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.

Long Noncoding RNA AROD Inhibits Host Antiviral Innate Immunity via the miR-324-5p-CUEDC2 Axis.

Long noncoding RNAs (lncRNAs) are a class of noncoding RNAs that are involved in multiple biological processes. Here, we report a mechanism through which the lnc-AROD-miR-324-5p-CUEDC2 axis regulates the host innate immune response, using influenza A virus (IAV) as a model. We identified that host lnc-AROD without protein-coding capability is composed of 975 nucleotides. Moreover, lnc-AROD inhibited interferon-ß expression, as well as interferon-stimulated genes ISG15 and MxA. Furthermore, in vivo assays confirmed that lnc-AROD overexpression increased flu virus pathogenicity and mortality in mice. Mechanistically, lnc-AROD interacted with miR-324-5p, leading to decreased binding of miR-324-5p to CUEDC2. Collectively, our findings demonstrated that lnc-AROD is a critical regulator of the host antiviral response via the miR-324-5p-CUEDC2 axis, and lnc-AROD functions as competing endogenous RNA. Our results also provided evidence that lnc-AROD serves as an inhibitor of the antiviral immune response and may represent a potential drug target. IMPORTANCE lnc-AROD is a potential diagnostic and discriminative biomarker for different cancers. However, so far the mechanisms of lnc-AROD regulating virus replication are not well understood. In this study, we identified that lnc-AROD is downregulated during RNA virus infection. We demonstrated that lnc-AROD enhanced CUEDC2 expression, which in turn inhibited innate immunity and favored IAV replication. Our studies indicated that lnc-AROD functions as a competing endogenous RNA that binds miR-324-5p and reduces its inhibitory effect on CUEDC2. Taken together, our findings reveal that lnc-AROD plays an important role during the host antiviral immune response.

Mitochondrial ROS driven by NOX4 upregulation promotes hepatocellular carcinoma cell survival after incomplete radiofrequency ablation by inducing of mitophagy via Nrf2/PINK1.

BACKGROUND: The recurrence of hepatocellular carcinoma (HCC) after radiofrequency ablation (RFA) remains a major clinical problem. Cells that survive the sublethal heat stress that is induced by incomplete RFA are the main source of HCC relapse. Heat stress has long been reported to increase intracellular reactive oxygen species (ROS) generation. Although ROS can induce apoptosis, a pro-survival effect of ROS has also been demonstrated. However, the role of ROS in HCC cells exposed to sublethal heat stress remains unclear. METHODS: HepG2 and HuH7 cells were used for this experiment. Insufficient RFA was performed in cells and in a xenograft model. ROS and antioxidant levels were measured. Apoptosis was analyed by Annexin-V/PI staining and flow cytometry. Protein expression was measured using western blotting. Colocalization of lysosomes and mitochondria was analyzed to assess mitophagy. Corresponding activators or inhibitors were applied to verify the function of specific objectives. RESULTS: Here,we showed that sublethal heat stress induced a ROS burst, which caused acute oxidative stress. This ROS burst was generated by mitochondria, and it was initiated by upregulated NOX4 expression in the mitochondria. N-acetylcysteine (NAC) decreased HCC cell survival under sublethal heat stress conditions in vivo and in vitro. NOX4 triggers the production of mitochondrial ROS (mtROS), and NOX4 inhibitors or siNOX4 also decreased HCC cell survival under sublethal heat stress conditions in vitro. Increased mtROS trigger PINK1-dependent mitophagy to eliminate the mitochondria that are damaged by sublethal heat stress and to protect cells from apoptosis. Nrf2 expression was elevated in response to this ROS burst and mediated the ROS burst-induced increase in PINK1 expression after sublethal heat stress. CONCLUSION: These data confirmed that the ROS burst that occurs after iRFA exerted a pro-survival effect. NOX4 increased the generation of ROS by mitochondria. This short-term ROS burst induced PINK1-dependent mitophagy to eliminate damaged mitochondria by increasing Nrf2 expression.

