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Virus genome recoding is an attenuation method that confers genetically stable attenuation by rewriting a virus genome with numerous silent mutations. Prior flavivirus genome recoding attempts utilised codon deoptimisation approaches. However, these codon deoptimisation approaches act in a species dependent manner and were unable to confer flavivirus attenuation in mosquito cells or in mosquito animal models. To overcome these limitations, we performed flavivirus genome recoding using the contrary approach of codon optimisation. The genomes of flaviviruses such as dengue virus type 2 (DENV2) and Zika virus (ZIKV) contain functional RNA elements that regulate viral replication. We hypothesised that flavivirus genome recoding by codon optimisation would introduce silent mutations that disrupt these RNA elements, leading to decreased replication efficiency and attenuation. We chose DENV2 and ZIKV as representative flaviviruses and recoded them by codon optimising their genomes for human expression. Our study confirms that this recoding approach of codon optimisation does translate into reduced replication efficiency in mammalian, human, and mosquito cells as well as in vivo attenuation in both mice and mosquitoes. In silico modelling and RNA SHAPE analysis confirmed that DENV2 recoding resulted in the extensive disruption of genomic structural elements. Serial passaging of recoded DENV2 resulted in the emergence of rescue or adaptation mutations, but no reversion mutations. These rescue mutations were unable to rescue the delayed replication kinetics and in vivo attenuation of recoded DENV2, demonstrating that recoding confers genetically stable attenuation. Therefore, our recoding approach is a reliable attenuation method with potential applications for developing flavivirus vaccines. Author summary: The mosquito-borne flaviviruses such as dengue virus (DENV) and Zika virus (ZIKV) have established themselves as major human pathogens. Live attenuated vaccines are seen as the most effective method for preventing flavivirus infection. Flavivirus genome recoding has emerged as a next-generation vaccine development method that acts by rewriting the flavivirus genome. Previous flavivirus genome recoding attempts were based on deoptimising the flavivirus genome. However, these deoptimised flaviviruses were found to be attenuated in a species dependent manner. For example, deoptimised DENV and ZIKV did not demonstrate attenuation in mosquito cells or mosquito animal models, which is undesirable because these mosquito-borne flaviviruses should be attenuated in their mosquito vector to prevent vaccine escape. To overcome these limitations, we adopted a flavivirus genome recoding approach based on the contrary approach of optimising the flavivirus genome and applied it to DENV2 and ZIKV. We found that this genome recoding approach of codon optimisation could confer attenuation in both mouse and mosquito animal models. This indicates that our flavivirus genome recoding approach may be used as a reliable method to construct attenuated vaccine backbones for the mosquito-borne-flaviviruses in general. [ABSTRACT FROM AUTHOR]

Dengue virus (DV) infection is the most prevalent mosquito-borne viral disease and its manifestation has been shown to be contributed in part by the host immune responses. In this study, pathogen recognition receptors, Toll-like receptor (TLR) 2 and TLR6 were found to be up-regulated in DV-infected human PBMC using immunofluorescence staining, flow cytometry and Western blot analyses. Using ELISA, IL-6 and TNF-α, cytokines downstream of TLR2 and TLR6 signaling pathways were also found to be up-regulated in DV-infected PBMC. IL-6 and TNF-α production by PBMC were reduced when TLR2 and TLR6 were blocked using TLR2 and TLR6 neutralizing antibodies during DV infection. These results suggested that signaling pathways of TLR2 and TLR6 were activated during DV infection and its activation contributed to IL-6 and TNF-α production. DV NS1 protein was found to significantly increase the production of IL-6 and TNF-α when added to PBMC. The amount of IL-6 and TNF-α stimulated by DV NS1 protein was reduced when TLR2 and TLR6 were blocked, suggesting that DV NS1 protein is the viral protein responsible for the activation of TLR2 and TLR6 during DV infection. Secreted alkaline phosphatase (SEAP) reporter assay was used to further confirm activation of TLR2 and TLR6 by DV NS1 protein. In addition, DV-infected and DV NS1 protein-treated TLR6-/- mice have higher survivability compared to DV-infected and DV NS1 protein-treated wild-type mice. Hence, activation of TLR6 via DV NS1 protein could potentially play an important role in the immunopathogenesis of DV infection. [ABSTRACT FROM AUTHOR]

