Inflammatory Mechanism of Brucella Infection in Placental Trophoblast Cells.

Brucellosis is a severe zoonotic infectious disease caused by the infection of the Brucella, which is widespread and causes considerable economic losses in underdeveloped areas. Brucella is a facultative intracellular bacteria whose main target cells for infection are macrophages, placental trophoblast cells and dendritic cells. The main clinical signs of Brucella infection in livestock are reproductive disorders and abortion. At present, the pathogenesis of placentitis or abortion caused by Brucella in livestock is not fully understood, and further research on the effect of Brucella on placental development is still necessary. This review will mainly introduce the research progress of Brucella infection of placental trophoblast cells as well as the inflammatory response caused by it, explaining the molecular regulation mechanism of Brucella leading to reproductive system disorders and abortion, and also to provide the scientific basis for revealing the pathogenesis and infection mechanism of Brucella.

The VirB System Plays a Crucial Role in Brucella Intracellular Infection.

Brucellosis is a highly prevalent zoonotic disease caused by Brucella. Brucella spp. are gram-negative facultative intracellular parasitic bacteria. Its intracellular survival and replication depend on a functional virB system, an operon encoded by VirB1-VirB12. Type IV secretion system (T4SS) encoded by the virB operon is an important virulence factor of Brucella. It can subvert cellular pathway and induce host immune response by secreting effectors, which promotes Brucella replication in host cells and induce persistent infection. Therefore, this paper summarizes the function and significance of the VirB system, focusing on the structure of the VirB system where VirB T4SS mediates biogenesis of the endoplasmic reticulum (ER)-derived replicative Brucella-containing vacuole (rBCV), the effectors of T4SS and the cellular pathways it subverts, which will help better understand the pathogenic mechanism of Brucella and provide new ideas for clinical vaccine research and development.

The Mechanism of Facultative Intracellular Parasitism of Brucella.

Brucellosis is a highly prevalent zoonotic disease characterized by abortion and reproductive dysfunction in pregnant animals. Although the mortality rate of Brucellosis is low, it is harmful to human health, and also seriously affects the development of animal husbandry, tourism and international trade. Brucellosis is caused by Brucella, which is a facultative intracellular parasitic bacteria. It mainly forms Brucella-containing vacuoles (BCV) in the host cell to avoid the combination with lysosome (Lys), so as to avoid the elimination of it by the host immune system. Brucella not only has the ability to resist the phagocytic bactericidal effect, but also can make the host cells form a microenvironment which is conducive to its survival, reproduction and replication, and survive in the host cells for a long time, which eventually leads to the formation of chronic persistent infection. Brucella can proliferate and replicate in cells, evade host immune response and induce persistent infection, which are difficult problems in the treatment and prevention of Brucellosis. Therefore, the paper provides a preliminary overview of the facultative intracellular parasitic and immune escape mechanisms of Brucella, which provides a theoretical basis for the later study on the pathogenesis of Brucella.

Up-regulation of TDAG51 is a dependent factor of LPS-induced RAW264.7 macrophages proliferation and cell cycle progression.

CONTEXT: As a component of the outer membrane in Gram-negative bacteria, lipopolysaccharide (LPS)-induced proliferation and cell cycle progression of monocytes/macrophages. It has been suggested that the proapoptotic T-cell death-associated gene 51 (TDAG51) might be associated with cell proliferation and cell cycle progression; however, its role in the interaction between LPS and macrophages remains unclear. OBJECTIVE: We attempted to elucidate the role(s) of TDAG51 played in the interaction between LPS and macrophages. MATERIALS AND METHODS: We investigated TDAG51 expression in RAW264.7 cells stimulated with LPS and examined the effects of RNA interference-mediated TDAG51 down-regulation. We used CCK-8 assay and flow cytometry analysis to evaluate the interaction between TDAG51 and LPS-induced proliferation and cell cycle progression in RAW264.7 cells. RESULTS: Our findings indicate that TDAG51 is up-regulated in LPS-stimulated RAW264.7 cells, the TDAG51 siRNA effectively reduced TDAG51 protein up-regulation following LPS stimulation in RAW264.7 cells, the significant changes of the proliferation and cell cycle progression of RAW264.7 cells in TDAG51 Knockdown RAW264.7 cells treated with LPS were observed. CONCLUSION: These findings suggested that TDAG51 up-regulation is a dependent event during LPS-mediated proliferation and cell cycle progression, and which increase our understanding of the interaction mechanism between LPS and macrophages.