Extraction of Keratin from Pig Nails and Electrospinning of Keratin/Nylon6 Nanofibers for Copper (II) Adsorption.

In this study, keratins were extracted from pig nail waste via the reduction method for the first time, using L-cysteine as the reductant and urea as the lytic agent. Nylon6 and pig nail keratin were successfully combined via electrospinning to generate a series of nylon6/pig nail keratin nanofibers with a variety of keratin concentrations (0% to 8%, w/w). From the results, it was found that the best concentration was 6% (w/w). The morphologies of the electrospun nanofibers were examined via scanning electron microscopy (SEM) and transmission electron microscopy (TEM). The structural properties were characterized using Fourier transform infrared spectroscopy (FTIR) and X-ray diffraction (XRD), and the thermal properties were described using thermo-gravimetric analysis (TGA). These results confirmed that the nanofibers were composed of both polymeric phases. Finally, copper (II) was used as a model ion, and the nanofiber membranes exhibited a strong adsorption affinity for metal ions in the water samples. This study provides an important foundation for the application of nanofiber membranes in metal adsorption.

Arsenic-Loaded Biomimetic Iron Oxide Nanoparticles for Enhanced Ferroptosis-Inducing Therapy of Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) has a poor response to most available systemic therapies due to intrinsic or acquired resistance to apoptosis. Ferroptosis-based therapy is expected to circumvent those limitations. Therefore, the rational design of ferroptosis-based therapies with targeted delivery of ferroptosis inducers for HCC is in need. In this study, we found that arsenic trioxide (ATO) can efficiently induce ferroptosis in HCC cells, and this effect could be reversed by the iron chelator deferoxamine. On this basis, a drug delivery system was constructed to enhance the therapeutic efficacy of ATO by camouflaging ATO-loaded magnetic nanoparticles (Fe3O4) with HCC cell membranes, termed AFN@CM. After AFN@CM treatment, glutathione peroxidase 4 was strongly inhibited and intracellular lipid peroxide species were significantly increased in HCC cells. In addition, enhanced ferroptosis and tumor suppression were observed both in vitro and in vivo. The bio-safety of AFN@CM was also demonstrated by the in vivo toxicity evaluation. In addition, benefiting from the cell membrane coating, AFN@CM showed enhanced accumulation at tumor sites and achieved continuous tumor elimination in the mouse model. In conclusion, AFN@CM exhibited satisfactory therapeutic efficacy in the treatment of HCC and provided a desirable ferroptosis-based strategy for safe and reliable HCC therapeutics.

Keratin-Based Composite Bioactive Films and Their Preservative Effects on Cherry Tomato.

In this study, keratins were extracted from pig nail waste through the reduction method using L-cysteine as a reductant. Curcumin was successively incorporated in a mixed solution including keratin, gelatin, and glycerin to prepare different kinds of keratin/gelatin/glycerin/curcumin composite films. The morphology of the keratin/ gelatin/glycerin/curcumin composite films were examined using scanning electron microscopy. The structures and the molecular interactions between curcumin, keratin, and pectin were examined using Fourier transform infrared spectroscopy and X-ray diffraction, and the thermal properties were determined through thermogravimetric analysis. The tensile strengths of keratin/gelatin/glycerin/curcumin and keratin/gelatin/curcumin composite films are 13.73 and 12.45 MPa, respectively, and their respective elongations at break are 56.7% and 4.6%. In addition, compared with the control group (no film wrapped on the surface of tomato), the ratio of weight loss of the keratin (7.0%)/gelatin (10%)/glycerin (2.0%)/curcumin (1.0%) experimental groups is 8.76 ± 0.2%, and the hardness value of the tomatoes wrapped with composite films is 11.2 ± 0.39 kg/cm3. Finally, the composite films have a superior antibacterial effect against Staphylococcus aureus and Escherichia coli because of the addition of curcumin. As the concentration of curcumin reaches 1.0%, the antibacterial activity effect of the film is significantly improved. The diameter of the inhibition zone of E. coli is (12.16 ± 0.53) mm, and that of S. aureus is (14.532 ± 0.97) mm. The multifunctional keratin/gelatin/glycerin/curcumin bioactive films have great potential application in the food packaging industry.

