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Lignosus rhinocerus (L. rhinocerus) is a rare in Southeast Asia and has been long used by the natives for various medicinal purposes. In the advent of the mushroom’s successful cultivation, a novel cultivar, named L.rhinocerus TM02®, was produced and has been tested for anti-inflammatory, anti-proliferative, antioxidant, and other medicinal properties. However, the antiviral effects of TM02® cultivar have not been demonstrated. In this study, the antiviral properties of cold-water extract (CWE) from the sclerotium of L.rhinocerus TM02® cultivar were investigated, in inhibiting dengue serotype 2 infection in Vero cells. The authors observed a dose-dependent inhibition on dengue virus replication, particularly during the early stage of dengue infection. The extract also exhibited a significant virucidal effect and displayed a mild prophylactic effect. Hence, in the absence of anti-dengue treatment and limited dengue vaccination, these results indicate that TM02® CWE may serve as an alternative medicine in reducing dengue infection. Further studies are required to identify and verify the bioactive compounds of TM02® CWE that mediate the antiviral activity.

Effective novel peptide inhibitors which targeted the domain III of the dengue envelope (E) protein by blocking dengue virus (DENV) entry into target cells, were identified. The binding affinities of these peptides towards E-protein were evaluated by using a combination of docking and explicit solvent molecular dynamics (MD) simulation methods. The interactions of these complexes were further investigated by using the Molecular Mechanics-Poisson Boltzmann Surface Area (MMPBSA) and Molecular Mechanics Generalized Born Surface Area (MMGBSA) methods. Free energy calculations of the peptides interacting with the E-protein demonstrated that van der Waals (vdW) and electrostatic interactions were the main driving forces stabilizing the complexes. Interestingly, calculated binding free energies showed good agreement with the experimental dissociation constant (Kd) values. Our results also demonstrated that specific residues might play a crucial role in the effective binding interactions. Thus, this study has demonstrated that a combination of docking and molecular dynamics simulations can accelerate the identification process of peptides as potential inhibitors of dengue virus entry into host cells.

Chikungunya virus (CHIKV) infection is a persistent problem worldwide due to efficient adaptation of the viral vectors, Aedes aegypti and Aedes albopictus mosquitoes. Therefore, the absence of effective anti-CHIKV drugs to combat chikungunya outbreaks often leads to a significant impact on public health care. In this study, we investigated the antiviral activity of drugs that are used to alleviate infection symptoms, namely, the non-steroidal anti-inflammatory drugs (NSAIDs), on the premise that active compounds with potential antiviral and anti-inflammatory activities could be directly subjected for human use to treat CHIKV infections. Amongst the various NSAID compounds, Mefenamic acid (MEFE) and Meclofenamic acid (MECLO) showed considerable antiviral activity against viral replication individually or in combination with the common antiviral drug, Ribavirin (RIBA). The 50% effective concentration (EC50) was estimated to be 13 μM for MEFE, 18 μM for MECLO and 10 μM for RIBA, while MEFE + RIBA (1:1) exhibited an EC50 of 3 μM, and MECLO + RIBA (1:1) was 5 μM. Because MEFE is commercially available and its synthesis is easier compared with MECLO, MEFE was selected for further in vivo antiviral activity analysis. Treatment with MEFE + RIBA resulted in a significant reduction of hypertrophic effects by CHIKV on the mouse liver and spleen. Viral titre quantification in the blood of CHIKV-infected mice through the plaque formation assay revealed that treatment with MEFE + RIBA exhibited a 6.5-fold reduction compared with untreated controls. In conclusion, our study demonstrated that MEFE in combination with RIBA exhibited significant anti-CHIKV activity by impairing viral replication in vitro and in vivo. Indeed, this finding may lead to an even broader application of these combinatorial treatments against other viral infections.

Dengue virus (DV) infection demonstrates an intriguing virus-induced intracellular membrane alteration that results in the augmentation of major histocompatibility complex (MHC) class I-restricted antigen presentation. As oppose to its biological function in attracting CD8(+) T-cells, this phenomenon appears to facilitate the immune evasion. However, the molecular events that attribute to the dysregulation of the antigen presenting mechanism (APM) by DV remain obscure. In this study, we aimed to characterize the host cell APM upon infection with all serotypes of whole DV. Cellular RNA were isolated from infected cells and the gene expressions of LMP2, LMP7, TAP1, TAP2, TAPBP, CALR, CANX, PDIA3, HLA-A and HLA-B were analyzed via quantitative PCR. The profiles of the gene expression were further validated. We showed that all four DV serotypes modulate host APM at the proteasomal level with DV2 showing the most prominent expression profile.

In this paper we report methacrylic microspheres (MMS), produced via free-radical polymerization between methyl methacrylate (MMA) and methacrylic acid (MAA), cross-linked with triethylene glycol dimethacrylate (TEGDMA). In particular, MAA monomer was chosen for structural carboxyl functionalities that exist on the surface of produced microspheres. Two different MMA/MAA molar ratios of MMA TEGDMA MAA microspheres were processed effectively and both compositions of MMS were analyzed with scanning electron microscopy (SEM), optical microscopy and X-ray photoelectron spectroscopy (XPS). Results confirmed uniform surface morphology and two ranges of particle size distributions for different macromolecular structures. Surface carboxyl groups (generated from MAA polymer units) were used as polymeric platforms for immobilization of Dengue antibody molecules. In order to examine the efficiency of antibody surface immobilization we have performed fluorescence enzyme-linked immunosorbent assay (ELISA). Dengue antigens were used for specific recognition of template proteins (antibodies) and results show more than 20 times higher fluorescence intensity in comparison to conventional ELISA assay used in contemporary clinical practice. Furthermore, our results show that the physical adsorption of template antibodies is almost as effective as covalent attachment through carbodiimide chemistry and formation of amide bonds between amine peptide groups and surface carboxyl groups on microspheres.