Identification of miR-221 and -222 as important regulators in genotype IV swine hepatitis E virus ORF3-expressing HEK 293 cells.

Hepatitis E virus (HEV) has emerged as an important cause of epidemic and sporadic acute viral hepatitis worldwide, which is a major public health challenge. A better understanding of the interaction between the virus and the host cell would be very helpful for its therapy. Swine HEV (SHEV) open reading frame 3 (ORF3) is a regulatory protein that alters the activity of selected transcription factors and cytoplasmic signaling pathways. MicroRNAs (miRNAs) are potent post-transcriptional regulators of protein-coding genes and represent an interesting lead to study SHEV infection and to identify new therapeutic targets. To explore how SHEV ORF3 affects miRNAs in host cells, we used miRNA array analysis to compare the expression patterns of miRNAs in stable cell lines that expressed or did not express SHEV ORF3. We found a significant down-regulation of miR-221 and -222 in ORF3 expressing human embryonic kidney 293 cell line. Among the 116 candidate targets genes of miR-221 and -222 that we detected in silico, we demonstrated that the expression of the cyclin-dependent kinase inhibitor 1B, also named p27(kip1), was directly regulated by these miRNAs. We hypothesize that SHEV ORF3-induced miR-221/222 downregulation enhances p27(kip1) expression in HEK293 cells. This provides new avenues for future exploration of the precise roles of miRNAs in SHEV infection.

The mechanism of chronic intracellular infection with Brucella spp.

Globally, brucellosis is a widespread zoonotic disease. It is prevalent in more than 170 countries and regions. It mostly damages an animal's reproductive system and causes extreme economic losses to the animal husbandry industry. Once inside cells, Brucella resides in a vacuole, designated the BCV, which interacts with components of the endocytic and secretory pathways to ensure bacterial survival. Numerous studies conducted recently have revealed that Brucella's ability to cause a chronic infection depends on how it interacts with the host. This paper describes the immune system, apoptosis, and metabolic control of host cells as part of the mechanism of Brucella survival in host cells. Brucella contributes to both the body's non-specific and specific immunity during chronic infection, and it can aid in its survival by causing the body's immune system to become suppressed. In addition, Brucella regulates apoptosis to avoid being detected by the host immune system. The BvrR/BvrS, VjbR, BlxR, and BPE123 proteins enable Brucella to fine-tune its metabolism while also ensuring its survival and replication and improving its ability to adapt to the intracellular environment.

siRNA targeting mCD14 inhibits TNF-α, MIP-2, and IL-6 secretion and NO production from LPS-induced RAW264.7 cells.

Innate immunity plays a key role in protecting a host against invading microorganism, including Gram-negative bacteria. Cluster of differentiation antigen 14 (CD14) is an important innate immunity molecule, existing as a soluble (sCD14) and membrane-associated (mCD14) protein. Endotoxin [lipopolysaccharide (LPS)] is recognized as a key molecule in the pathogenesis of sepsis and septic shock caused by Gram negative bacteria. Emerging evidences indicate that upstream inhibition of bacterial LPS/Toll-like receptor 4(TLR4)/CD14-mediated inflammation pathway is an effective therapeutic approach for attenuating damaging immune activation. RNA interference (RNAi) provides a promising approach to down-regulate gene expression specifically. To explore the possibility of using RNAi against mCD14 as a strategy for inhibiting the secretion of cytokines and the nitric oxide (NO) production from LPS-activated RAW264.7 cells, four different short interfering RNA (siRNA) molecules corresponding to the sequence of mCD14 gene were designed and synthesized. We then tested the inhibition effects of these siRNA molecules on mCD14 expression by real-time quantitative RT-PCR and Western blot. After effective siRNA molecule (mCD14-siRNA-224), which is capable of reducing messenger RNA (mRNA) accumulation and protein expression of mCD14 specifically, was identified, RAW264.7 cells pretreated with mCD14-siRNA-224 were stimulated with LPS, and the secretion of tumor necrosis factor alpha (TNF-α), macrophage inflammatory protein-2 (MIP-2) and interleukin-6 (IL-6) and the NO production were evaluated. The results indicated that mCD14-siRNA-224 effectively inhibited TNF-α, MIP-2, and IL-6 release and NO production from LPS-stimulated RAW 264.7 cells by down-regulating mRNA accumulation and protein expression of mCD14 specifically. These findings provide useful information for the development of RNAi-based prophylaxis and therapy for endotoxin-related diseases.

