TIM-1 Promotes Japanese Encephalitis Virus Entry and Infection.

Japanese encephalitis virus (JEV) is a mosquito-borne Flavivirus, the leading cause of viral-induced encephalitis. Several host molecules have been identified as the JEV attachment factor; however, the molecules involved in JEV entry remain poorly understood. In the present study, we demonstrate that TIM-1 is important for efficient infection by JEV. Firstly, three TIM-1 variants (V1, V2, and V3) were cloned from A549 cells, and we revealed that only ectopically TIM-1 V2 expression in 293T cells significantly promotes JEV attachment, entry and infection. Point mutation of phosphatidylserine (Ptdser) binding pocket in the TIM-1 IgV domain dampened JEV entry, indicating that TIM-1-mediated JEV infection is Ptdser-dependent. Furthermore, we found the cytoplasmic domain of TIM-1 is also required for enhancing JEV entry. Additionally, knock down of TIM-1 expression in A549 cells impaired JEV entry and infection, but not attachment, suggesting that additional factors exist in A549 cells that allow the virus to bind. In conclusion, our findings demonstrate that TIM-1 promotes JEV infection as an entry cofactor, and the polymorphism of TIM-1 is associated with JEV susceptibility to host cells.

Identification of a novel porcine OASL variant exhibiting antiviral activity.

2', 5'-Oligoadenylate synthetase-lilke (OASL) protein is an atypical oligoadenylate synthetase (OAS) family member, which possesses antiviral activity but lacks 2', 5'-oligoadenylate synthetase activity. Here, a novel variant of porcine OASL (pOASL2) was identified through RT-PCR amplification. This gene is distinguishable from the previously described wild-type porcine OASL (pOASL1). The gene appears to be derived from a truncation of exon 4 plus 8 nucleotides of exon 5 with a premature termination, measuring only 633 bp in length, although its position corresponds to that of pOASL1. Given this novel gene appears to be a variant of pOASL, we assayed for antiviral activity of the protein. We demonstrated that pOASL2 could inhibit Japanese encephalitis virus (JEV) proliferation as well as pOASL1 in a transient overexpression assay of pOASL1 and pOASL2 in PK-15 and Vero cells. In addition to JEV, pOASL1 and pOASL2 also decreased the proliferations of Porcine reproductive and respiratory syndrome virus (PRRSV) and vesicular stomatitis virus (VSV), but did not exhibit antiviral activity against pseudorabies virus (PRV). Structural analysis showed that the pOASL2 gene retained only the first three exons at the 5'-. To investigate the role of the αN4 helix in pOASL in antiviral responses like that in hOASL, we mutated key residues in the anchor domain of the αN4 helix in pOASL2, based on the domain's location in hOASL. However, the antiviral activity of pOASL2 was not affected. Thus, the αN4 helix of pOASL likely does not play a significant role in its antiviral activity. In conclusion, pOASL2 acts as a new splice isoform of pOASL that plays a role in resistance to infection of several kinds of RNA viruses.

The immunomodulatory peptide bursopentin (BP5) enhances proliferation and induces sIgM expression in DT40 cells.

