Analysis of the recombination and evolution of the new type mutant pseudorabies virus XJ5 in China.

Pseudorabies have caused enormous economic losses in China's pig industry and have recurred on many large pig farms since late 2011. The disease is caused by highly pathogenic, antigenic variant pseudorabies virus (vPRV) strains. Our laboratory isolated a pseudorabies virus in 2015 and named it XJ5. The pathogenic ability of this mutant strain was much stronger than that of the original isolate. After we sequenced its whole genome (GenBank accession number: OP512542), we found that its overall structure was not greatly changed compared with that of the previous strain Ea (KX423960.1). The whole genome alignment showed that XJ5 had a strong genetic relationship with the strains isolated in China after 2012 reported in GenBank. Based on the isolation time of XJ5 and the mutation and recombination analysis of programs, we found that the whole genome homology of XJ5 and other strains with Chinese isolates was greater than 95%, while the homology with strains outside Asia was less than 94%, which indicated that there may be some recombination and mutation patterns. We found that virulent PRV isolates emerged successively in China in 2011 and formed two different evolutionary clades from foreign isolates. At the same time, this may be due to improper immunization and the presence of wild strains in the field, and recent reports have confirmed that Bartha vaccine strains recombine with wild strains to obtain new pathogenic strains. We performed genetic evolution analysis of XJ5 isolated and sequenced in our laboratory to trace its possible mutations and recombination. We found that XJ5 may be the result of natural mutation of a virus in a branch of mutant strains widely existing in China.

A pseudorabies outbreak in hunting dogs in Campania region (Italy): a case presentation and epidemiological survey.

BACKGROUND: Pseudorabies is an infection of domestic and wild pigs that has occasionally been reported in dogs with fatal encephalitis. Hunting dogs are predisposed to pseudorabies exposure due to incorrect practices (administration of raw infected meat) or close contact with infected wild boars. This study described an outbreak of pseudorabies in two hunting dogs in the Campania region, southern Italy. CASE PRESENTATION: Two hunting dogs were hospitalized after a hunting trip, with fever, itching, and self-inflicted lesions. Laboratory tests showed mild anemia and marked leukocytosis. Despite conservative therapy, both animals died 48 h after the presentation of symptoms. One of the carcasses was sent to the Department of Veterinary Medicine and Animal Production in Naples to confirm the suspicion of pseudorabies. DNA was extracted from different matrices and used as a template for real-time PCR to detect PRV. Several samples (brain, cerebellum, brainstem, lung, and liver) tested positive. Subsequent sequence analyses of glycoprotein E from DNA extracted from the brain stem revealed a sequence similarity to those described in previous cases of pseudorabies in dogs in Italy, France and Belgium. One month after the outbreak, blood samples were collected from 42 dogs belonging to the same hunting team and from 245 dogs (cohort population) living in the Campania region. All samples were tested with two commercial ELISAs to detect seroconversion against glycoproteins B and E. A seroprevalence of 19% was observed in the hunting team affected by the outbreak, while only 0.8% was observed in the regional dog population. CONCLUSIONS: The data reported in this study demonstrate potential exposure to PRV by dead-end hosts, particularly hunting dogs. The sequencing results indicated the homogeneity of PRV strains circulating in the different Italian regions.

Immunological characteristics of a recombinant alphaherpesvirus with an envelope-embedded Cap protein of circovirus.