RNA interference induces effective inhibition of mRNA accumulation and protein expression of SHEV ORF3 gene in vitro.

RNA interference (RNAi) provides a powerful promising approach to inhibit viral infection specifically. To explore the possibility of using RNAi as a strategy against HEV infection, which is a serious public health problem in developing countries of Asia, Middle East, Africa, and in Mexico, after the fusion protein expression plasmids pEGFP-ORF3 which contain the EGFP reporter gene and SHEV ORF3 as silencing target, were constructed; EGFP-ORF3 fusion protein expressing HEK 293 cell lines were established; and four siRNAs targeting ORF3 gene were designed, synthesized, and used to transfect the stable cell lines. At 24, 48, and 72 h after transfection, flow cytometry, real-time quantitative PCR, and Western blot were used to assess the expression level of ORF3. The results demonstrated that specific siRNAs which are sequence dependant effectively inhibited mRNA accumulation and protein expression of SHEV ORF3 in HEK 293 cells. These findings provide useful information for the development of RNAi-based prophylaxis and therapy for SHEV infection.

Identifying Circular RNAs in HepG2 Expressing Genotype IV Swine Hepatitis E Virus ORF3 Via Whole Genome Sequencing.

Swine hepatitis E (SHE) is a new type of zoonotic infectious disease caused by swine hepatitis E virus (SHEV). Open reading frame 3 (ORF3) is a key regulatory and virulent protein of SHEV. Circular RNAs (circRNAs) are a special kind of non-coding RNA molecule, which has a closed ring structure. In this study, to identify the circRNA profile in host cells affected by SHEV ORF3, adenovirus ADV4-ORF3 mediated the overexpression of ORF3 in HepG2 cells, whole genome sequencing was used to investigate the differentially expressed circRNAs, GO and KEGG were performed to enrichment analyze of differentially expressed circRNA-hosting gene, and Targetscan and miRanda softwares were used to analyze the interaction between circRNA and miRNA. The results showed adenovirus successfully mediated the overexpression of ORF3 in HepG2 cells, 1,105 up-regulation circRNAs and 1,556 down-regulation circRNAs were identified in ADV4-ORF3 infection group compared with the control. GO function enrichment analysis of differentially expressed circRNAs-hosting genes classified three main categories (cellular component, biological process and molecular function). KEGG pathway enrichment analysis scatter plot showed the pathway term of top20. The circRNAs with top10 number of BS sites for qRT-PCR validation were selected to confirmed, the results indicated that the up-regulated hsa_circ_0001423 and hsa_circ_0006404, and down-regulated of hsa_circ_0004833 and hsa_circ_0007444 were consistent with the sequencing data. Our findings first preliminarily found that ORF3 protein may affect triglyceride activation (GO:0006642) and riboflavin metabolism (ko00740) in HepG2 cells, which provides a scientific basis for further elucidating the effect of ORF3 on host lipid metabolism and the mechanism of SHEV infection.

Deep Insight Into Long Non-coding RNA and mRNA Transcriptome Profiling in HepG2 Cells Expressing Genotype IV Swine Hepatitis E Virus ORF3.