BACKGROUND: In the recent past, many studies have been focused on extracts of BF and multiple biologically active factors and their effects on humoral immune system in chickens and birds. However, the mechanism of those immunomodulatory peptides on the B lineage cells proliferation and antibody production in chicken is fairly unknown. DT40 cell line, an avian leucosis virus-induced chicken pre-B cell line, expresses immunoglobulin M (IgM) isotype B cell reporter in the plasma membrane. There are many evidences suggesting that DT40 cells are best characterized as a bursal stem cell line. Because of the unique characteristics of DT40 cell line, it has been widely used to observe biological processes of pre-B lymphocyte cell within living cells. METHODS: The chicken B cell line DT40 was cultured in Roswell Park Memorial Institute (RPMI) 1640 medium and cytotoxicity was studied. Also, effect of BP5 on cell proliferation and cell cycle distribution of DT40 cells was studied. Also, the effect of BP5 on sIgM mRNA expression was studied by using real-time PCR. OBJECTIVES: To investigat the effects of Bursopentin (Cys-Lys-Arg-Val-Tyr, BP5) on a chicken promyelocyte cell line DT40, assays of cell proliferation, cell cycle distribution, detection of surface immunoglobulin G (sIgM) mRNA expression and gene microarray analysis were performed. RESULTS: The results showed that BP5 displayed concentration-dependent effects on the proliferation, cell cycle, and sIgM mRNA expression in DT40 cells. And the analysis of expression profiles identified a signature set of 3022 genes (1254 up regulated genes, 1762 down regulated genes), which clearly discriminated the BP5-treated DT40 cells from control with high certainty (P≤0.02). The results of microarray analysis were confirmed by quantitative reverse transcription-polymerase chain reaction for 12 of the differentially expressed genes. CONCLUSION: Theses findings showed the immuno-activity effect of BP5 on B lymphocyte and indicated that BP5 treatment regulated eight signaling pathways, in which Toll-like signaling pathway was the most significant enrichment pathway.

Rab5 and Rab11 Are Required for Clathrin-Dependent Endocytosis of Japanese Encephalitis Virus in BHK-21 Cells.

During infection Japanese encephalitis virus (JEV) generally enters host cells via receptor-mediated clathrin-dependent endocytosis. The trafficking of JEV within endosomes is controlled by Rab GTPases, but which Rab proteins are involved in JEV entry into BHK-21 cells is unknown. In this study, entry and postinternalization of JEV were analyzed using biochemical inhibitors, RNA interference, and dominant negative (DN) mutants. Our data demonstrate that JEV entry into BHK-21 cells depends on clathrin, dynamin, and cholesterol but not on caveolae or macropinocytosis. The effect on JEV infection of dominant negative (DN) mutants of four Rab proteins that regulate endosomal trafficking was examined. Expression of DN Rab5 and DN Rab11, but not DN Rab7 and DN Rab9, significantly inhibited JEV replication. These results were further tested by silencing Rab5 or Rab11 expression before viral infection. Confocal microscopy showed that virus particles colocalized with Rab5 or Rab11 within 15 min after virus entry, suggesting that after internalization JEV moves to early and recycling endosomes before the release of the viral genome. Our findings demonstrate the roles of Rab5 and Rab11 on JEV infection of BHK-21 cells through the endocytic pathway, providing new insights into the life cycle of flaviviruses.IMPORTANCE Although Japanese encephalitis virus (JEV) utilizes different endocytic pathways depending on the cell type being infected, the detailed mechanism of its entry into BHK-21 cells is unknown. Understanding the process of JEV endocytosis and postinternalization will advance our knowledge of JEV infection and pathogenesis as well as provide potential novel drug targets for antiviral intervention. With this objective, we used systematic approaches to dissect this process. The results show that entry of JEV into BHK-21 cells requires a low-pH environment and that the process occurs through dynamin-, actin-, and cholesterol-dependent clathrin-mediated endocytosis that requires Rab5 and Rab11. Our work provides a detailed picture of the entry of JEV into BHK-21 cells and the cellular events that follow.

mosGCTL-7, a C-Type Lectin Protein, Mediates Japanese Encephalitis Virus Infection in Mosquitoes.