Introduction: Variant pseudorabies virus (PRV) is a newly emerged zoonotic pathogen that can cause human blindness. PRV can take advantage of its large genome and multiple non-essential genes to construct recombinant attenuated vaccines carrying foreign genes. However, a major problem is that the foreign genes in recombinant PRV are only integrated into the genome for independent expression, rather than assembled on the surface of virion. Methods: We reported a recombinant PRV with deleted gE/TK genes and an inserted porcine circovirus virus 2 (PCV2) Cap gene into the extracellular domain of the PRV gE gene using the Cre-loxP recombinant system combined with the CRISPR-Cas9 gene editing system. This recombinant PRV (PRV-Cap), with the envelope-embedded Cap protein, exhibits a similar replication ability to its parental virus. Results: An immunogenicity assay revealed that PRV-Cap immunized mice have 100% resistance to lethal PRV and PCV2 attacks. Neutralization antibody and ELISPOT detections indicated that PRV-Cap can enhance neutralizing antibodies to PRV and produce IFN-γ secreting T cells specific for both PRV and PCV2. Immunological mechanistic investigation revealed that initial immunization with PRV-Cap stimulates significantly early activation and expansion of CD69+ T cells, promoting the activation of CD4 Tfh cell dependent germinal B cells and producing effectively specific effector memory T and B cells. Booster immunization with PRV-Cap recalled the activation of PRV-specific IFN-γ+IL-2+CD4+ T cells and IFN-γ+TNF-α+CD8+ T cells, as well as PCV2-specific IFN-γ+TNF-α+CD8+ T cells. Conclusion: Collectively, our data suggested an immunological mechanism in that the recombinant PRV with envelope-assembled PCV2 Cap protein can serve as an excellent vaccine candidate for combined immunity against PRV and PCV2, and provided a cost-effective method for the production of PRV- PCV2 vaccine.

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

IMPORTANCE: 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. OBJECTIVE: This study examined the function of EP0 to provide a direction for its in-depth analysis. METHODS: 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. RESULTS: 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. CONCLUSIONS AND RELEVANCE: 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.

The inhibitory effect of swine TAB1 on the replication of pseudorabies virus.

TAK1-binding protein 1 (TAB1) assembles with TAK1 through its C-terminal domain, leading to the self-phosphorylation and activation of TAK1, which plays an important role in the activation of NF-κB and MAPK signaling pathway. Pseudorabies virus (PRV) is the pathogen of Pseudorabies (PR), which belongs to the Alphaherpesvirus subfamily and causes serious economic losses to the global pig industry. However, the impact of swine TAB1 (sTAB1) on PRV infection has not been reported. In this study, evidence from virus DNA copies, virus titer and western blotting confirmed that sTAB1 could inhibit PRV replication and knockout of sTAB1 by CRISPR-Cas9 gene editing system could promote PRV replication. Further mechanistic studies by real-time PCR and luciferase reporter gene assay demonstrated that sTAB1 could enhance the production of inflammatory factors and chemokines, IFN-ß transcription level and IFN-ß promoter activity after PRV infection. In summary, we clarify the underlying mechanism of sTAB1 in inhibiting PRV replication for the first time, which provides a new idea for preventing PRV infection and lays a foundation for PRV vaccine development.

HIF-1 Transcriptionally Regulates Basal Expression of STING to Maintain Cellular Innate Immunity.

Stimulator of IFN genes (STING) is a critical component of the innate immune system, playing an essential role in defending against DNA virus infections. However, the mechanisms governing basal STING regulation remain poorly understood. In this study, we demonstrate that the basal level of STING is critically maintained by hypoxia-inducible factor 1 (HIF-1)α through transcription. Under normal conditions, HIF-1α binds constitutively to the promoter region of STING, actively promoting its transcription. Knocking down HIF-1α results in a decrease in STING expression in multiple cell lines and zebrafish, which in turn reduces cellular responses to synthetic dsDNAs, including cell signaling and IFN production. Moreover, this decrease in STING levels leads to an increase in cellular susceptibility to DNA viruses HSV-1 and pseudorabies virus. These findings unveil a (to our knowledge) novel role of HIF-1α in maintaining basal STING levels and provide valuable insights into STING-mediated antiviral activities and associated diseases.

Rapid, sensitive, and visual detection of pseudorabies virus with an RPA-CRISPR/EsCas13d-based dual-readout portable platform.

