Determinants of duck Tembusu virus NS2A/2B polyprotein procession attenuated viral replication and proliferation in vitro.
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ABSTRACT:
SUBMITTER: Jiang B
PROVIDER: S-EPMC7381675 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Duck Tembusu virus (DTMUV), a member of Flaviviridae family, causes acute egg-drop syndrome in ducks. MicroRNAs (miRNAs) have been found to be involved in various biological processes, including tumor genesis, viral infection, and immune response. However, the functional effect of miRNAs on DTMUV replication remains largely unclear. This study aimed to elucidate the role of host microRNA-221-3p (miR-221-3p) in regulating DTMUV replication. Here, we indicated that the expression of miR-221-3p was significantly upregulated in duck embryo fibroblasts (DEFs) during DTMUV infection. Transfection of miR-221-3p mimic significantly reduced interferon (IFN) ? production, whereas transfection of miR-221-3p inhibitor conversely significantly increased the expression of IFN-? in DTMUV-infected DEF. Moreover, we found that viral RNA copies, viral E protein expression level, and virus titer, which represent the replication and proliferation of virus, were all enhanced when transfecting the miR-221-3p mimic into DEF; reverse results were also observed by transfecting the miR-221-3p inhibitor. We also found that the expression of suppressor of cytokine signaling 5 (SOCS5) was downregulated in DEF infected with DTMUV. Besides, we further proved that SOCS5 is a target of miR-221-3p and that miR-221-3p could negatively modulate SOCS5 expression at both mRNA and protein levels. Finally, our results showed that overexpression of SOCS5 inhibited DTMUV replication and knockdown of SOCS5 enhanced DTMUV replication. Thus, our findings reveal a novel host evasion mechanism adopted by DTMUV via miR-221-3p, which may hew out novel strategies for designing miRNA-based vaccines and therapies.
| S-EPMC7186361 | biostudies-literature
Project description:Duck Tembusu virus (DTMUV) is a newly emerging pathogenic flavivirus that has caused massive economic losses to the duck industry in China. The cellular factors required for DTMUV replication have been poorly studied. The ubiquitin-proteasome system (UPS), the major intracellular proteolytic pathway, mediates diverse cellular processes, including endocytosis and signal transduction, which may be involved in the entry of virus. In the present study, we explored the interplay between DTMUV replication and the UPS in BHK-21?cells and found that treatment with proteasome inhibitor (MG132 and lactacystin) significantly decreased the DTMUV progency at the early infection stage. We further revealed that inhibition of the UPS mainly occurs on the level of viral protein expression and RNA transcription. In addition, using specific siRNAs targeting ubiquitin reduces the production of viral progeny. In the presence of MG132 the staining for the envelope protein of DTMUV was dramatically reduced in comparison with the untreated control cells. Overall, our observations reveal an important role of the UPS in multiple steps of the DTMUV infection cycle and identify the UPS as a potential drug target to modulate the impact of DTMUV infection.
| S-EPMC7126904 | biostudies-literature
Project description:Duck Tembusu virus (DTMUV), a neurotropic flavivirus, is a causative agent of severe neurological diseases in different birds. No approved vaccines or antiviral therapeutic treatments are available to date. The poultry industry experiences significant economic losses due to DTMUV infections. Minocycline is a second-generation semi-synthetic tetracycline analogue that is commonly used as an antimicrobial treatment. Experimental studies have indicated the successful protective effects of minocycline against neuronal cell death from neurodegenerative diseases and viral encephalitis. The aim of this study was to investigate the effects of minocycline on DTMUV infection in neurons. Primary duck neurons were treated with minocycline, which exhibited neuroprotective effects via anti-apoptotic function rather than through viral replication inhibition. Minocycline might serve as a potential effective drug in DTMUV infection.
| S-EPMC8111341 | biostudies-literature
Project description:Duck Tembusu virus (DTMUV) has recently appeared in ducks in China and the key cellular determiners for DTMUV replication in host cells remain unknown. Autophagy is an evolutionarily conserved cellular process that has been reported to facilitate flavivirus replication. In this study, we utilized primary duck embryo fibroblast (DEF) as the cell model and found that DTMUV infection triggered LC3-II increase and polyubiquitin-binding protein sequestosome 1 (p62) decrease, confirming that complete autophagy occurred in DEF cells. The induction of autophagy by pharmacological treatment increased DTMUV replication in DEF cells, whereas the inhibition of autophagy with pharmacological treatments or RNA interference decreased DTMUV replication. Inhibiting autophagy enhanced the activation of the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-?B) and interferon regulatory factor 7 (IRF7) pathways and increased the p62 protein level in DTMUV-infected cells. We further found that the overexpression of p62 decreased DTMUV replication and inhibited the activation of the NF-?B and IRF7 pathways, and changes in the NF-?B and IRF7 pathways were consistent with the level of phosphorylated TANK-binding kinase 1 (p-TBK1). Opposite results were found in p62 knockdown cells. In summary, we found that autophagy-mediated p62 degradation acted as a new strategy for DTMUV to evade host innate immunity.