Japanese encephalitis virus (JEV) is an arthropod-borne flavivirus prevalent in Asia and the Western Pacific and is the leading cause of viral encephalitis. JEV is maintained in a transmission cycle between mosquitoes and vertebrate hosts, but the molecular mechanisms by which the mosquito vector participates in transmission are unclear. We investigated the expression of all C-type lectins during JEV infection in Aedes aegypti The C-type lectin mosquito galactose-specific C-type lectin 7 (mosGCTL-7) (VectorBase accession no. AAEL002524) was significantly upregulated by JEV infection and facilitated infection in vivo and in vitro mosGCTL-7 bound to the N-glycan at N154 on the JEV envelope protein. This recognition of viral N-glycan by mosGCTL-7 is required for JEV infection, and we found that this interaction was Ca2+ dependent. After mosGCTL-7 bound to the glycan, mosPTP-1 bound to mosGCTL-7, promoting JEV entry. The viral burden in vivo and in vitro was significantly decreased by mosPTP-1 double-stranded RNA (dsRNA) treatment, and infection was abolished by anti-mosGCTL-7 antibodies. Our results indicate that the mosGCTL-7/mosPTP-1 pathway plays a key role in JEV infection in mosquitoes. An improved understanding of the mechanisms underlying flavivirus infection in mosquitoes will provide further opportunities for developing new strategies to control viral dissemination in nature.IMPORTANCE Japanese encephalitis virus is a mosquito-borne flavivirus and is the primary cause of viral encephalitis in the Asia-Pacific region. Twenty-four countries in the WHO Southeast Asia and Western Pacific regions have endemic JEV transmission, which exposes >3 billion people to the risks of infection, although JEV primarily affects children. C-type lectins are host factors that play a role in flavivirus infection in humans, swine, and other mammals. In this study, we investigated C-type lectin functions in JEV-infected Aedes aegypti and Culex pipiens pallens mosquitoes and cultured cells. JEV infection changed the expression of almost all C-type lectins in vivo and in vitro, and mosGCTL-7 bound to the JEV envelope protein via an N-glycan at N154. Cell surface mosPTP-1 interacted with the mosGCTL-7-JEV complex to facilitate virus infection in vivo and in vitro Our findings provide further opportunities for developing new strategies to control arbovirus dissemination in nature.

Mx Is Not Responsible for the Antiviral Activity of Interferon-α against Japanese Encephalitis Virus.

Mx proteins are interferon (IFN)-induced dynamin-like GTPases that are present in all vertebrates and inhibit the replication of myriad viruses. However, the role Mx proteins play in IFN-mediated suppression of Japanese encephalitis virus (JEV) infection is unknown. In this study, we set out to investigate the effects of Mx1 and Mx2 expression on the interferon-α (IFNα) restriction of JEV replication. To evaluate whether the inhibitory activity of IFNα on JEV is dependent on Mx1 or Mx2, we knocked down Mx1 or Mx2 with siRNA in IFNα-treated PK-15 cells and BHK-21 cells, then challenged them with JEV; the production of progeny virus was assessed by plaque assay, RT-qPCR, and Western blotting. Our results demonstrated that depletion of Mx1 or Mx2 did not affect JEV restriction imposed by IFNα, although these two proteins were knocked down 66% and 79%, respectively. Accordingly, expression of exogenous Mx1 or Mx2 did not change the inhibitory activity of IFNα to JEV. In addition, even though virus-induced membranes were damaged by Brefeldin A (BFA), overexpressing porcine Mx1 or Mx2 did not inhibit JEV proliferation. We found that BFA inhibited JEV replication, not maturation, suggesting that BFA could be developed into a novel antiviral reagent. Collectively, our findings demonstrate that IFNα inhibits JEV infection by Mx-independent pathways.

Entry of Classical Swine Fever Virus into PK-15 Cells via a pH-, Dynamin-, and Cholesterol-Dependent, Clathrin-Mediated Endocytic Pathway That Requires Rab5 and Rab7.

