SIGNIFICANCE AND IMPACT OF THE STUDY: The study has demonstrated, for the first time, the expression of nonstructural 1 (NS1) protein of influenza A virus H5N1 on the cell wall of Lactobacillus casei using the pSGANC332 expression plasmid. Display of NS1 protein on the bacterial cell wall was evident under an immunofluorescence microscopic observation. Lactobacillus casei carrying the NS1 protein could be developed into a universal oral influenza vaccine since the NS1 is highly conserved among influenza viruses.
METHODS: The effect of geraniin on DENV-2 RNA synthesis in infected Vero cells was tested using quantitative RT-PCR. The in vivo efficacy of geraniin in inhibiting DENV-2 infection was then tested using BALB/c mice with geraniin administered at three different times. The differences in spleen to body weight ratio, DENV-2 RNA load and liver damage between the three treatment groups as compared to DENV-2 infected mice without geraniin administration were determined on day eight post-infection.
RESULTS: Quantitative RT-PCR confirmed the decrease in viral RNA synthesis of infected Vero cells when treated with geraniin. Geraniin seemed to provide a protective effect on infected BALB/c mice liver when given at 24 h pre- and 24 h post-infection as liver damage was observed to be very mild even though a significant reduction of DENV-2 RNA load in serum was not observed in these two treatment groups. However, when administered at 72 h post-infection, severe liver damage in the form of necrosis and haemorrhage had prevailed despite a substantial reduction of DENV-2 RNA load in serum.
CONCLUSIONS: Geraniin was found to be effective in reducing DENV-2 RNA load when administered at 72 h post-infection while earlier administration could prevent severe liver damage caused by DENV-2 infection. These results provide evidence that geraniin is a potential candidate for the development of anti-dengue drug.
METHODS: Geraniin was prepared from Nephelium lappaceum rind by reverse phase C-18 column chromatography. Cytotoxicity of geraniin towards Vero cells was evaluated using MTT assay while IC50 value was determined by plaque reduction assay. The mode-of-action of geraniin was characterized using the virucidal, attachment, penetration and the time-of-addition assays'. Docking experiments with geraniin molecule and the DENV envelope (E) protein was also performed. Finally, recombinant E Domain III (rE-DIII) protein was produced to physiologically test the binding of geraniin to DENV-2 E-DIII protein, through ELISA competitive binding assay.
RESULTS: Cytotoxicity assay confirmed that geraniin was not toxic to Vero cells, even at the highest concentration tested. The compound exhibited DENV-2 plaque formation inhibition, with an IC50 of 1.75 μM. We further revealed that geraniin reduced viral infectivity and inhibited DENV-2 from attaching to the cells but had little effect on its penetration. Geraniin was observed to be most effective when added at the early stage of DENV-2 infection. Docking experiments showed that geraniin binds to DENV E protein, specifically at the DIII region, while the ELISA competitive binding assay confirmed geraniin's interaction with rE-DIII with high affinity.
CONCLUSIONS: Geraniin from the rind of Nephelium lappaceum has antiviral activity against DENV-2. It is postulated that the compound inhibits viral attachment by binding to the E-DIII protein and interferes with the initial cell-virus interaction. Our results demonstrate that geraniin has the potential to be developed into an effective antiviral treatment, particularly for early phase dengue viral infection.
METHODS: Using a mouse model, the miRNA expressions in liver during DENV-1 infection was investigated using high throughput miRNA sequencing. The differential expressions of miRNAs were then validated by qPCR, followed by target genes prediction. The identified miRNA targets were subjected to gene ontology (GO) annotation and pathway enrichment analysis to elucidate the potential biological pathways and molecular mechanisms associated with DENV-1 infection.
RESULTS: A total of 224 and 372 miRNAs out of 433 known mouse miRNAs were detected in the livers of DENV-1-infected and uninfected mice, respectively; of these, 207 miRNAs were present in both libraries. The miR-148a-3p and miR-122-5p were the two most abundant miRNAs in both groups. Thirty-one miRNAs were found to have at least 2-fold change in upregulation or downregulation, in which seven miRNAs were upregulated and 24 miRNAs were downregulated in the DENV-1-infected mouse livers. The miR-1a-3p was found to be the most downregulated miRNA in the DENV-1-infected mouse livers, with a significant fold change of 0.10. To validate the miRNA sequencing result, the expression pattern of 12 miRNAs, which were highly differentially expressed or most abundant, were assessed by qPCR and nine of them correlated positively with the one observed in deep sequencing. In silico functional analysis revealed that the adaptive immune responses involving TGF-beta, MAPK, PI3K-Akt, Rap1, Wnt and Ras signalling pathways were modulated collectively by 23 highly differentially expressed miRNAs during DENV-1 infection.
CONCLUSION: This study provides the first insight into the global miRNA expressions of mouse livers in response to DENV-1 infection in vivo and the possible roles of miRNAs in modulating the adaptive immune responses during DENV-1 infection.