Pseudorabies viruses (PRV) pose a major threat to the global pig industry and public health. Rapid, intuitive, affordable, and accurate diagnostic testing is critical for controlling and eradicating infectious diseases. In this study, a portable detection platform based on RPA-CRISPR/EsCas13d was developed. The platform exhibits high sensitivity (1 copy/µL), good specificity, and no cross-reactivity with common pathogens. The platform uses rapid preamplification technology to provide visualization results (lateral flow assays or visual fluorescence) within 1 h. Fifty pig samples (including tissues, oral fluids, and serum) were tested using this platform and real-time quantitative polymerase chain reaction (qPCR), showing 34.0 % (17 of 50) PRV positivity with the portable CRISPR/EsCas13d dual-readout platform, consistent with the qPCR results. These results highlight the stability, sensitivity, efficiency, and low equipment requirements of the portable platform. Additionally, a novel point-of-care test is being developed for clinical use in remote rural and resource-limited areas, which could be a prospective measure for monitoring the progression of pseudorabies and other infectious diseases worldwide.

An atlas of brain-bone sympathetic neural circuits in mice.

There is clear evidence that the sympathetic nervous system (SNS) mediates bone metabolism. Histological studies show abundant SNS innervation of the periosteum and bone marrow-these nerves consist of noradrenergic fibers that immunostain for tyrosine hydroxylase, dopamine beta-hydroxylase, or neuropeptide Y. Nonetheless, the brain sites that send efferent SNS outflow to the bone have not yet been characterized. Using pseudorabies (PRV) viral transneuronal tracing, we report, for the first time, the identification of central SNS outflow sites that innervate bone. We find that the central SNS outflow to bone originates from 87 brain nuclei, sub-nuclei, and regions of six brain divisions, namely the midbrain and pons, hypothalamus, hindbrain medulla, forebrain, cerebral cortex, and thalamus. We also find that certain sites, such as the raphe magnus (RMg) of the medulla and periaqueductal gray (PAG) of the midbrain, display greater degrees of PRV152 infection, suggesting that there is considerable site-specific variation in the levels of central SNS outflow to the bone. This comprehensive compendium illustrating the central coding and control of SNS efferent signals to bone should allow for a greater understanding of the neural regulation of bone metabolism, and importantly and of clinical relevance, mechanisms for central bone pain.

Pseudorabies virus tegument protein US2 antagonizes antiviral innate immunity by targeting cGAS-STING signaling pathway.

Background: The cGAS-STING axis-mediated type I interferon pathway is a crucial strategy for host defense against DNA virus infection. Numerous evasion strategies developed by the pseudorabies virus (PRV) counteract host antiviral immunity. To what extent PRV-encoded proteins evade the cGAS-STING signaling pathway is unknown. Methods: Using US2 stably expressing cell lines and US2-deficient PRV model, we revealed that the PRV tegument protein US2 reduces STING protein stability and downregulates STING-mediated antiviral signaling. Results: To promote K48-linked ubiquitination and STING degradation, US2 interacts with the LBD structural domain of STING and recruits the E3 ligase TRIM21. TRIM21 deficiency consistently strengthens the host antiviral immune response brought on by PRV infection. Additionally, US2-deficient PRV is less harmful in mice. Conclusions: Our study implies that PRV US2 inhibits IFN signaling by a new mechanism that selectively targets STING while successfully evading the host antiviral response. As a result, the present study reveals a novel strategy by which PRV evades host defense and offers explanations for why the Bartha-K61 classical vaccine strain failed to offer effective defense against PRV variant strains in China, indicating that US2 may be a key target for developing gene-deficient PRV vaccines.

Leveraging biotin-based proximity labeling to identify cellular factors governing early alphaherpesvirus infection.