UNLABELLED: Classical swine fever virus (CSFV), a member of the genus Pestivirus within the family Flaviviridae, is a small, enveloped, positive-strand RNA virus. Due to its economic importance to the pig industry, the biology and pathogenesis of CSFV have been investigated extensively. However, the mechanisms of CSFV entry into cells are not well characterized. In this study, we used systematic approaches to dissect CSFV cell entry. We first observed that CSFV infection was inhibited by chloroquine and NH4Cl, suggesting that viral entry required a low-pH environment. By using the specific inhibitor dynasore, or by expressing the dominant negative (DN) K44A mutant, we verified that dynamin is required for CSFV entry. CSFV particles were observed to colocalize with clathrin at 5 min postinternalization, and CSFV infection was significantly reduced by chlorpromazine treatment, overexpression of a dominant negative form of the EPS15 protein, or knockdown of the clathrin heavy chain by RNA interference. These results suggested that CSFV entry depends on clathrin. Additionally, we found that endocytosis of CSFV was dependent on membrane cholesterol, while neither the overexpression of a dominant negative caveolin mutant nor the knockdown of caveolin had an effect. These results further suggested that CSFV entry required cholesterol and not caveolae. Importantly, the effect of DN mutants of three Rab proteins that regulate endosomal traffic on CSFV infection was examined. Expression of DN Rab5 and Rab7 mutants, but not the DN Rab11 mutant, significantly inhibited CSFV replication. These results were confirmed by silencing of Rab5 and Rab7. Confocal microscopy showed that virus particles colocalized with Rab5 or Rab7 during the early phase of infection within 45 min after virus entry. These results indicated that after internalization, CSFV moved to early and late endosomes before releasing its RNA. Taken together, our findings demonstrate for the first time that CSFV enters cells through the endocytic pathway, providing new insights into the life cycle of pestiviruses. IMPORTANCE: Bovine viral diarrhea virus (BVDV), a single-stranded, positive-sense pestivirus within the family Flaviviridae, is internalized by clathrin-dependent receptor-mediated endocytosis. However, the detailed mechanism of cell entry is unknown for other pestiviruses, such as classical swine fever (CSF) virus (CSFV). CSFV is the etiological agent of CSF, a highly contagious disease of swine that causes numerous deaths in pigs and enormous economic losses in China. Understanding the entry pathway of CSFV will not only advance our knowledge of CSFV infection and pathogenesis but also provide novel drug targets for antiviral intervention. Based on this objective, we used systematic approaches to dissect the pathway of entry of CSFV into PK-15 cells. This is the first report to show that the entry of CSFV into PK-15 cells requires a low-pH environment and involves dynamin- and cholesterol-dependent, clathrin-mediated endocytosis that requires Rab5 and Rab7.

mRNA expression in different developmental stages of the chicken bursa of Fabricius.

The bursa of Fabricius, the central humoral immune organ unique to birds, plays an important role in B-lymphocyte differentiation. In order to gain a better understanding of the molecular mechanism of critical biological processes like B-cell immigration, differentiation, and final emigration, the transcriptional changes during embryonic and posthatch development of this organ were investigated. We generated a cDNA library from total RNA isolated from 3 representative developmental stages (embryonic day [ED] 10, posthatch d 2 and d 21). We generated over 70 million high-quality reads from the cDNA library by using deep sequencing. The uniquely mapped sequences of ED 10, d 2 and d 21 were 71087280, 59167491 and 70263675 respectively. All of the differential expressed genes were involved in Vitamin A metabolism, regulation of actin cytoskeleton, Wnt signaling pathway, MAPK signaling pathway, Jak-STAT signaling pathway, Notch signaling pathway, Toll-like receptor signaling pathway. The RNA-seq analysis provides a powerful method for analyzing the transcriptome and investigating the transcriptional changes of different development stages of bursa of Fabricius. The assembled bursa transcriptome provides an essential resource for future investigations about chicken Bursa development.

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.

Porcine 2', 5'-oligoadenylate synthetases inhibit Japanese encephalitis virus replication in vitro.

