle, cats, dogs, and wolves, with diverse clinical symptoms. Notably, approximately more than 20 cases of human PRV infection have been reported in recent years, with fever, seizures, human encephalitis, intraocular inflammation, and severe central nervous system symptoms. However, whether PRV can infect humans or belongs to a zoonotic virus is still controversial. In this study, human neuronal cells were infected with PRV and blindly passaged to obtain human cell‐adapted PRV, followed by comparing the characteristics of human cell‐adapted PRV and pig‐derived PRV in vitro and in vivo, to determine whether PRV has the potential to infect humans. The results showed that PRV could be stably passaged in human cells and produced progeny viruses similar to the parental virus, including morphology, infectivity, and pathogenicity. The human cell‐adapted PRV can also cross‐transmit to cells from other origins, including humans, mice, pigs, and monkeys, causing different cytopathic effects. Moreover, multiple tissue damage can be detected in mice infected with human cell‐adapted PRV. These results demonstrate that PRV is a potential zoonotic virus, and it is necessary to pay close attention to the spread and variation of the virus in animals and humans.

Development and immunogenicity evaluation of a quadruple-gene-deleted pseudorabies virus strain.

Since 2011, the emergence of Pseudorabies virus (PRV) variants has led to significant vaccine failures, resulting in severe economic losses in China's swine industry. Conventional PRV vaccines have shown limited efficacy against these emergent variants, underscoring the urgent need for novel immunization strategies. This study aimed to develop and evaluate a novel recombinant PRV vaccine candidate with improved safety and immunogenicity profiles. Utilizing the homology-directed repair (HDR)-CRISPR/Cas9 system, we generated a recombinant PRV strain, designated PRV SX-10ΔgI/gE/TK/UL24, with deletions in the gI, gE, TK, and UL24 genes. In vitro analyses demonstrated that the recombinant virus exhibited similar replication kinetics and growth curves comparable to the parental strain. The immunological properties of the recombinant PRV were assessed in murine and porcine models. All animals inoculated with PRV SX-10ΔgI/gE/TK/UL24 survived without exhibiting significant clinical signs or pathological alterations. Immunological assays revealed that PRV SX-10ΔgI/gE/TK/UL24 elicited significantly higher levels of gB-specific antibodies, neutralizing antibodies, and cytokines (including IFN-γ, IL-2, and IL-4) compared to both the Bartha-K61 and PRV SX-10ΔgI/gE/TK strains. Notably, both murine and porcine subjects immunized with PRV SX-10ΔgI/gE/TK/UL24 demonstrated enhanced protection against challenges with the variant PRV SX-10 strain, compared to other vaccine strains. These findings suggest that PRV SX-10ΔgI/gE/TK/UL24 represents a promising PRV vaccine candidate strain, offering valuable insights for the prevention and control of PRV in clinical applications.

Comparative proteomic analysis of PK-15 cells infected with wild-type strain and its EP0 gene-deleted mutant strain of pseudorabies virus.

As one of the main etiologic agents of infectious diseases in pigs, pseudorabies virus (PRV) infections have caused enormous economic losses worldwide. EP0, one of the PRV early proteins (EP) plays a vital role in PRV infections, but the mechanisms are unclear. This study examined the function of EP0 to provide a direction for its in-depth analysis. In this study, the EP0-deleted PRV mutant was obtained, and Tandem Mass Tag-based proteomic analysis was used to screen the differentially expressed proteins (DEPs) quantitatively in EP0-deleted PRV- or wild-type PRV-infected porcine kidney 15 cells. This study identified 7,391 DEPs, including 120 and 21 up-regulated and down-regulated DEPs, respectively. Western blot analysis confirmed the changes in the expression of the selected proteins, such as speckled protein 100. Comprehensive analysis revealed 141 DEPs involved in various biological processes and molecular functions, such as transcription regulator activity, biological regulation, and localization. These results holistically outlined the functions of EP0 during a PRV infection and might provide a direction for more detailed function studies of EP0 and the stimulation of lytic PRV infections.

Vigilância ativa para Doença de Aujeszky e aspectos sobre a biosseguridade de granjas suínas no Estado do Espírito Santo, Brasil

ABSTRACT: Aujeszky’s Disease (AD) is a highly infectious swine disease caused by Suid herpesvirus 1 (SuHV 1). The present study evaluated the biosafety of commercial swine farms and carried out the first serological survey for Aujeszky’s Disease in Espírito Santo State, Brazil. A total of 55 farms in 27 municipalities of the state were selected, where 416 swine serum samples were collected to be submitted to serological diagnosis for AD. Finally, a survey was carried out using a questionnaire to obtain information on the biosecurity of the farms. The results by the ELISA technique indicated the absence of antibodies for AD. Regarding the biosecurity of the farms, 56.4% of the farms did not meet the requirement of internal isolation, 67.3% did not have changing rooms, 72.7% did not provide specific clothing for employees and visitors, 85.5% did not require visitors bathe to access the sheds and only 10.9% of the farms controlled the flow of visitors and vehicles accessing the property. Another noteworthy factor is that only 25.5% of the farms had an area to quarantine replacement animals and 69.1% had an access ramp located close to the animal pens. With the results, it was concluded that there is a need to improve biosecurity measures on commercial farms in the state of Espírito Santo and that there was no presence of Aujeszky’s Disease in the evaluated animals.

