DHAV-1 2A1 Peptide – A Newly Discovered Co-expression Tool That Mediates the Ribosomal “Skipping” Function

Duck hepatitis A virus 1 (DHAV-1) belongs to the genus Avihepatovirus in the family Picornaviridae. Little research has been carried out on the non-structural proteins of this virus. This study reports that 2A1 protein, the first non-structural protein on the DHAV-1 genome, has a ribosomal “skipping” function mediated by a “-GxExNPGP-” motif. In addition, we prove that when the sequence is extended 10aa to VP1 from the N-terminal of 2A1, the ribosome “skips” completely. However, as the N-terminus of 2A is shortened, the efficiency of ribosomal “skipping” reduces. When 2A1 is shortened to 10aa, it does not function. In addition, we demonstrate that N18, P19 G20, and P21 have vital roles in this function. We find that the expression of upstream and downstream proteins linked by 2A1 is different, and the expression of the upstream protein is much greater than that of the downstream protein. In addition, we demonstrate that it is the nature of 2A1 that is responsible for the expression imbalance. We also shows that the protein “cleavage” is not due to RNA “cleavage” or RNA transcription abnormalities, and the expressed protein level is independent of RNA transcriptional level. This study provides a systematic analysis of the activity of the DHAV-1 2A1 sequence and, therefore, adds to the “tool-box” that can be deployed for the co-expression applications. It provides a reference for how to apply 2A1 as a co-expression tool

A proposed disease classification system for duck viral hepatitis

The nomenclature of duck viral hepatitis (DVH) was historically not a problem. However, 14 hepatotropic viruses among 10 different genera are associated with the same disease name, DVH. Therefore, the disease name increasingly lacks clarity and may no longer fit the scientific description of the disease. Because one disease should not be attributed to 10 genera of viruses, this almost certainly causes misunderstanding regarding the disease-virus relationship. Herein, we revisited the problem and proposed an update to DVH disease classification. This classification is based on the nomenclature of human viral hepatitis and the key principle of Koch's postulates (“one microbe and one disease”). In total, 10 types of disease names have been proposed. These names were literately matched with hepatitis-related viruses. We envision that this intuitive nomenclature system will facilitate scientific communication and consistent interpretation in this field, especially in the Asian veterinary community, where these diseases are most commonly reported

DHAV-1 Inhibits Type I Interferon Signaling to Assist Viral Adaption by Increasing the Expression of SOCS3

Duck hepatitis A virus type 1 (DHAV-1) is one of the most lethal pathogens in the duck industry. The attenuated vaccine (the CH60 strain) is cultivated through serial passage in chicken embryos and is widely used for the prevention and control of the disease. However, the specific mechanism underlying its adaptation in chicken embryos has not been fully elucidated. In this study, we first infected chicken embryo fibroblasts (CEFs) with the DHAV-1 CH60 strain. The peak of viral proliferation occurred within 36–48 h post-infection. The different DHAV-1 strains significantly induced the expression of IFNα, IFNγ, and Suppressor of cytokine signaling 3 (SOCS3) in CEFs, and we found that SOCS3 overexpression significantly promoted viral replication. Furthermore, SOCS3 overexpression significantly inhibited the expression of IFNα but promoted the expression of IFNγ. In addition, SOCS3 overexpression clearly decreased the mRNA levels of STAT1 and STAT3 in the Janus kinase (JAK)-STAT signaling pathway and inhibited the expression of the antiviral proteins MX1 and OASL. Immune-precipitation assays indicated that SOCS3 and IFNα do not physically interact. Subcellular localization of SOCS3 and IFNα revealed that SOCS3 was mainly located in the nucleus and cytoplasm, while IFNα was located only in the cytoplasm. Co-localization of these two proteins was not observed in the cytoplasm. In conclusion, the DHAV-1 CH60 strain may inhibit the expression of IFNα by increasing the SOCS3 protein and SOCS3 can in turn decrease STAT1 and STAT3 mRNA levels, thereby inhibiting the antiviral protein MX1 and ultimately promoting viral proliferation, indirectly assisting in viral adaptation in chicken embryos