The 2', 5'-oligoadenylate synthetases (OAS) are antiviral proteins and several isoforms have been identified as flavivirus-resistance biomarkers in human and mouse. The expression kinetics and antiviral functions of porcine OAS family (OAS1, OAS2, and OASL) in PK-15 cells following infection by Japanese encephalitis virus (JEV) were evaluated in the present study. The endogenous expression of the three OAS genes was efficiently induced by IFN-α treatment in PK-15 cells. However, expression of pOAS1 and pOAS2 responded more quickly than pOASL. Infection by JEV also induced the expression of the pOAS isoforms, but at a significantly lower level than that observed following IFN-α stimulation. Transient overexpression of pOASL and pOAS1 inhibited JEV replication more efficiently than OAS2 overexpression. Interestingly, knockdown of pOAS2 expression by siRNA treatment led to the highest increase in JEV multiplication. Co-silencing of RNase L and each pOAS revealed that the anti-JEV function of pOAS1 and pOAS2 were RNase L dependent, while the antiviral activity of pOASL was not. In conclusion, all pOAS isoforms play a significant role in the response to JEV infection, and are differentially induced by different stimuli. The alternative pathways of antiviral activity stimulated by OASL require further study.

[Hsp70 Fused with the Envelope Glycoprotein E0 of Classical Swine Fever Virus Enhances Immune Responses in Balb/c Mice].

Heat-shock protein (Hsp) 70 potentiates specific immune responses to some antigenic peptides fused to it. Here, the prokaryotic plasmids harboring the envelope glycoprotein E0 gene of classical swine fever virus (CSFV) and/or the Hsp70 gene of Haemophilus parasuis were constructed and expressed in Escherichia coli Rosseta 2(R2). The fusion proteins were then purified. Groups of Balb/c mice were immunized with these fusion proteins, respectively, and sera collected 7 days after the third immunization. Immune effects were determined via an enzyme-linked immunosorbent assay and flow cytometric analyses. E0-Hsp70 fusion protein and E0+Hsp70 mixture significantly improved the titer of E-specific antibody, levels of CD4+ and CD8+ T cells, and release of interferon-γ. These findings suggested that Hsp70 can significantly enhance the immune effects of the envelope glycoprotein E0 of CSFV, thereby laying the foundation of further application in pigs.

[Expression and adjuvant effects of the fusion peptide TBP5].

Thymopentin (TP5) and bursopentin (BP5) are both immunopotentiators. To explore whether the TP5-BP5 fusion peptide (TBP5) has adjuvant activity or not, we cloned the TBP5 gene and confirmed that the TBP5 gene in a recombinant prokaryotic expression plasmid was successfully expressed in Escherichia coli BL21. TBP5 significantly promoted the proliferation of thymic and splenic lymphocytes of mice. The potential adjuvant activity of the TBP5 was examined in mice by coinjecting TBP5 and H9N2 avian influenza virus (AIV) inactivated vaccine. HI antibody titers, HA antibodies and cytokines levels (IL-4 and IFN-γ) were determined. We found that TBP5 markedly elevated serum HI titers and HA antibody levels, induced the secretion of both IL-4 and IFN-γ cytokines. Furthermore, virus challenge experiments confirmed that TBP5 contributed to inhibition replication of the virus [H9N2 AIV (A/chicken/Jiangsu/NJ07/05)] from mouse lungs. Altogether, these findings suggest that TBP5 may be an effective adjuvant for avian vaccine and that this study provides a reference for further research on new vaccine adjuvants.

Porcine Mx1 fused to HIV Tat protein transduction domain (PTD) inhibits classical swine fever virus infection in vitro and in vivo.