Neurotropic alphaherpesviruses, including herpes simplex virus type 1 and pseudorabies virus, establish a lifelong presence within the peripheral nervous system of their mammalian hosts. Upon entering cells, two conserved tegument proteins, pUL36 and pUL37, traffic DNA-containing capsids to nuclei. These proteins support long-distance retrograde axonal transport and invasion of the nervous system in vivo. To better understand how pUL36 and pUL37 function, recombinant viral particles carrying BioID2 fused to these proteins were produced to biotinylate cellular proteins in their proximity (<10 nm) during infection. Eighty-six high-confidence host proteins were identified by mass spectrometry and subsequently targeted by CRISPR-Cas9 gene editing to assess their contributions to early infection. Proteins were identified that both supported and antagonized infection in immortalized human epithelial cells. The latter included zyxin, a protein that localizes to focal adhesions and regulates actin cytoskeletal dynamics. Zyxin knockout cells were hyper-permissive to infection and could be rescued with even modest expression of GFP-zyxin. These results provide a resource for studies of the virus-cell interface and identify zyxin as a novel deterrent to alphaherpesvirus infection.IMPORTANCENeuroinvasive alphaherpesviruses are highly prevalent with many members found across mammals [e.g., herpes simplex virus type 1 (HSV-1) in humans and pseudorabies virus in pigs]. HSV-1 causes a range of clinical manifestations from cold sores to blindness and encephalitis. There are no vaccines or curative therapies available for HSV-1. A fundamental feature of these viruses is their establishment of lifelong infection of the nervous system in their respective hosts. This outcome is possible due to a potent neuroinvasive property that is coordinated by two proteins: pUL36 and pUL37. In this study, we explore the cellular protein network in proximity to pUL36 and pUL37 during infection and examine the impact of knocking down the expression of these proteins upon infection.

The global landscapes of lysine crotonylation in pseudorabies virus infection.

Lysine crotonylation is a common occurrence in eukaryotic cells, regulating various physiological functions, including chromatin remodeling, cellular growth, and development. However, its involvement in viral infections has rarely been documented. In this study, we reveal that pseudorabies virus (PRV) infection significantly alters the global lysine crotonylation levels in porcine kidney PK-15 cells. Specifically, we identified a few viral proteins, including UL54, gM, gD, UL19, UL37, and UL46, which undergo crotonylation modification. Our observations indicate that at 20 h post-infection (hpi), 551 crotonylation sites were reduced across 345 proteins, while 47 new sites emerged in 37 proteins compared to the control group. By 40 hpi, 263 sites had decreased in 190 proteins, while 389 new sites appeared in 240 proteins. Deeper analysis revealed that the proteins with altered crotonylation levels were primarily involved in binding, catalytic activity, biosynthetic processes, ribosome activity, and metabolic processes. Additionally, our findings underscored the significance of ribosomes and the endoplasmic reticulum (ER), which were enriched with proteins exhibiting altered crotonylation. Overall, our study for the first time offers new insights into the relationship between crotonylation and herpes virus infection, paving the way for future investigations into the role of crotonylation in viral infections.

Novel Multiparametric Bioelectronic Measurement System for Monitoring Virus-Induced Alterations in Functional Neuronal Networks.

Development and optimisation of bioelectronic monitoring techniques like microelectrode array-based field potential measurement and impedance spectroscopy for the functional, label-free and non-invasive monitoring of in vitro neuronal networks is widely investigated in the field of biosensors. Thus, these techniques were individually used to demonstrate the capabilities of, e.g., detecting compound-induced toxicity in neuronal culture models. In contrast, extended application for investigating the effects of central nervous system infecting viruses are rarely described. In this context, we wanted to analyse the effect of herpesviruses on functional neuronal networks. Therefore, we developed a unique hybrid bioelectronic monitoring platform that allows for performing field potential monitoring and impedance spectroscopy on the same microelectrode. In the first step, a neuronal culture model based on primary hippocampal cells from neonatal rats was established with reproducible and stable synchronised electrophysiological network activity after 21 days of cultivation on microelectrode arrays. For a proof of concept, the pseudorabies model virus PrV Kaplan-ΔgG-GFP was applied and the effect on the neuronal networks was monitored by impedance spectroscopy and field potential measurement for 72 h in a multiparametric mode. Analysis of several bioelectronic parameters revealed a virus concentration-dependent degeneration of the neuronal network within 24-48 h, with a significant early change in electrophysiological activity, subsequently leading to a loss of activity and network synchronicity. In conclusion, we successfully developed a microelectrode array-based hybrid bioelectronic measurement platform for quantitative monitoring of pathologic effects of a herpesvirus on electrophysiological active neuronal networks.