Swine hepatitis E (swine HE) is a new type of zoonotic infectious disease caused by the swine hepatitis E virus (swine HEV). Open reading frame 3 (ORF3) is an important virulent protein of swine HEV, but its function still is mainly unclear. In this study, we generated adenoviruses ADV4-ORF3 and ADV4 negative control (ADV4-NC), which successfully mediated overexpression of enhanced green fluorescent protein (EGFP)-ORF3 and EGFP, respectively, in HepG2 cells. High-throughput sequencing was used to screen for differentially expressed long non-coding RNAs (lncRNAs) and messenger RNAs (mRNAs). The cis-target genes of lncRNAs were predicted, functional enrichment (Gene Ontology [GO] and Kyoto Encyclopedia of Genes and Genomes [KEGG]) was performed, and 12 lncRNAs with statistically significant different expressions (p ≤ 0.05 and q ≤ 1) were selected for further quantitative real-time reverse transcription (qRT-PCR) validation. In HepG2 cells, we identified 62 significantly differentially expressed genes (DEGs) (6,564 transcripts) and 319 lncRNAs (124 known lncRNAs and 195 novel lncRNAs) that were affected by ORF3, which were involved in systemic lupus erythematosus, Staphylococcus aureus infection, signaling pathways pluripotency regulation of stem cells, the peroxisome proliferator-activated receptor (PPAR) signaling pathway, and platinum drug resistance pathways. Cis-target gene prediction identified 45 lncRNAs corresponding to candidate mRNAs, among which eight were validated by qRT-PCR: LINC02476 (two transcripts), RAP2C-AS1, AC016526, AL139099, and ZNF337-AS1 (3 transcripts). Our results revealed that the lncRNA profile in host cells affected by ORF3, swine HEV ORF3, might affect the pentose and glucuronate interconversions and mediate the formation of obstructive jaundice by influencing bile secretion, which will help to determine the function of ORF3 and the infection mechanism and treatment of swine HE.

Immunological pathways of macrophage response to Brucella ovis infection.

As the molecular mechanisms of Brucella ovis pathogenicity are not completely clear, we have applied a transcriptome approach to identify the differentially expressed genes (DEGs) in RAW264.7 macrophage infected with B. ovis. The DEGs related to immune pathway were identified by Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) functional enrichment analysis. Quantitative real-time PCR (qRT-PCR) was performed to validate the transcriptome sequencing data. In total, we identified 337 up-regulated and 264 down-regulated DEGs in B. ovis-infected group versus mock group. Top 20 pathways were enriched by KEGG analysis and 20 GO by functional enrichment analysis in DEGs involved in the molecular function, cellular component, and biological process and so on, which revealed multiple immunological pathways in RAW264.7 macrophage cells in response to B. ovis infection, including inflammatory response, immune system process, immune response, cytokine activity, chemotaxis, chemokine-mediated signaling pathway, chemokine activity, and CCR chemokine receptor binding. qRT-PCR results showed Ccl2 (ENSMUST00000000193), Ccl2 (ENSMUST00000124479), Ccl3 (ENSMUST00000001008), Hmox1 (ENSMUST00000005548), Hmox1 (ENSMUST00000159631), Cxcl2 (ENSMUST00000075433), Cxcl2 (ENSMUST00000200681), Cxcl2 (ENSMUST00000200919), and Cxcl2 (ENSMUST00000202317). Our findings firstly elucidate the pathways involved in B. ovis-induced host immune response, which may lay the foundation for revealing the bacteria-host interaction and demonstrating the pathogenic mechanism of B. ovis.

Integrative Bioinformatics Indentification of the Autophagic Pathway-Associated miRNA-mRNA Networks in RAW264.7 Macrophage Cells Infected with ∆Omp25 Brucella melitensis.