BACKGROUND: Classical swine fever (CSF) caused by CSF virus (CSFV) is highly contagious andcauses significant economic losses in the pig industry throughout the world. Previously we demonstrated that porcine Mx1 (poMx1), when fused to HIV Tat protein transduction domain (PTD), inhibits CSFV propagation in PK-15 cells, but it is unknown whether PTD-poMx1 exhibits antiviral activity in other porcine lines and it is efficacious for controlling CSFV infection in pigs in China. METHODS: Two porcine cell lines, ST and 3D4/21, were used to investigate in vitro antiviral activity of PTD-poMx1 against CSFV using confocal microscopy, western blot, flow cytometry, and real-time RT-PCR. Furthermore, in vivo antiviral activity of PTD-poMx1 was assessed by means of rectal temperature, clinical score, pathological lesion, white blood cell count, viral load, etc. RESULTS: PTD-poMx1 entered both cell lines within 3 h and maintained for 16 h, but did not affect CSFV binding and uptake. Viral titers and qRT-PCR data showed that PTD-poMx1 inhibited CSFV replication in both cell lines, showing significant antiviral activity after infection. Injection of PTD-poMx1 into CSFV-challenged pigs attenuated CSFV symptoms and viremia in dose-dependent manner but did not completely block virus replication within 14 days post challenge, suggesting that PTD-poMx1 confers partial protection against a lethal challenge. CONCLUSION: We demonstrated the anti-CSFV activity of PTD-poMx1 in vitro and in vivo. The results have shown that treatment with PTD-poMx1 alleviated symptoms and viral load in infected pigs. The results support our previous in vitro studies and suggest that PTD-poMx1 could be promising in reducing the clinical signs caused by CSFV.

The A66G back mutation in NS2A of JEV SA14-14-2 strain contributes to production of NS1' protein and the secreted NS1' can be used for diagnostic biomarker for virulent virus infection.

Japanese encephalitis virus (JEV) is the most common cause of the prevalent encephalitis in Asia-Pacific region and poses a serious risk to public health. Here, we developed a reliable reverse genetics system based on the JEV SA14-14-2 strain to further explore the mechanism for the synthesis of NS1' protein and to investigate the function of NS1' protein during virus infection. NS1' is an additional form of NS1 protein with 52 amino acid carboxy-terminal extension and is expressed by the members of the Japanese encephalitis (JE) serogroup due to the translation frameshift. A66G substitution in NS2A gene of JEV SA14-14-2 strain contributed to recover the GC-rich pseudoknot and resulted in the formation of the NS1'. The NS1' protein has no significant effect on the virus replication properties in BHK-21 cells. Animal experiments demonstrated that the NS1' protein had a rather minor effect on neurovirulence of JEV SA14-14-2 strain. But the NS1'-expressing virus (rA66G) could induce a higher humoral immune response than the NS1'-non-expressing virus (rSA14-14-2). NS1' protein can be detected in the serum of JEV rA66G infected animal and in the cultural media of that infected mammalian cells. Interesting, only the dimer of NS1' can be detected in the cultural media of the infected BHK-21 cells and the amount of the secreted NS1' was in agreement with that of the secreted virion. In comparison with the live-attenuated JE vaccine strain which is incapable of formation of NS1', most of the virulent JEV strains produce the NS1' protein. And the secreted NS1' may serve as an early surrogate biomarker for viremia to distinguish the field infection from the vaccine inoculation. In total, in the present study, we identified the nt 66 in the viral NS2A gene as one of the critical site for the -1 programmed ribosomal frameshift to produce the NS1' protein and demonstrated the secreted NS1' could be used for diagnostic biomarker during JEV infection.

Isolation and immunomodulatory activity of bursal peptide, a novel bursal peptide from the chicken bursa of Fabricius.

The bursa of Fabricius (BF), which is unique to birds, serves as the central humoral immune organ and plays a significant role in B lymphocyte differentiation. In this study, a new bursal peptide (BP-IV) was isolated from BF, which promoted colony-forming unit pre-B formation and regulated B cell differentiation. BP-IV also exerted immunomodulatory effects on antigen-specific immune responses via both humoral and cellular immunity in chicken and mice that had been immunized with inactivated avian influenza virus (AIV; H9N2 subtype), including enhancing AIV-specific antibody and cytokine production. The results of this study provided novel insights into the use of a potential candidate reagent for B cell development and future immuno-pharmacological use.

[Antitumor mechanism of Bursopentin (BP5)].