First Report of Integrative Conjugative Elements in Riemerella anatipestifer Isolates From Ducks in China

We report for the first time the occurrence of integrative conjugative elements (ICEs) in Riemerella anatipestifer (R.anatipestifer) isolated from diseased ducks in China. For this purpose, a total of 48 genome sequences were investigated, which comprised 30 publicly available R. anatipestifer genome sequences, and 18 clinical isolates genomes sequences. Two ICEs, named ICERanRCAD0133-1 and ICERanRCAD0179-1 following the classic nomenclature system, were identified in R. anatipestifer through the use of bioinformatics tools. Comparative analysis revealed that three ICEs in Ornithobacterium rhinotracheale showed a high degree of conservation with the core genes of ICERanRCAD0133-1, while 13 ICEs with high similarity to ICERanRCAD0179-1 were found in Bacteroidetes. Based on the definition of ICE family, ICERanRCAD0179-1 was grouped in CTnDOT/ERL family; however, ICERanRCAD0133-1, which had no significant similarity with known ICEs, might be classified into a novel ICE family. The sequences of ICERanRCAD0133-1 and ICERanRCAD0179-1 were 70890 bp and 49166 bp in length, had 33.14 and 50.34% GC content, and contained 77 CDSs and 51 CDSs, respectively. Cargo genes carried by these two ICEs were predicted to encode: R-M systems, IS elements, a putative TonB-dependent receptor, a bacteriocin/lantibiotic efflux ABC transporter, a tetracycline resistance gene and more. In addition, phylogenetic analyses revealed that ICERanRCAD0179-1 and related ICEs were derived from a common ancestor, which may have undergone divergence prior to integartation into the host bacterial chromosome, and that the core genes co-evolved via a related evolutionary process or experienced only a low degree of recombination events during spread from a common CTnDOT/ERL family ancestor. Collectively, this study is the first identification and characterization of ICEs in R. anatipestifer; and provides new insights into the genetic diversity, evolution, adaptation, antimicrobial resistance, and virulence of R. anatipestifer

DprA Is Essential for Natural Competence in Riemerella anatipestifer and Has a Conserved Evolutionary Mechanism

Riemerella anatipestifer ATCC11845 (RA ATCC11845) is naturally competent. However, the genes involved in natural transformation in this species remain largely unknown. Bioinformatic analysis predicts that DprA of RA (DprARa) has three domains: a sterile alpha motif (SAM), a Rossmann fold (RF) domain and a Z-DNA-binding domain (Zα). Inactivation of dprA abrogated natural transformation in RA ATCC11845, and this effect was restored by the expression of dprA in trans. The dprA with SAM and RF domains of Streptococcus pneumoniae and the dprA with RF and Zα domains of Helicobacter pylori was able to restore natural transformation in the RA ATCC11845 dprA mutant. An Arg123 mutation in the RF domain of R. anatipestifer was not able to restore natural transformation of the RA ATCC11845 dprA mutant. Furthermore, DprAR123E abolished its ability to bind DNA, suggesting that the RF domain is essential for the function of DprA. Finally, the dprA of Fusobacterium naviforme which has not been reported to be natural competent currently was partially able to restore natural transformation in RA ATCC11845 dprA mutant. These results collectively suggest that DprA has a conserved evolutionary mechanism

Mechanism of herpesvirus UL24 protein regulating viral immune escape and virulence

Herpesviruses have evolved a series of abilities involved in the process of host infection that are conducive to virus survival and adaptation to the host, such as immune escape, latent infection, and induction of programmed cell death for sustainable infection. The herpesvirus gene UL24 encodes a highly conserved core protein that plays an important role in effective viral infection. The UL24 protein can inhibit the innate immune response of the host by acting on multiple immune signaling pathways during virus infection, and it also plays a key role in the proliferation and pathogenicity of the virus in the later stage of infection. This article reviews the mechanism by which the UL24 protein mediates herpesvirus immune escape and its effects on viral proliferation and virulence by influencing syncytial formation, DNA damage and the cell cycle. Reviewing these studies will enhance our understanding of the pathogenesis of herpesvirus infection and provide evidence for new strategies to combat against viral infection