MIL-53(Al)-oil/water emulsion composite as an adjuvant promotes immune responses to an inactivated pseudorabies virus vaccine in mice and pigs.

Although vaccination with inactivated vaccines is a popular preventive method against pseudorabies virus (PRV) infection, inactivated vaccines have poor protection efficiency because of their weak immunogenicity. The development of an effective adjuvant is urgently needed to improve the efficacy of inactivated PRV vaccines. In this study, a promising nanocomposite adjuvant named as MIL@A-SW01-C was developed by combining polyacrylic acid-coated metal-organic framework MIL-53(Al) (MIL@A) and squalene (oil)-in-water emulsion (SW01) and then mixing it with a carbomer solution. One part of the MIL@A was loaded onto the oil/water interface of SW01 emulsion via hydrophobic interaction and coordination, while another part was dispersed in the continuous water phase using carbomer. MIL@A-SW01-C showed good biocompatibility, high PRV (antigen)-loading capability, and sustained antigen release. Furthermore, the MIL@A-SW01-C adjuvanted PRV vaccine induced high specific serum antibody titers, increased splenocyte proliferation and cytokine secretion, and a more balanced Th1/Th2 immune response compared with commercial adjuvants, such as alum and biphasic 201. In the mouse challenge experiment, two- and one-shot vaccinations resulted in survival rates of 73.3 % and 86.7 %, respectively. After one-shot vaccination, the host animal pigs were also challenged with wild PRV. A protection rate of 100 % was achieved, which was much higher than that observed with commercial adjuvants. This study not only establishes the superiority of MIL@A-SW01-C composite nanoadjuvant for inactivated PRV vaccine in mice and pigs but also presents an effective method for developing promising nanoadjuvants. STATEMENT OF SIGNIFICANCE: We have developed a nanocomposite of MIL-53(Al) and oil-in-water emulsion (MIL@A-SW01-C) as a promising adjuvant for the inactivated PRV vaccines. MIL@A-SW01-C has good biocompatibility, high PRV (antigen) loading capability, and prolonged antigen release. The developed nanoadjuvant induced much higher specific IgG antibody titers, increased splenocyte proliferation and cytokine secretion, and a more balanced Th1/Th2 immune response than commercial adjuvants alum and biphasic 201. In mouse challenge experiments, survival rates of 73.3 % and 86.7 % were achieved from two-shot and one-shot vaccinations, respectively. At the same time, a protection rate of 100 % was achieved with the host animal pigs challenged with wild PRV.

Neuronal expression of herpes simplex virus-1 VP16 protein induces pseudorabies virus escape from silencing and reactivation.

Alpha herpesvirus (α-HV) particles enter their hosts from mucosal surfaces and efficiently maintain fast transport in peripheral nervous system (PNS) axons to establish infections in the peripheral ganglia. The path from axons to distant neuronal nuclei is challenging to dissect due to the difficulty of monitoring early events in a dispersed neuron culture model. We have established well-controlled, reproducible, and reactivateable latent infections in compartmented rodent neurons by infecting physically isolated axons with a small number of viral particles. This system not only recapitulates the physiological infection route but also facilitates independent treatment of isolated cell bodies or axons. Consequently, this system enables study not only of the stimuli that promote reactivation but also the factors that regulate the initial switch from productive to latent infection. Adeno-associated virus (AAV)-mediated expression of herpes simplex-1 (HSV-1) VP16 alone in neuronal cell bodies enabled the escape from silencing of incoming pseudorabies virus (PRV) genomes. Furthermore, the expression of HSV VP16 alone reactivated a latent PRV infection in this system. Surprisingly, the expression of PRV VP16 protein supported neither PRV escape from silencing nor reactivation. We compared transcription transactivation activity of both VP16 proteins in primary neurons by RNA sequencing and found that these homolog viral proteins produce different gene expression profiles. AAV-transduced HSV VP16 specifically induced the expression of proto-oncogenes including c-Jun and Pim2. In addition, HSV VP16 induces phosphorylation of c-Jun in neurons, and when this activity is inhibited, escape of PRV silencing is dramatically reduced.IMPORTANCEDuring latency, alpha herpesvirus genomes are silenced yet retain the capacity to reactivate. Currently, host and viral protein interactions that determine the establishment of latency, induce escape from genome silencing or reactivation are not completely understood. By using a compartmented neuronal culture model of latency, we investigated the effect of the viral transcriptional activator, VP16 on pseudorabies virus (PRV) escape from genome silencing. This model recapitulates the physiological infection route and enables the study of the stimuli that regulate the initial switch from a latent to productive infection. We investigated the neuronal transcriptional activation profiles of two homolog VP16 proteins (encoded by HSV-1 or PRV) and found distinct gene activation signatures leading to diverse infection outcomes. This study contributes to understanding of how alpha herpesvirus proteins modulate neuronal gene expression leading to the initiation of a productive or a latent infection.