Brucellosis is a zoonotic infectious disease caused by Brucella infection. Outer membrane protein 25 (Omp25) is closely related to the virulence and immunogenicity of Brucella. However, the molecular mechanism of Omp25 affecting Brucella-mediated macrophage autophagy remains unclear. Our previous study reported that four miRNAs (the upregulation of mmu-miR-146a-5p and mmu-miR-155-5p and downregulation of mmu-miR-149-3p and mmu-miR-5126) were confirmed and revealed the differentially expressed genes (DEGs) profile in RAW264.7 macrophage cells infected with Brucella melitensis Omp25 deletion mutant (∆Omp25 B. melitensis). Here, we predicted the target genes of the four miRNAs by TargetScan, miRanda, and PicTar. GO and KEGG were used for functional enrichment analysis of DEGs profile to reveal the autophagic pathway-associated genes. The overlapped genes, which drawn the autophagic pathway-associated miRNA-mRNA networks by cytoscape software, were identified by intersecting with the predicted target genes and autophagic pathway-associated DEGs. qRT-PCR was performed to validate the mRNAs of networks. The results showed that the autophagic pathway-associated networks of mmu-miR-149-3p-Ptpn5, mmu-miR-149-3p-Ppp2r3c, and mmu-miR-146a-5p-Dusp16 were identified in RAW264.7 macrophage cells infected with ∆Omp25 B. melitensis. Our findings are of great significance in elucidating the function of Omp25, revealing the infection mechanism of Brucella and prophylaxising and treating brucellosis.

Transcriptome Landscape of Intracellular Brucella ovis Surviving in RAW264.7 Macrophage Immune System.

Brucella ovis infection results in genital damage and epididymitis in rams, placental inflammation and rare abortion in ewes, and neonatal mortality in lambs. However, the mechanism underlying B. ovis infection remains unclear. In the present study, we used prokaryotic transcriptome sequencing to identify the differentially expressed genes (DEGs) between wild-type B. ovis and intracellular B. ovis in RAW264.7 macrophages. Gene ontology (GO) term enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were performed, and quantitative reverse transcriptase PCR (qRT-PCR) was used to validate the top 10 upregulated and downregulated DEGs. The results showed that 212 genes were differentially expressed, including 68 upregulated and 144 downregulated genes, which were mainly enriched in 30 GO terms linked to biological process, cellular component, and molecular function. KEGG analysis showed that the DEGs were enriched in the hypoxia-inducible factor 1 (HIF-1) signaling pathway, mitogen-activated protein kinase (MAPK) signaling pathway, beta-alanine metabolism, and quorum sensing pathway. BME_RS01160, BME_RS04270, BME_RS08185, BME_RS12880, BME_RS25875, predicted_RNA865, and predicted_RNA953 were confirmed with the transcriptome sequencing data. Hence, our findings not only reveal the intracellular parasitism of B. ovis in the macrophage immune system, but also help to understand the mechanism of chronic B. ovis infection.

MicroRNA expression profiles from HEK293 cells expressing H5N1 avian influenza virus non-structural protein 1.

H5N1 avian influenza poses a serious threat to the poultry industry and human health. Non-structural protein 1 (NS1) plays an important role in the replication and pathogenesis of avian influenza virus (AIV). However, the function of the NS1 gene is still unclear. In this study, illumina genome analyzer iix screening was used to identify the differentially expressed microRNAs (miRNAs) in HEK293 cells expressing H5N1 AIV NS1. There were 13 differentially expressed miRNAs (hsa-miR-17-5p, hsa-miR-221-3p, hsa-miR-22-3p, hsa-miR-31-5p, hsa-miR-20a-5p, hsa-miR-222-3p, hsa-miR-24-3p, hsa-miR-3613-3p, hsa-miR-3178, hsa-miR-4505, hsa-miR-345-3p, hsa-miR-3648, and hsa-miR-455-3p) ( P < 0.01). The qRT-PCR validation results demonstrated that hsa-miR-221-3p, hsa-miR-22-3p, hsa-miR-20a-5p, and hsa-miR-3613-3p were upregulated, while hsa-miR-3178 and hsa-miR-4505 were down-regulated. The softwares targetscan and miranda were further used to predict their target genes, and the gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis results showed that 20 GO terms and 20 KEGG pathways were significantly enriched. Our findings are the first to report expression profiling of miRNA and their functions in H5N1 AIV NS1-expressing HEK293 cells, and pave the way to further elucidating the accurate interaction mechanism between NS1 and virus replication, thus providing brand new insight into the prophylaxis and treatment of H5N1 AIV.