| S-EPMC7157248 | biostudies-literature
Project description:BACKGROUND: Duck Tembusu virus is a member of the Ntaya group in the genus Flavivirus. The virus has been responsible for severe duck egg-drop syndrome in China since 2010. Its emergence and rapid spread have caused great economic loss for the poultry industry. The epidemiology of the virus infection and the potential threat to public health is of great concern because of the infective and zoonotic nature of flaviviruses. RESULTS: In this study, the pathogenicity of duck Tembusu virus in BALB/c mice was investigated. Infected mice developed clinical signs, including loss of appetite, ruffled hair, weight loss, disorientation, blindness and paralysis of hind limbs from six days post- infection following intracerebral inoculation. Morbidity was 100%, with mortality ranging from 20 to 80% in three- to eight-week-old mice. High virus titers were recovered from the brain, and the virus was distributed in several organs. Histologically, there was widespread non-suppurative encephalitis in the brain. Lymphocyte depletion in the spleen was observed, along with fatty degeneration in the liver and kidney. CONCLUSIONS: Our results demonstrate, for the first time, that duck Tembusu virus is highly neurovirulent in BALB/c mice. The mouse model used in this work was able to produce Tembusu virus infection and could be useful for elucidating some of the aspects of the pathophysiology of other flavivirus infections.
| S-EPMC3765187 | biostudies-literature
Project description:Duck Tembusu virus (DTMUV) can cause serious disease in ducks, characterized by reduced egg production. Although the virus has been isolated and detection methods developed, the host immune responses to DTMUV infection are unclear. Therefore, we systematically examined the expression of immune-related genes and the viral distribution in DTMUV-infected ducks, using quantitative real-time PCR. Our results show that DTMUV replicates quickly in many tissues early in infection, with the highest viral titers in the spleen 1 day after infection. Rig-1, Mda5, and Tlr3 are involved in the host immune response to DTMUV, and the expression of proinflammatory cytokines (Il-1?, -2, -6, Cxcl8) and antiviral proteins (Mx, Oas, etc.) are also upregulated early in infection. The expression of Il-6 increased most significantly in the tissues tested. The upregulation of Mhc-I was observed in the brain and spleen, but the expression of Mhc-II was upregulated in the brain and downregulated in the spleen. The expression of the interferons was also upregulated to different degrees in the spleen but that of the brain was various. Our study suggests that DTMUV replicates rapidly in various tissues and that the host immune responses are activated early in infection. However, the overexpression of cytokines may damage the host. These results extend our understanding of the immune responses of ducks to DTMUV infection, and provide insight into the pathogenesis of DTMUV attributable to host factors.
| S-EPMC4424876 | biostudies-literature
Project description:Duck tembusu virus (DTMUV) was firstly identified in 2010 in China; since then, it has caused enormous economic loss to breeding industry. Great efforts have been made to develop drugs and vaccines against DTMUV. However, current available vaccines or anti-DTMUV drugs are consistently inefficient. Hence, various more broadly effective drugs have become important for the treatment of DTMUV infection; among these, lycorine, one of the important sources of active alkaloids, is a promising example. Nevertheless, it is not known whether lycorine has any antiviral activities against DTMUV. Therefore, the purpose of the present study is to investigate the anti-DTMUV abilities of lycorine. The cytotoxicity of lycorine was evaluated on BHK-21 cells by CCK-8 assay, and its antiviral effect against DTMUV was examined by real-time PCR assays, virus titer determination, Western blot and immunofluorescence (IFA) assays, respectively. Furthermore, the underlying mechanisms of the anti-DTMUV effects of lycorine were also investigated. The results indicated that the highest nontoxicity concentration of lycorine on BHK-21 cells was 5 µM. Lycorine possessed the antiviral ability against DTMUV on BHK-21 cells, as demonstrated by the reduction of virus titers and copy numbers in vitro. Western blot and IFA analysis showed the inhibitory effect of lycorine on DTMUV envelope (E) protein expression. Moreover, using time-of-addition assays, we found that lycorine displays its antivirus and virucidal activities through blocking viral internalization and entry in vitro. Taken together, our findings firstly demonstrate the antiviral activities of lycorine against DTMUV, suggesting that lycorine can be a potential drug for the treatment of DTMUV infection.