Simvastatin, a Novel Stimulator of Eryptosis, the Suicidal Erythrocyte Death

Background/Aims: The 3-hydroxy-3-methyl-glutaryl-Coenzyme A (HMG-CoA) reductase inhibitor simvastatin has been shown to trigger apoptosis of several cell types. The substance has thus been proposed as an additional treatment of malignancy. Similar to apoptosis of nucleated cells, erythrocytes may enter eryptosis, the suicidal erythrocyte death. Hallmarks of eryptosis include cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the extracellular face of the erythrocyte cell membrane. Signaling contributing to stimulation of eryptosis include increase of cytosolic Ca2+ activity ([Ca2+]i), induction of oxidative stress, increase of ceramide abundance, and activation of SB203580-sensitive p38 kinase. The present study explored, whether simvastatin induces eryptosis and aimed to shed light on cellular mechanisms involved. Methods: Flow cytometry was employed to quantify phosphatidylserine exposure at the cell surface from annexin-V-binding, cell volume from forward scatter, [Ca2+]i from Fluo3-fluorescence, reactive oxygen species (ROS) abundance from DCFDA dependent fluorescence, and ceramide abundance utilizing specific antibodies. Hemolysis was estimated from hemoglobin concentration in the supernatant. Results: A 48 h exposure of human erythrocytes to simvastatin (1 µg/ml) significantly decreased the forward scatter, significantly augmented the percentage of annexin-V-binding cells, significantly increased Fluo3-fluorescence, and significantly enhanced DCFDA fluorescence. Simvastatin tended to increase ceramide abundance, an effect, however, escaping statistical significance. The effect of simvastatin on annexin-V-binding was significantly blunted by removal of extracellular Ca2+ and by addition of SB203580 (2 µM). Conclusions: Simvastatin stimulates eryptosis, an effect at least in part due to Ca2+ entry, oxidative stress, and p38 kinase

1α,25(OH)2D3 Induces Actin Depolymerization in Endometrial Carcinoma Cells by Targeting RAC1 and PAK1

Background: Cell proliferation and motility require actin reorganization, which is under control of various signalling pathways including ras-related C3 botulinum toxin substrate 1 (RAC1), p21 protein-activated kinase 1 (PAK1) and actin related protein 2 (ARP2). Tumour cell proliferation is modified by 1α,25-Dihydroxy-Vitamin D3 (1α,25(OH)2D3), a steroid hormone predominantly known for its role in calcium and phosphorus metabolism. The present study explored whether 1α,25(OH)2D3 modifies actin cytoskeleton in Ishikawa cells, a well differentiated endometrial carcinoma cell line. Methods: To this end, actin cytoskeleton was visualized by confocal microscopy. Globular over filamentous actin ratio was determined utilizing Western blotting and flow cytometry, transcript levels by qRT-PCR and protein abundance by immunoblotting. Results: A 24 hour treatment with 1α,25(OH)2D3 (100 nM) significantly decreased RAC1 and PAK1 transcript levels and activity, decreased ARP2 protein levels and depolymerized actin. The effect of 1α,25(OH)2D3 on actin polymerization was mimicked by pharmacological inhibition of RAC1 and PAK1. Conclusions: 1α,25(OH)2D3 leads to disruption of RAC1 and PAK1 activity with subsequent actin depolymerization of endometrial carcinoma cells

Data from: Downregulation of endometrial mesenchymal marker SUSD2 causes cell senescence and cell death in endometrial carcinoma cells