Coronaviruses Nsp5 Antagonizes Porcine Gasdermin D-Mediated Pyroptosis by Cleaving Pore-Forming p30 Fragment.

Coronaviruses (CoVs) are a family of RNA viruses that typically cause respiratory, enteric, and hepatic diseases in animals and humans. Here, we use porcine epidemic diarrhea virus (PEDV) as a model of CoVs to illustrate the reciprocal regulation between CoV infection and pyroptosis. For the first time, we elucidate the molecular mechanism of porcine gasdermin D (pGSDMD)-mediated pyroptosis and demonstrate that amino acids R238, T239, and F240 within pGSDMD-p30 are critical for pyroptosis. Furthermore, 3C-like protease Nsp5 from SARS-CoV-2, MERS-CoV, PDCoV, and PEDV can cleave pGSDMD at the Q193-G194 junction to produce two fragments unable to trigger pyroptosis. The two cleaved fragments could not inhibit PEDV replication. In addition, Nsp5 from SARS-CoV-2 and MERS-CoV also cleave human GSDMD (hGSDMD). Therefore, we provide clear evidence that PEDV may utilize the Nsp5-GSDMD pathway to inhibit pyroptosis and, thus, facilitate viral replication during the initial period, suggesting an important strategy for the coronaviruses to sustain their infection. IMPORTANCE Recently, GSDMD has been reported as a key executioner for pyroptosis. This study first demonstrates the molecular mechanism of pGSDMD-mediated pyroptosis and that the pGSDMD-mediated pyroptosis protects host cells against PEDV infection. Notably, PEDV employs its Nsp5 to directly cleave pGSDMD in favor of its replication. We found that Nsp5 proteins from other coronaviruses, such as porcine deltacoronavirus, severe acute respiratory syndrome coronavirus 2, and Middle East respiratory syndrome coronavirus, also had the protease activity to cleave human and porcine GSDMD. Thus, we provide clear evidence that the coronaviruses might utilize Nsp5 to inhibit the host pyroptotic cell death and facilitate their replication during the initial period, an important strategy for their sustaining infection. We suppose that GSDMD is an appealing target for the design of anticoronavirus therapies.

A Previously Undiscovered Circular RNA, circTNFAIP3, and Its Role in Coronavirus Replication.

Circular RNAs (circRNAs) are a newly discovered class of noncoding RNAs (ncRNAs) present in various tissues and cells. However, the functions of most circRNAs have not been verified experimentally. Here, using deltacoronavirus as a model, differentially expressed circRNAs in cells with or without deltacoronavirus infection were analyzed by RNA sequencing to characterize the cellular responses to RNA virus infection. More than 57,000 circRNA candidates were detected, and seven significantly dysregulated circRNAs were quantitated by real-time reverse transcription-PCR. We discovered a previously unidentified circRNA derived from the TNFAIP3 gene, named circTNFAIP3, which is distributed and expressed widely in various tissues. RNA viruses, including deltacoronaviruses, rather than DNA viruses tend to activate the expression of endogenous circTNFAIP3. Overexpression of circTNFAIP3 promoted deltacoronavirus replication by reducing the apoptosis, while silencing of circTNFAIP3 inhibited deltacoronavirus replication by enhancing the apoptosis. In summary, our work provides useful circRNA-related information to facilitate investigation of the underlying mechanism of deltacoronavirus infection and identifies a novel circTNFAIP3 that promotes deltacoronavirus replication via regulating apoptosis. IMPORTANCE CircRNAs, a new class of ncRNAs, play important roles in cell growth, neural development, carcinogenesis, and anticarcinogenesis. Porcine deltacoronavirus is an emerging enteropathogenic coronavirus that causes diarrhea, but the role of host circRNAs in regulating its infection is unknown. Here, we performed expression profiling of circRNAs in mock- and deltacoronavirus- infected cells and identified the novel differentially expressed circular RNA circTNFAIP3. We demonstrate that circTNFAIP3 promotes deltacoronavirus replication by inhibiting apoptosis. Our findings first illustrate that circRNA can act as an apoptosis negative regulator during RNA virus infection and help to explore the underlying mechanism of deltacoronavirus infection.