Alterations of microRNAs and their predicted targeting mRNAs expression in RAW264.7 macrophages infected with Omp25 mutant Brucella melitensis.

Brucellosis is a worldwide zoonosis caused by Brucella species and represents a serious threat to both human and animal health. Omp25 is an important immunogenic and protective antigen in Brucella species; however, the functional mechanism of Omp25 in macrophages has not yet been elucidated. Here, we constructed a Brucella melitensis omp25 deletion mutant (M5-90-Δ omp25) and performed microRNA (miRNA) profiling of infected RAW264.7 cells. Eight differentially expressed miRNAs ( mmu-miR-146a-5p, mmu-miR-155-5p, mmu-miR-3473a, mmu-miR-149-3p, mmu-miR-671-5p, mmu-miR-1224-5p, mmu-miR-1895, and mmu-miR-5126) were identified, with quantitative real-time PCR (qRT-PCR) analysis confirming the up-regulation of mmu-miR-146-a-5p and mmu-miR-155-5p and down-regulation of mmu-miR-149-3p and mmu-miR-5126. mRNA profiling of B. melitensis M5-90-Δo mp25-infected RAW264.7 cells identified 967 differentially expressed genes (DEGs) (fold change ≥ 2). Among these, we focused on genes that were predicted by TargetScan, miRanda, and PicTar to be the potential targets of the differentially expressed miRNAs. The results suggested that 17 separate genes are potentially targeted by mmu-miR-149-3p, with one of these genes, Tbr1, also targeted by mmu-miR-5126. qRT-PCR analysis confirmed the up-regulation of nine of the predicted target genes. Our findings provide important information about the functional molecules in host cells, including miRNA and their target genes, affected by Omp25 from Brucella. This information is particularly valuable for the prophylaxis and treatment of brucellosis.

CD14 gene silencing alters the microRNA expression profile of RAW264.7 cells stimulated by Brucella melitensis infection.

Innate recognition of Brucella spp. is a key step in the activation of inflammation. CD14 binds PAMPs and is involved in LPS-induced pro-inflammatory cytokine release. Previously we showed that knock down of CD14 in RAW264.7 macrophages disrupted Brucella-host interactions. However, its effect on the macrophage microRNA (miRNA) expression profile, especially after stimulation by Brucella infection, is still unclear. To identify miRNAs involved in the macrophage response to Brucella infection, we performed miRNA expression profiling of CD14 knock-down RAW264.7 (224.3) macrophages infected with Brucella melitensis, and demonstrated, for the first time, that CD14 knock down significantly up-regulated the expression of mmu-miR-199a-3p and mmu-miR-183-5p in these conditions. These miRNAs have a well-characterized association with the target genes involved in immune response, inflammatory response, innate immune response, apoptosis processes, anti-apoptosis, cytokine production and cytokine-mediated signaling pathways. Among the 104 inflammation-related candidate target genes of mmu-miR-199a-3p and mmu-miR-183-5p in the 224.3+ B. melitensis group cells, the expression of the Cbl-b, a potential target of mmu-miR-199a-3p, was confirmed to be down-regulated using qRT-PCR and Western blot analysis. Our findings suggest that CD14 functions in the Brucella-host interaction may be through altered miRNA expression, and regulation of Cbl-b proteins.

In Vitro and In Vivo Immunomodulatory Activity of Okra (Abelmoschus esculentus L.) Polysaccharides.