The cause of death among the majority of endometrial cancer patients involves migration of cancer cells within the peritoneal cavity and subsequent implantation of cancer spheroids into neighbouring organs. It is, thereby, important to identify factors that mediate metastasis. Cell adhesion and migration are modified by the mesenchymal stem cell (MSC) marker Sushi domain containing 2 (SUSD2), a type I transmembrane protein that participates in the orchestration of cell adhesion and migration through interaction with its partner Galactosidase-binding soluble-1 (LGALS1). MSCs have emerged as attractive targets in cancer therapy. Human endometrial adenocarcinoma (Ishikawa) cells were treated with TGFβ (10ng/ml) for 72h. SUSD2, LGALS1 and MKI67 transcript levels were quantified using qRT-PCR. The proportion of SUSD2 positive (SUSD2+) cells and SMAD2/3 abundance were quantified by FACS and Western blotting, respectively. Senescent cells were identified with β-galactosidase staining; cell cycle and cell death were quantified using Propidium Iodide staining. Treatment of endometrial cancer cells (Ishikawa cells) with TGFβ (10ng/ml) significantly decreased SUSD2 transcript levels and the proportion of SUSD2 positive cells. Silencing of SUSD2 using siRNA resulted in senescence and cell death of Ishikawa cells via activation of SMAD2/3. These findings suggest that SUSD2 counteracts senescence and cell death and is thus a potential chemotherapeutic target in human endometrial cancer

1α,25(OH) 2D3 Sensitive Cytosolic pH Regulation and Glycolytic Flux in Human Endometrial Ishikawa Cells

Background/Aims: Tumor cell proliferation is modified by 1,25-Dihydroxy-Vitamin D3 (1,25(OH)2D3), a steroid hormone predominantly known for its role in calcium and phosphorus metabolism. Key properties of tumor cells include enhanced glycolytic flux with excessive consumption of glucose and formation of lactate. As glycolysis is highly sensitive to cytosolic pH, maintenance of glycolysis requires export of H+ ions and lactate, which is in part accomplished by Na+/H+ exchangers, such as NHE1 and monocarboxylate transporters, such as MCT4. An effect of 1,25(OH)2D3 on those transport processes has, however, never been reported. As cytosolic pH impacts on apoptosis, the study further explored the effect of 1,25(OH)2D3 on apoptosis and on the apoptosis regulating kinase AKT, transcription factor Forkhead box O-3 (FOXO3A) and B-cell lymphoma protein BCL-2. Methods: In human endometrial adenocarcinoma (Ishikawa) cells, cytosolic pH (pHi) was determined utilizing (2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein [BCECF] fluorescence, Na+/H+ exchanger activity from Na+ dependent realkalinization after an ammonium pulse, NHE1 and MCT4 transcript levels using qRT-PCR, NHE1, MCT4, total & phospho AKT, total & phospho-FOXO3A and BCL-2 protein abundance by Western blotting, lactate concentration in the supernatant utilizing a colorimetric enzyme assay and cell death quantification using CytoTox 96®, Annexin V and Propidium Iodide staining. Results: A 24 hours treatment with 1,25(OH)2D3 (100 nM) significantly increased cytosolic pH (pHi), significantly decreased Na+/H+ exchanger activity, NHE1 and MCT4 transcript levels as well as protein abundance and significantly increased lactate concentration in the supernatant. Treatment of Ishikawa cells with 1,25(OH)2D3 (100 nM) further triggered apoptosis, an effect paralleled by decreased phosphorylation of AKT and FOXO3A as well as decreased abundance of BCL-2. Conclusions: In Ishikawa cells 1,25(OH)2D3 is a powerful stimulator of glycolysis, an effect presumably due to cytosolic alkalinization. Despite stimulation of glycolysis, 1,25(OH)2D3 stimulates slightly but significantly suicidal cell death, an effect presumably in part due to decreased activation of AKT with decreased inhibition of pro-apoptotic transcription factor FOXO3A and downregulation of the anti-apoptotic protein BCL-2