Purification, characterization, antioxidant and immunological activity of polysaccharide from Sagittaria sagittifolia L.

As a healthy food and traditional Chinese medicine, sagittaria sagittifolia L. has been used for a long history. Nevertheless, reports on the bioactivity and chemical characterization of S. sagittifolia L. polysaccharides are still very rare. In this paper, ultrasound-assisted method (UAE) was used to extract S. sagittifolia L. polysaccharides, after alcohol precipitation and column chromatography isolation, the structural characteristics, antioxidant and immunological activities of the purified polysaccharide (SPU60-W) were preliminarily investigated. The results indicated SPU60-W (16.62 kDa) was a pyranoid polysaccharide containing α-glycosidic bond composed of mannose, xylose, and glucose with a molar ratio of 2.69: 2.04: 95.27. It consisted of slender wormlike strands, which may involve some degree of aggregation of helices, as well as a small proportion of irregular spherical structures. Furthermore, antioxidant activity analysis showed that SPU60-W possess excellent hydroxyl and ABTS radical scavenging activity comparable to vitamin C (Vc), and moderate DPPH radical scavenging activity. Immunity tests suggested that SPU60-W significantly promoted the proliferation, phagocytosis and NO production of mouse macrophage RAW264.7. According to this study, SPU60-W might be utilized as a potent antioxidant and immunomodulator in food and medicinal industry.

Structure and functional properties of soy protein isolate-lentinan conjugates obtained in Maillard reaction by slit divergent ultrasonic assisted wet heating and the stability of oil-in-water emulsions.

Effects of a novel slit divergent ultrasound (SDU) treatment on soybean protein isolate (SPI)-lentinan conjugates via Maillard reaction was investigated. Besides, the stability of emulsions prepared by SPI and SPI-lentinan conjugates (ultrasound and un-ultrasound) as emulsifiers was compared. The results showed that ultrasonic treatment (40 min) markedly increased the degree of grafting (26.48%) by 1.91 times comparing with traditional heating method (2 h, 13.89%). In addition, structural analysis showed that the conjugates obtained by SDU treatment changed the secondary structure and had higher surface hydrophobicity and fluorescence intensity than those obtained by traditional heating method. Apart from this, SDU treatment could significantly improve the functional properties (solubility, foaming, emulsifying capacity, thermal stability, and viscosity) of conjugates. Furthermore, the emulsions prepared by the SPI-lentinan conjugates (ultrasound) as emulsifiers possessed the highest stability against environmental stresses. Taken together, SDU-assisted heating could be an excellent method to improve the functional properties of conjugates.

Porcine Epidemic Diarrhea Virus Deficient in RNA Cap Guanine-N-7 Methylation Is Attenuated and Induces Higher Type I and III Interferon Responses.