Crude okra (Abelmoschus esculentus L.) polysaccharide (RPS) was obtained by water extraction and alcohol precipitation. Three purified fractions of RPS, designated RPS-1, RPS-2, and RPS-3, were fractioned by diethylaminoethyl (DEAE)-cellulose chromatography. Their molecular weights, monosaccharide compositions, infrared (Fourier transform infrared [FT-IR]) spectra, and nuclear magnetic resonance (NMR) spectra were analyzed. Their immunomodulatory activity was evaluated with an in vitro cell model (RAW264.7 cells). In vivo immunomodulatory activity of RPS-2 was evaluated in normal and cyclophosphamide-induced immunosuppressed mice. The results showed that the molecular weights of RPS-1, RPS-2, and RPS-3 were 600, 990, and 1300 kDa, respectively. RPS-1 and RPS-2 were mainly composed of galactose, rhamnose, galacturonic acid, and glucuronic acid, while RPS-3 was mainly composed of galactose, rhamnose, galacturonic acid, glucuronic acid, and glucose. FT-IR and NMR spectrum data indicated a rhamnogalacturonan I characteristic of polysaccharide. Both RPS and its purified fractions RPS-1, RPS-2, and RPS-3 significantly increased RAW264.7 cell proliferation, nitric oxide (NO) production, inducible nitric oxide synthase (iNOS) expression, and tumor necrosis factor (TNF)-α, interferon (IFN)-γ, and interleukin (IL)-10 secretion (P < .05). The purified fraction RPS-2 also increased the spleen index, splenocyte proliferation, and cytokine secretion in vivo. These results indicate that okra polysaccharides may potentially serve as novel immunomodulators.

Transcriptome Analysis of HepG2 Cells Expressing ORF3 from Swine Hepatitis E Virus to Determine the Effects of ORF3 on Host Cells.

Hepatitis E virus- (HEV-) mediated hepatitis has become a global public health problem. An important regulatory protein of HEV, ORF3, influences multiple signal pathways in host cells. In this study, to investigate the function of ORF3 from the swine form of HEV (SHEV), high-throughput RNA-Seq-based screening was performed to identify the differentially expressed genes in ORF3-expressing HepG2 cells. The results were validated with quantitative real-time PCR and gene ontology was employed to assign differentially expressed genes to functional categories. The results indicated that, in the established ORF3-expressing HepG2 cells, the mRNA levels of CLDN6, YLPM1, APOC3, NLRP1, SCARA3, FGA, FGG, FGB, and FREM1 were upregulated, whereas the mRNA levels of SLC2A3, DKK1, BPIFB2, and PTGR1 were downregulated. The deregulated expression of CLDN6 and FREM1 might contribute to changes in integral membrane protein and basement membrane protein expression, expression changes for NLRP1 might affect the apoptosis of HepG2 cells, and the altered expression of APOC3, SCARA3, and DKK1 may affect lipid metabolism in HepG2 cells. In conclusion, ORF3 plays a functional role in virus-cell interactions by affecting the expression of integral membrane protein and basement membrane proteins and by altering the process of apoptosis and lipid metabolism in host cells. These findings provide important insight into the pathogenic mechanism of HEV.

Mmu-miR-27a-5p-Dependent Upregulation of MCPIP1 Inhibits the Inflammatory Response in LPS-Induced RAW264.7 Macrophage Cells.

Lipopolysaccharide (LPS) stimulates macrophages to release proinflammatory cytokines. MicroRNAs (miRNAs) are short noncoding RNAs that are involved in inflammatory reaction. Our previously study identified the downregulated expression of mmu-miR-27a-5p in RAW267.4 cells treated with LPS. To dissect the mechanism that mmu-miR-27a-5p regulates target genes and affects proinflammatory cytokine secretion more clearly, based on previous bioinformatics prediction data, one of the potential target genes, MCPIP1 was observed to be upregulated with qRT-PCR and western blot. Luciferase reporter assays were performed to further confirm in silico prediction and determine that MCPIP1 is the target of mmu-miR-27-5p. The results suggested that mmu-miR-27a-5p directly targeted the 3'-UTR of MCPIP1 and the interaction between mmu-miR-27-5p and the 3'-UTR of MCPIP1 is sequence-specific. MCPIP1 overexpression decreased the secretion of IL-6, IL-1ß, and IL-10 in macrophage cells stimulated with LPS. Our findings may provide the important information for the precise roles of mmu-miR-27a-5p in the macrophage inflammatory response to LPS stimulation in the future.

Complete genome sequence of bovine papillomavirus genotype 13 from local yellow cattle in hainan province, china.

Here, we present the complete genome sequence of bovine papillomavirus genotype 13 isolated from local yellow cattle in Hainan, China. The genome is 7,961 bp and contains six early genes and two late genes. This analysis provides important information for the research of bovine papillomavirus (BPV) in China.