Human Enterovirus 71 (EV71) commonly causes Hand, Foot and Mouth Disease in young children, and occasional occurrences of neurological complications can be fatal. In this study, a high-throughput cell-based screening on the serine/threonine kinase siRNA library was performed to identify potential antiviral agents against EV71 replication. Among the hits, Misshapen/NIKs-related kinase (MINK) was selected for detailed analysis due to its strong inhibitory profile and novelty. In the investigation of the stage at which MINK is involved in EV71 replication, virus RNA transfection in MINK siRNA-treated cells continued to cause virus inhibition despite bypassing the normal entry pathway, suggesting its involvement at the post-entry stage. We have also shown that viral RNA and protein expression level was significantly reduced upon MINK silencing, suggesting its involvement in viral protein synthesis which feeds into viral RNA replication process. Through proteomic analysis and infection inhibition assay, we found that the activation of MINK was triggered by early replication events, instead of the binding and entry of the virus. Proteomic analysis on the activation profile of p38 Mitogen-activated Protein Kinase (MAPK) indicated that the phosphorylation of p38 MAPK was stimulated by EV71 infection upon MINK activation. Luciferase reporter assay further revealed that the translation efficiency of the EV71 internal ribosomal entry site (IRES) was reduced after blocking the MINK/p38 MAPK pathway. Further investigation on the effect of MINK silencing on heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) localisation demonstrated that cytoplasmic relocalisation of hnRNP A1 upon EV71 infection may be facilitated via the MINK/p38 MAPK pathway which then positively regulates the translation of viral RNA transcripts. These novel findings hence suggest that MINK plays a functional role in the IRES-mediated translation of EV71 viral RNA and may provide a potential target for the development of specific antiviral strategies against EV71 infection. [ABSTRACT FROM AUTHOR]

A close relative of poliovirus, enterovirus 71 (EV71) is regarded as an important neurotropic virus of serious public health concern. EV71 causes Hand, Foot and Mouth Disease and has been associated with neurological complications in young children. Our limited understanding of the mechanisms involved in its neuropathogenesis has hampered the development of effective therapeutic options. Here, using a two-dimensional proteomics approach combined with mass spectrometry, we have identified a unique panel of host proteins that were differentially and dynamically modulated during EV71 infection of motor-neuron NSC-34 cells, which are found at the neuromuscular junctions where EV71 is believed to enter the central nervous system. Meta-analysis with previously published proteomics studies in neuroblastoma or muscle cell lines revealed minimal overlapping which suggests unique host-pathogen interactions in NSC-34 cells. Among the candidate proteins, we focused our attention on prohibitin (PHB), a protein that is involved in multiple cellular functions and the target of anti-cancer drug Rocaglamide (Roc-A). We demonstrated that cell surface-expressed PHB is involved in EV71 entry into neuronal cells specifically, while membrane-bound mitochondrial PHB associates with the virus replication complex and facilitates viral replication. Furthermore, Roc-A treatment of EV71-infected neuronal cells reduced significantly virus yields. However, the inhibitory effect of Roc-A on PHB in NSC-34 cells was not through blocking the CRAF/MEK/ERK pathway as previously reported. Instead, Roc-A treated NSC-34 cells had lower mitochondria-associated PHB and lower ATP levels that correlated with impaired mitochondria integrity. In vivo, EV71-infected mice treated with Roc-A survived longer than the vehicle-treated animals and had significantly lower virus loads in their spinal cord and brain, whereas virus titers in their limb muscles were comparable to controls. Together, this study uncovers PHB as the first host factor that is specifically involved in EV71 neuropathogenesis and a potential drug target to limit neurological complications. [ABSTRACT FROM AUTHOR]

Dengue virus (DENV) is one of the most important arthropod-borne pathogens that cause life-threatening diseases in humans. However, no vaccine or specific antiviral is available for dengue. As seen in other RNA viruses, the innate immune system plays a key role in controlling DENV infection and disease outcome. Although the interferon (IFN) response, which is central to host protective immunity, has been reported to limit DENV replication, the molecular details of how DENV infection is modulated by IFN treatment are elusive. In this study, by employing a gain-of-function screen using a type I IFN-treated cell-derived cDNA library, we identified a previously uncharacterized gene, C19orf66, as an IFN-stimulated gene (ISG) that inhibits DENV replication, which we named epressor of ield of V (RyDEN). Overexpression and gene knockdown experiments revealed that expression of RyDEN confers resistance to all serotypes of DENV in human cells. RyDEN expression also limited the replication of hepatitis C virus, Kunjin virus, Chikungunya virus, herpes simplex virus type 1, and human adenovirus. Importantly, RyDEN was considered to be a crucial effector molecule in the IFN-mediated anti-DENV response. When affinity purification-mass spectrometry analysis was performed, RyDEN was revealed to form a complex with cellular mRNA-binding proteins, poly(A)-binding protein cytoplasmic 1 (PABPC1), and La motif-related protein 1 (LARP1). Interestingly, PABPC1 and LARP1 were found to be positive modulators of DENV replication. Since RyDEN influenced intracellular events on DENV replication and, suppression of protein synthesis from DENV-based reporter construct RNA was also observed in RyDEN-expressing cells, our data suggest that RyDEN is likely to interfere with the translation of DENV via interaction with viral RNA and cellular mRNA-binding proteins, resulting in the inhibition of virus replication in infected cells. [ABSTRACT FROM AUTHOR]