The 5' cap methylation of viral RNA plays important roles in RNA stability, efficient translation, and immune evasion. Thus, RNA cap methylation is an attractive target for antiviral discovery and development of new live attenuated vaccines. For coronaviruses, RNA cap structure is first methylated at the guanine-N-7 (G-N-7) position by nonstructural protein 14 (nsp14), which facilitates and precedes the subsequent ribose 2'-O methylation by the nsp16-nsp10 complex. Using porcine epidemic diarrhea virus (PEDV), an Alphacoronavirus, as a model, we showed that G-N-7 methyltransferase (G-N-7 MTase) of PEDV nsp14 methylated RNA substrates in a sequence-unspecific manner. PEDV nsp14 can efficiently methylate RNA substrates with various lengths in both neutral and alkaline pH environments and can methylate cap analogs (GpppA and GpppG) and single-nucleotide GTP but not ATP, CTP, or UTP. Mutations to the S-adenosyl-l-methionine (SAM) binding motif in the nsp14 abolished the G-N-7 MTase activity and were lethal to PEDV. However, recombinant rPEDV-D350A with a single mutation (D350A) in nsp14, which retained 29.0% of G-N-7 MTase activity, was viable. Recombinant rPEDV-D350A formed a significantly smaller plaque and had significant defects in viral protein synthesis and viral replication in Vero CCL-81 cells and intestinal porcine epithelial cells (IPEC-DQ). Notably, rPEDV-D350A induced significantly higher expression of both type I and III interferons in IPEC-DQ cells than the parental rPEDV. Collectively, our results demonstrate that G-N-7 MTase activity of PEDV modulates viral replication, gene expression, and innate immune responses.IMPORTANCE Coronaviruses (CoVs) include a wide range of important human and animal pathogens. Examples of human CoVs include severe acute respiratory syndrome coronavirus (SARS-CoV-1), Middle East respiratory syndrome coronavirus (MERS-CoV), and the most recently emerged SARS-CoV-2. Examples of pig CoVs include porcine epidemic diarrhea virus (PEDV), porcine deltacoronavirus (PDCoV), and swine enteric alphacoronavirus (SeACoV). There are no vaccines or antiviral drugs for most of these viruses. All known CoVs encode a bifunctional nsp14 protein which possesses ExoN and guanine-N-7 methyltransferase (G-N-7 MTase) activities, responsible for replication fidelity and RNA cap G-N-7 methylation, respectively. Here, we biochemically characterized G-N-7 MTase of PEDV nsp14 and found that G-N-7 MTase-deficient PEDV was defective in replication and induced greater responses of type I and III interferons. These findings highlight that CoV G-N-7 MTase may be a novel target for rational design of live attenuated vaccines and antiviral drugs.

Comparison of characterization, antioxidant and immunological activities of three polysaccharides from Sagittaria sagittifolia L.

To investigate and compare the preliminary structural characteristics and biological activity in vitro of polysaccharides from Sagittaria sagittifolia L. (SSs) by different extration methods, three polysaccharides (SSW, SSU, and SSP) were obtained with hot water, ultrasound-assisted, and subcritical water extraction. Their structural features were elucidated using High Performance Liquid Chromatography (HPLC), Gas Chromatography (GC), Scanning Electron Microscopy (SEM), Infrared Spectroscopy (IR), Atomic Force Microscopy (AFM), Zeta Potential and Congo red methods. Furthermore, the antioxidant activity and immunostimulatory effects were investigated in vitro. Molecular weight and monosaccharide composition analysis exhibited that SSW (2275.0 kDa), SSU (148.7 kDa), and SSP (1984.0 kDa) were heteropolysaccharide with dramatically different monosaccharide species and mole ratios. In addition, SSP exhibited stronger antioxidant activity in vitro and more potent immunomodulatory activity than SSW and SSU. SSP has greater potential to be explored as biologicalagents for use in complementary medicine or functional foods.

Identification of functional lncRNAs in pseudorabies virus type II infected cells.

Long noncoding RNAs (lncRNAs) play important roles in the antiviral responses. However, little is known about the identification and functions of swine lncRNAs in response to pseudorabies virus type II (PRV-II). Here, we detected the expression profiles of host lncRNAs from a wild-type (PRV-II DX) and gE/TK deficient (gE-TK-PRV) PRV-II infected cells. RNA-seq identified 664 differentially expressed (DE) lncRNAs from PRV-DX infected cells, 654 DE lncRNAs from gE-TK-PRV infected cells and 276 DE lncRNAs between PRV-DX and gE-TK-PRV infected cells. The potential functions of the significant differentially expressed (SDE) lncRNAs were involved in interleukin secretion, axon extension and metabolic process based on the gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases. Moreover, the expression patterns of sixteen SDE lncRNAs determined by RT-qPCR exhibited high correlation (r > 0.95) with those by RNA-seq results. Western blotting assay displayed the lncA02830 did not code for protein, and the silencing of lncA02830 could significantly up-regulate the transcription levels of IRF3, IFNß as well as MX1 and inhibit the replication of PRV-II. Taken together, these data highlighted the potentials of lncRNA as targets for antiviral therapy and provided some novel knowledge of the mechanisms underlying the host interaction with PRV-II.