Enhanced in vivo antiviral activity against pseudorabies virus through transforming gallic acid into graphene quantum dots with stimulation of interferon-related immune responses.

With the urgent need for antiviral agents, antiviral materials with high biocompatibility and antiviral effects have attracted a lot of attention. In this study, gallic acid, a natural polyphenolic compound, was transformed into biocompatible graphene quantum dots (GAGQDs) which exhibit enhanced antiviral activity against pseudorabies virus (PRV). The as-prepared GAGQDs inhibit PRV proliferation with a 104-fold reduction in viral titers. Investigation of the antiviral mechanism revealed that GAGQDs inhibit the adsorption, invasion and replication of PRV infection. Treatment with GAGQDs regulates the expression levels of interferon-related antiviral proteins, including mitochondrial antiviral-signaling protein (MAVS), signal transducer and activator of transcription 1 (STAT1) and 2',5'-oligoadenylate synthetase 1 (OAS1), suggesting that GAGQDs can stimulate innate antiviral immune responses, resulting in enhanced antiviral effects. More importantly, GAGQD treatments alleviate clinical symptoms and reduce mortality in PRV-infected mice. Our results reveal the enhanced therapeutic effects of GAGQDs against PRV infection in vitro and in vivo, suggesting the potential of GAGQDs as a promising novel antiviral agent.

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.

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.

Immunosuppression of the Nasal Cavity by a Novel Pathogenic Pseudorabies Virus Isolation from Cattle in China

Immunosuppression of the Nasal Cavity by a Novel Pathogenic Pseudorabies Virus Isolation from Cattle in China

Pseudorabies virus causes splenic injury via inducing oxidative stress and apoptosis related factors in mice

Pseudorabies virus (PRV) is an immunosuppressive virus that causes significant damage to the pig industry. This study aimed to investigate the effects of PRV on oxidative stress and apoptotic related in the spleen of mice to provide basis knowledge for further research on the pathogenesis of PRV in mice model. 36 mice were randomly two groups, the control group which only received 200 μL PBS and infection group which was subcutaneously infected with 200 μL of 1 × 103 TCID50/100 μL PRV, respectively. Spleen tissues in each group were collected for further experiments at 48, 72, and 96 h post-infection (hpi). Pathological observation was performed by hematoxylin and eosin Y staining. Biochemical and Flow cytometry methods were used to determine the reactive oxygen species profile and apoptosis of the spleen post-infection and apoptosis detection. In addition, q-PCR and Western blot were adopted to measure the apoptotic conditions of the spleen infected with PRV. The results indicated that the reactive oxygen species (ROS) level in the PRV infection group was remarkedly increased (p < 0.01) at a time-dependent pattern. Furthermore, the Malondialdehyde levels in the spleen of mice in the infection group increased (p < 0.01) in a time-dependent mode. However, the activity of Catalase, Superoxide dismutase, and glutathione peroxidase and the content of Glutathione in the infection group were decreased with the control group (p < 0.01) at a time-dependent manner. In addition, the ratio of splenocyte apoptosis in the infection group significantly increased (p < 0.01) in a time-dependent manner. In conclusion, PRV infection causes apoptosis of the spleen via oxidative stress in mice.

Development of a double-antibody sandwich enzyme-linked immunosorbent assay for rapid detection of VZV

BackgroundVaricella zoster virus (VZV) is the pathogen of varicella and herpes zoster, it is necessary to develop a rapid, sensitive and specific detection method for the prevention and control of related diseases. MethodsWe inserted the gB protein extracellular region gene (gB-ex, 1–2208 bp) of VZV into lentivirus vector, and then obtained the recombinant gB protein through mammalian expression system. BALB/c mice were immunized multiple times with purified gB protein as immunogen. Then four strains of high affinity monoclonal antibodies targeting gB protein were prepared by cell fusion technique. Monoclonal antibodies 5G4 and HRP-4E9 were selected as capture and detection antibodies respectively, and a double-antibody sandwich ELISA method was established for detection. ResultsThe detection limit of the DAS-ELISA was 156 PFU/mL, and there was no cross-reaction with Herpes simplex virus-1/Herpes simplex virus-2/Pseudorabies virus. The coefficients of variation of intra-assay and inter-assay repeatability were less than 5%. ConclusionsIn this study, a double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) was established for the detection of VZV. The assay has good sensitivity, specificity and repeatability, which provides strong technical support and product guarantee for the rapid clinical detection of VZV.

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

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