OBJECTIVE: Bursopentin (BP5) is a multi-functional bioactive peptide with functions of immunomodulatory, antioxidant and antitumor. However, the antitumor mechanism of BP5 is still unclear. METHODS: We constructed T7 phage cDNA library of DT40 cells, and the proteins interacted with BP5 were identified. Then, the expression profile of BP5-treated DT40 cells were analyzed using gene microarray, p53 Luciferase activity was detected. RESULTS: The results of the expression profiling revealed that BP5 regulated expression of 1078 genes, of which 537 were up-regulated and 541 were down-regulated. Differentially expressed genes involved in various pathways were identified, of which 25 pathways were associated with immune responses and tumorigenic processes, including the p53 signaling. Furththmore, BP5 significantly enhanced p53 luciferase activity and stimulated expression of p53 protein in HCT116 cells. CONCLUSION: These results suggest that BP5 exerted antitumor activity through p53 signaling and that this study provides novel insights on the antitumor mechanism of BP5.

Integration analysis of miRNA and mRNA expression profiles in swine testis cells infected with Japanese encephalitis virus.

To elucidate the role of microRNAs (miRNA) in the regulation of gene expression in Japanese encephalitis virus (JEV) infected swine testis (ST) cells, we analyzed miRNA and mRNA expression profiles of JEV infected ST cells by high-throughput sequencing technology as compared to uninfected controls. The results showed that 104 known miRNAs and 9 new miRNA candidates were differentially expressed in ST cells after JEV infection. We identified 396 differentially expressed mRNAs. Bioinformatics analysis identified 435 known miRNA-mRNA interaction pairs and 94 novel miRNA-mRNA interaction pairs involving miRNAs inversely correlated with the expression of their predicted target mRNAs. The known miRNAs inversely correlated with their target genes were involved in the biological processes of immunity, cytokine production, inflammation, and apoptosis. Selected miRNA-mRNA interactions were validated by luciferase reporter assay. Overall, our findings indicate that miRNAs may play critical roles in the pathogenesis of JEV infection.

The potential mechanism of bursal-derived BP8 on B cell developments.

The bursa of Fabricius, the key humoral immune organ unique to birds, is critical for B cell differentiation and antibody production. BP8 (AGHTKKAP) is a novel immunomodulatory peptide that regulates B-cell development. Gene microarray was used to investigate the mechanism of BP8 on B cell development. BP8 regulated expressions of 1,570 genes that were involved in retinol metabolism, the Wnt signaling pathway, MAPK pathway, Jak-Stat pathway, Notch signaling pathway, cytokine-cytokine receptor interaction, and Ca(2+) signals. Finally, BP8 triggered ADH7 and RDH10 expression, interacted with retinol binding protein, and regulated retinol uptake in vitro and vivo. These data reveal a bursal-derived multifunctional factor, BP8, as a novel biomaterial which is essential for the development of the immune system and represents an important linker between the B cell development and retinol metabolism. This study elucidates the mechanisms involved in humoral immune system and has implications in treating human diseases.

Isolation and genome characterization of a novel duck Tembusu virus with a 74 nucleotide insertion in the 3' non-translated region.

During investigations into the outbreak of duck Tembusu virus (DTMUV) infection in 2011 in China, a DTMUV strain (DTMUV-AH2011) was isolated from the affected ducks. The length of the genome of the DTMUV-AH2011 strain was found to be 11,064 nucleotides and to possess 10,278 nucleotides of one open reading frame (ORF), flanked by 94 nucleotides of the 5' non-translated region (NTR) and 692 nucleotides of the 3' NTR. In comparison with five fully sequenced TMUV genomes, the genome of DTMUV-AH2011 had a 74 nucleotide insertion in the 3' NTR. Comparison of the DTMUV-AH2011 fully deduced amino acid sequences with those of other Tembusu virus strains reported recently in China showed they had a highly conserved polyprotein precursor, sharing 98.9% amino acid identities, at least. The overall divergences of amino acid substitutions were randomly distributed among viral proteins except for the protein NS4B, the protein NS4B was unchanged. Knowledge of the biological characters of DTMUV and the potential role of the insertion in the 3' NTR in RNA replication will be useful for further studies of the mechanisms of virus replication and pathogenesis.