Yeast ß-glucan promotes antiviral type I interferon response via dectin-1.

Pseudorabies virus (PRV), an alphaherpesvirus, is a neglected zoonotic pathogen. Dectin-1 sensing of ß-glucan (BG) induces trained immunity, which can possibly form a new strategy for the prevention of viral infection. However, alphaherpesvirus including PRV have received little to no investigation in the context of trained immunity. Here, we found that BG pretreatment improved the survival rate, weight loss outcomes, alleviated histological injury and decreased PRV copy number of tissues in PRV-infected mice. Type I interferons (IFNs) including IFN-α/ß levels in serum were significantly increased by BG. However, these effects were abrogated in the presence of Dectin-1 antagonist. Dectin-1-mediated effect of BG was also confirmed in porcine and murine macrophages. These results suggested that BG have effects on type I IFNs with antiviral property involved in Dectin-1. In piglets, oral or injected immunization with BG and PRV vaccine could significantly elevated the level of PRV-specific IgG and type I IFNs. And it also increased the antibody levels of porcine reproductive and respiratory syndrome virus vaccine and classical swine fever vaccine that were later immunized, indicating a broad-spectrum effect on improving vaccine immunity. On the premise that the cost was greatly reducing, the immunological effect of oral was better than injection administration. Our findings highlighted that BG induced type I IFNs related antiviral effect against PRV involved in Dectin-1 and potential application value as a feed additive to help control the spread of PRV and future emerging viruses.

MARCH1 and MARCH2 inhibit pseudorabies virus replication by trapping the viral cell-to-cell fusion complex in trans-Golgi network.

The membrane-associated RING-CH (MARCH) family of proteins are members of the E3 ubiquitin ligase family and are essential for a variety of biological functions. Currently, MARCH proteins are discovered to execute antiviral functions by directly triggering viral protein degradation or blocking the furin cleavage of viral class I fusion proteins. Here, we report a novel antiviral mechanism of MARCH1 and MARCH2 (MARCH1/2) in the replication of Pseudorabies virus (PRV), a member of the Herpesviridae family. We discovered MARCH1/2 restrict PRV replication at the cell-to-cell fusion step. Furthermore, MARCH1/2 block gB cleavage, and this is dependent on their E3 ligase activity. Interestingly, the blocking of gB cleavage by MARCH1/2 does not contribute to their antiviral activity in vitro. We discovered that MARCH1/2 are associated with the cell-to-cell fusion complex of gB, gD, gH, and gL and trap these viral proteins in the trans-Golgi network (TGN) rather than degrading them. Overall, we conclude that MARCH1/2 inhibit PRV by trapping the viral cell-to-cell fusion complex in TGN.

The heat shock protein DNAJB8 inhibits pseudorabies virus replication by autophagy.