Structural characterization and immunostimulatory activity of a novel polysaccharide isolated with subcritical water from Sagittaria sagittifolia L.

In the present study, we obtained polysaccharides from Sagittaria sagittifolia L. (SSP) with subcritical water extraction (SWE). Two water-soluble polysaccharides (SSP-W1 and SSP-S1) from the acquired SSP were isolated with DEAE-52 and Sephadex G-100. Besides, the structural characteristics and immunostimulatory activity were also investigated. The results showed that both SSP-W1 and SSP-S1 were homogeneous polysaccharides and the molecular weight was 62.03 KDa and 15.2 KDa, respectively. In addition, both SSP-W1 and SSP-S1 are heteropolysaccharides. Moreover, FT-IR analysis showed that SSP-W1 was α-pyranose polysaccharide, while SSP-S1 was a typical ß-pyranose polysaccharide. Congo red staining showed that there was no triple helix structure in both SSP-W1 and SSP-S1. Furthermore, both SSP-W1 and SSP-S1 could promote the proliferation, production of NO, and secretion of TNF-α and IL-10 of macrophages RAW 264.7, significantly. Therefore, the polysaccharides extracted from Sagittaria sagittifolia L. with SWE have the potential to be used as immunoreactive agent in medicine and functional foods.

Effects of subcritical water extraction microenvironment on the structure and biological activities of polysaccharides from Lentinus edodes.

In the present study, we investigated the effects of subcritical water microenvironment on the structure and biological activities of polysaccharides from Lentinus edodes. The results showed that, under different SWE conditions (100-150 °C for 15 min, and 150 °C for 10-30 min), all the polysaccharides were heteropolysaccharides and consisted of glucose, mannose and galactose with different molar ratios. In addition, FT-IR results showed that all samples were typical ß-pyranose and protein-free polysaccharides. Moreover, Congo red staining and atomic force microscope (AFM) analyses showed that when the temperature was above 150 °C for 15 min, the triple-helix structure no longer existed. Furthermore, various polysaccharides had varying degrees of inhibitory effect on HepG2 cells and MCF-7 cells in vitro. These findings indicated that subcritical water microenvironment has an extremely important effect on the structure and bioactivities of polysaccharides. The polysaccharides may be extracted more effectively by changing the subcritical water microenvironment.

Three amino acid substitutions in the NS1 protein change the virus replication of H5N1 influenza virus in human cells.

Influenza A viruses have sophisticated strategies to promote their own replication. Here, we found that three H5N1 influenza viruses display different replication patterns in human A549 and macrophage cells. The HN01 virus displayed poor replication compared to HN021 and JS01. In addition, the HN01 virus was unable to counteract the interferon response and block general gene expression. This capability was restored by three amino acid substitutions on the NS1 protein: K55E, K66E, and C133F, resulting in recovered binding to CPSF30 and decreased interferon response activity. Furthermore, a recombinant HN01 virus expressing either NS1-C133F or the triple mutation replicate with higher titers in human A549 cells and macrophages compared to the parent virus. These three amino acid mutations reveal a new pathway to alter H5N1 virus replication.

BECN1-dependent CASP2 incomplete autophagy induction by binding to rabies virus phosphoprotein.