| S-EPMC8414183 | biostudies-literature
Project description:Duck Tembusu virus (DTMUV) is a recently emerging pathogenic flavivirus that has resulted in a huge economic loss in the duck industry. However, no vaccine is currently available to control this pathogen. Consequently, a practical strategy to construct a vaccine against this pathogen should be determined. In this study, duck enteritis virus (DEV) was examined as a candidate vaccine vector to deliver the envelope (E) of DTMUV. A modified mini-F vector was inserted into the SORF3 and US2 gene junctions of the attenuated DEV vaccine strain C-KCE genome to generate an infectious bacterial artificial chromosome (BAC) of C-KCE (vBAC-C-KCE). The envelope (E) gene of DTMUV was inserted into the C-KCE genome through the mating-assisted genetically integrated cloning (MAGIC) strategy, resulting in the recombinant vector, pBAC-C-KCE-E. A bivalent vaccine C-KCE-E was generated by eliminating the BAC backbone. Immunofluorescence and western blot analysis results indicated that the E proteins were vigorously expressed in C-KCE-E-infected chicken embryo fibroblasts (CEFs). Duck experiments demonstrated that the insertion of the E gene did not alter the protective efficacy of C-KCE. Moreover, C-KCE-E-immunized ducks induced neutralization antibodies against DTMUV. These results demonstrated, for the first time, that recombinant C-KCE-E can serve as a potential bivalent vaccine against DEV and DTMUV.
| S-EPMC4074935 | biostudies-literature
Project description:Palmitoylation of viral proteins is crucial for host-virus interactions. In this study, we examined the palmitoylation of Japanese encephalitis virus (JEV) nonstructural protein 2A (NS2A) and observed that NS2A was palmitoylated at the C221 residue of NS2A. Blocking NS2A palmitoylation by introducing a cysteine-to-serine mutation at C221 (NS2A/C221S) impaired JEV replication in vitro and attenuated the virulence of JEV in mice. NS2A/C221S mutation had no effect on NS2A oligomerization and membrane-associated activities, but reduced protein stability and accelerated its degradation through the ubiquitin-proteasome pathway. These observations suggest that NS2A palmitoylation at C221 played a role in its protein stability, thereby contributing to JEV replication efficiency and virulence. Interestingly, the C221 residue undergoing palmitoylation was located at the C-terminal tail (amino acids 195 to 227) and is removed from the full-length NS2A following an internal cleavage processed by viral and/or host proteases during JEV infection. IMPORTANCE An internal cleavage site is present at the C terminus of JEV NS2A. Following occurrence of the internal cleavage, the C-terminal tail (amino acids 195 to 227) is removed from the full-length NS2A. Therefore, it was interesting to discover whether the C-terminal tail contributed to JEV infection. During analysis of viral palmitoylated protein, we observed that NS2A was palmitoylated at the C221 residue located at the C-terminal tail. Blocking NS2A palmitoylation by introducing a cysteine-to-serine mutation at C221 (NS2A/C221S) impaired JEV replication in vitro and attenuated JEV virulence in mice, suggesting that NS2A palmitoylation at C221 contributed to JEV replication and virulence. Based on these findings, we could infer that the C-terminal tail might play a role in the maintenance of JEV replication efficiency and virulence despite its removal from the full-length NS2A at a certain stage of JEV infection.
| S-EPMC10308905 | biostudies-literature
Project description:Duck Tembusu virus disease, caused by the duck Tembusu virus (DTMUV), can lead to a severe reduction in egg production and growth retardation in laying ducks and ducklings, respectively. In this study, we engineered a novel recombinant adenovirus expressing the E protein of DTMUV (rAd-E) in AAV-293 cells (analyzed by western blot and indirect immunofluorescence assays). Intramuscular immunization of Cherry Valley ducks with rAd-E was performed to evaluate host cellular and humoral immune responses. Compared to the phosphate-buffered saline administered group and the negative control wild-type adenovirus (wtAd) group, the rAd-E vaccinated group showed increased cellular and humoral responses. The results from the cytokine release and lymphocyte proliferation assays showed that rAd-E induced a stronger cellular immune response than the control group (P<0.01), 4 weeks after primary immunization. The results of enzyme-linked immunosorbent and virus neutralization assays showed that rAd-E induced higher titers of specific neutralizing antibodies, 2 weeks after primary immunization. The DTMUV challenge experiment showed a higher survival rate (80%) of ducks in the rAd-E group, when challenged with 0.5 ml (ELD50=10-2.67/0.2 ml) of the DTMUV strain AH-F10. These results indicate that rAd-E effectively protects ducks against DTMUV infection. Therefore, rAd-E could be a vaccine candidate to provide an effective and safe method for prevention and control of DTMUV infection.
| S-EPMC6395196 | biostudies-literature