Pseudorabies virus (PRV) effectively utilizes numerous host proteins and pathways to establish a successful infection. Consequently, it becomes imperative to investigate novel host factors implicated in viral infections to gain a deeper understanding of PRV pathogenesis. In this study, we reveal that the host heat shock protein, DNAJB8, functions as a negative regulator in PRV replication. Our findings indicated that both mRNA and protein levels of DNAJB8 were downregulated in cells infected with PRV. Further analysis demonstrated that overexpressing DNAJB8 suppressed PRV replication, whereas its knockdown enhanced viral replication. From a mechanistic perspective, DNAJB8 promoted cellular autophagy, subsequently impeding viral replication. Additionally, we discovered that the transcription factor SOX30 regulated DNAJB8 expression, thereby influencing viral replication. Collectively, these findings enhance our comprehension of the roles played by DNAJB8 and SOX30 in viral replication, broadening our knowledge of virus-host interactions.

MARCH8 inhibits pseudorabies virus replication by trapping the viral cell-to-cell fusion complex in the trans-Golgi network.

The membrane-associated RING-CH 8 protein (MARCH8), a member of the E3 ubiquitin ligase family, has broad-spectrum antiviral activity. However, some viruses hijack MARCH8 to promote virus replication, highlighting its dual role in the viral lifecycle. Most studies on MARCH8 have focused on RNA viruses, leaving its role in DNA viruses largely unexplored. Pseudorabies virus (PRV) is a large DNA virus that poses a potential threat to humans. In this study, we found that MARCH8 inhibited PRV replication at the cell-to-cell fusion stage. Interestingly, our findings proved that MARCH8 blocks gB cleavage by recruiting furin but this activity does not inhibit viral infection in vitro. Furthermore, we confirmed that MARCH8 inhibits cell-to-cell fusion independent of its E3 ubiquitin ligase activity but dependent on the interaction with the cell-to-cell fusion complex (gB, gD, gH, and gL). Finally, we discovered that the distribution of the cell-to-cell fusion complex is significantly altered and trapped within the trans-Golgi network. Overall, our results indicate that human MARCH8 acts as a potent antiviral host factor against PRV via trapping the cell-to-cell fusion complex in the trans-Golgi network.

Rehmmannia glutinosa polysaccharide exerts antiviral activity against pseudorabies virus and antioxidant activity.

Pseudorabies virus (PRV) is an important pathogen harming the global pig industry. Vaccines available for swine cannot protect against PRV completely. Furthermore, no antiviral drugs are available to treat PRV infections. Rehmmannia glutinosa polysaccharide (RGP) possesses several medicinal properties. However, its antiviral activity is not reported. In the present study, we found that RGP can inhibit PRV/XJ5 infection by western blotting, immunofluorescent assay (IFA), and TCID50 assay quantitative polymerase chain reaction (qPCR). We revealed RGP can inhibit virus adsorption and invasion into PK-15 cells in a dose-dependent manner via western blotting, IFA, TCID50 assay, and quantitative polymerase chain reaction (qPCR), and suppressed PRV/XJ5 replication through western blotting, and qPCR. Additionally, it also reduced PRV/XJ5-induced ROS, lipid oxidation, and improved SOD levels in PK-15 cells, which was observed by using corresponding test kits. To conclude, our findings suggest that RGP might be a novel therapeutic agent for preventing and controlling PRV infection and antioxidant agent.

Isolation, structural determination, and antiviral activities of a novel alanine-conjugated polyketide from Talaromyces sp.

Antiviral agents are highly sought after. In this study, a novel alkylated decalin-type polyketide, alaspelunin, was isolated from the culture broth of the fungus Talaromyces speluncarum FMR 16671, and its structure was determined using spectroscopic analyses (1D/2D NMR and MS). The compound was condensed with alanine, and its absolute configuration was determined using Marfey's method. Furthermore, the antiviral activity of alaspelunin against various viruses was evaluated, and it was found to be effective against both severe acute respiratory syndrome coronavirus 2 and pseudorabies (Aujeszky's disease) virus, a pathogen affecting pigs. Our results suggest that this compound is a potential broad-spectrum antiviral agent.