Autophagy is an essential component of host immunity and used by viruses for survival. However, the autophagy signaling pathways involved in virus replication are poorly documented. Here, we observed that rabies virus (RABV) infection triggered intracellular autophagosome accumulation and results in incomplete autophagy by inhibiting autophagy flux. Subsequently, we found that RABV infection induced the reduction of CASP2/caspase 2 and the activation of AMP-activated protein kinase (AMPK)-AKT-MTOR (mechanistic target of rapamycin) and AMPK-MAPK (mitogen-activated protein kinase) pathways. Further investigation revealed that BECN1/Beclin 1 binding to viral phosphoprotein (P) induced an incomplete autophagy via activating the pathways CASP2-AMPK-AKT-MTOR and CASP2-AMPK-MAPK by decreasing CASP2. Taken together, our data first reveals a crosstalk of BECN1 and CASP2-dependent autophagy pathways by RABV infection.

MicroRNA transcriptome profiling of mice brains infected with Japanese encephalitis virus by RNA sequencing.

Japanese encephalitis (JE) is a mosquito borne viral disease, caused by Japanese encephalitis virus (JEV) infection producing severe neuroinflammation in the central nervous system (CNS) with the associated disruption of the blood brain barrier. MicroRNAs (miRNAs) are a family of 21-24 nt small non-coding RNAs that play important post-transcriptional regulatory roles in gene expression and have critical roles in virus pathogenesis. We examined the potential roles of miRNAs in JEV-infected suckling mice brains and found that JEV infection changed miRNA expression profiles when the suckling mice began showing nervous symptoms. A total of 1062 known and 71 novel miRNAs were detected in JEV-infected group, accompanied with 1088 known and 75 novel miRNAs in mock controls. Among these miRNAs, one novel and 25 known miRNAs were significantly differentially expressed, including 18 up-regulated and 8 down-regulated miRNAs which were further confirmed by real-time PCR. Gene ontology (GO) and signaling pathway analysis of the predicted target mRNAs of the modulated miRNAs showed that they are correlated with the regulation of apoptosis, neuron differentiation, antiviral immunity and infiltration of mouse brain, and the validated targets of 12 differentially expressed miRNAs were enriched for the regulation of cell programmed death, proliferation, transcription, muscle organ development, erythrocyte differentiation, gene expression, plasma membrane and protein domain specific binding. KEGG analysis further reveals that the validated target genes were involved in the Pathways in cancer, Neurotrophin signaling pathway, Toll like receptor signaling pathway, Endometrial cancer and Jak-STAT signaling pathway. We constructed the interaction networks of miRNAs and their target genes according to GO terms and KEGG pathways and the expression levels of several target genes were examined. Our data provides a valuable basis for further studies on the regulatory roles of miRNAs in JE pathogenesis.

In Vitro Coinfection and Replication of Classical Swine Fever Virus and Porcine Circovirus Type 2 in PK15 Cells.

Increasing clinical lines of evidence have shown the coinfection/superinfection of porcine circovirus type 2 (PCV2) and classical swine fever virus (CSFV). Here, we investigated whether PCV2 and CSFV could infect the same cell productively by constructing an in vitro coinfection model. Our results indicated that PCV2-free PK15 cells but not ST cells were more sensitive to PCV2, and the PK15 cell line could stably harbor replicating CSFV (PK15-CSFV cells) with a high infection rate. Confocal and super-resolution microscopic analysis showed that PCV2 and CSFV colocalized in the same PK15-CSFV cell, and the CSFV E2 protein translocated from the cytoplasm to the nucleus in PK15-CSFV cells infected with PCV2. Moreover, PCV2-CSFV dual-positive cells increased gradually in PK15-CSFV cells in a PCV2 dose-dependent manner. In PK15-CSFV cells, PCV2 replicated well, and the production of PCV2 progeny was not influenced by CSFV infection. However, CSFV reproduction decreased in a PCV2 dose-dependent manner. In addition, cellular apoptosis was not strengthened in PK15-CSFV cells infected with PCV2 in comparison with PCV2-infected PK15 cells. Moreover, using this coinfection model we further demonstrated PCV2-induced apoptosis might contribute to the impairment of CSFV HCLV strain replication in coinfected cells. Taken together, our results demonstrate for the first time the coinfection/superinfection of PCV2 and CSFV within the same cell, providing an in vitro model to facilitate further investigation of the underlying mechanism of CSFV and PCV2 coinfection.