Chikungunya virus (CHIKV) outbreaks have led to a serious economic burden, as the available treatment strategies can only alleviate disease symptoms, and no effective therapeutics or vaccines are currently available for human use. Here, we report the use of a new cost-effective approach involving production of a recombinant antiviral peptide-fusion protein that is scalable for the treatment of CHIKV infection. A peptide-fusion recombinant protein LATA-PAP1-THAN that was generated by joining Latarcin (LATA) peptide with the N-terminus of the PAP1 antiviral protein, and the Thanatin (THAN) peptide to the C-terminus, was produced in Escherichia coli as inclusion bodies. The antiviral LATA-PAP1-THAN protein showed 89.0% reduction of viral plaque formation compared with PAP1 (46.0%), LATA (67.0%) or THAN (79.3%) peptides alone. The LATA-PAP1-THAN protein reduced the viral RNA load that was 0.89-fold compared with the untreated control cells. We also showed that PAP1 resulted in 0.44-fold reduction, and THAN and LATA resulting in 0.78-fold and 0.73-fold reductions, respectively. The LATA-PAP1-THAN protein inhibited CHIKV replication in the Vero cells at an EC50 of 11.2μg/ml, which is approximately half of the EC50 of PAP1 (23.7μg/ml) and protected the CHIKV-infected mice at the dose of 0.75mg/ml. We concluded that production of antiviral peptide-fusion protein in E. coli as inclusion bodies could accentuate antiviral activities, enhance cellular internalisation, and could reduce product toxicity to host cells and is scalable to epidemic response quantities.

Two major flaviviruses, dengue virus (DENV) and Zika virus (ZIKV), cause severe health and economic burdens worldwide. Recently, genome-wide screenings have uncovered the importance of regulators of the Hrd1 ubiquitin ligase-mediated endoplasmic reticulum (ER)-associated degradation (ERAD) pathway for flavivirus replication in host cells. Here we report the identification of the compound Bardoxolone methyl (CDDO-me) as a potent inhibitor of the Hrd1 ubiquitin ligase-mediated ERAD, which possesses a broad-spectrum activity against both DENV and ZIKV. Cellular thermal shift assay (CETSA) suggested that CDDO-me binds to grp94, a key component of the Hrd1 pathway, at a low nanomolar concentration, whereas interaction was not detected with its paralog Hsp90. CDDO-me and the grp94 inhibitor PU-WS13 substantially suppressed DENV2 replication and the cytopathic effects caused by DENV and ZIKV infection. The antiviral activities of both compounds were demonstrated for all four DENV serotypes and four ZIKV strains in multiple human cell lines. This study defines grp94 as a crucial host factor for flavivirus replication and identified CDDO-me as a potent small molecule inhibitor of flavivirus infection. Inhibition of grp94 may contribute to the antiviral activity of CDDO-me. Further investigation of grp94 inhibitors may lead to a new class of broad-spectrum anti-flaviviral medications.

Boesenbergia rotunda (L.) cyclohexenyl chalcone derivatives, 4-hydroxypanduratin A and panduratin A, showed good competitive inhibitory activities towards dengue 2 virus NS3 protease with the Ki values of 21 and 25 microM, respectively, whilst those of pinostrobin and cardamonin were observed to be non-competitive. NMR and GCMS spectroscopic data formed the basis of assignment of structures of the six compounds isolated.

Dengue virus type 2 NS3, a multifunctional protein, has a serine protease domain (NS3pro) that requires the conserved hydrophilic domain of NS2B for protease activity in cleavage of the polyprotein precursor at sites following two basic amino acids. In this study, we report the expression of the NS2B-NS3pro precursor in Escherichia coli as a fusion protein with a histidine tag at the N terminus. The precursor was purified from insoluble inclusion bodies by Ni(2+) affinity and gel filtration chromatography under denaturing conditions. The denatured precursor was refolded to yield a purified active protease complex. Biochemical analysis of the protease revealed that its activity toward either a natural substrate, NS4B-NS5 precursor, or the fluorogenic peptide substrates containing two basic residues at P1 and P2, was dependent on the presence of the NS2B domain. The peptide with a highly conserved Gly residue at P3 position was 3-fold more active as a substrate than a Gln residue at this position. The cleavage of a chromogenic substrate with a single Arg residue at P1 was NS2B-independent. These results suggest that heterodimerization of the NS3pro domain with NS2B generates additional specific interactions with the P2 and P3 residues of the substrates.

Background High-risk HPV E6 and E7 oncoprotein expression is important for malignant transformation of cervical cancer cells. In particular, the E7 protein of high-risk HPV binds to pRB family members (pRB, p107, and p130) for degradation. p107 and p130 pocket proteins are members of mammalian DREAM complexes, which are temporally regulated during cell cycle. E7 targets p130 for HPV16 to promote the host cell to exit from quiescence (G 0 ) state and enter S phase. However, p130 is also degraded in HPV48 even though HPV48 E7 does not binding directly to the pocket protein. Therefore we explored the mechanisms responsible for the activities of HPV16 E7 and HPV48 E7 on disruption of p130/DREAM complex. Methods The experiments were done with a p130 mutant that is defective in binding the E7 LXCXE motif (p130mE7) and another mutant that cannot be phosphorylated by CDK (p130PM22). In addition, a double mutant (p130PM22/mE7) was constructed. The p130 mutants and the wild-type p130 were expressed in HPV-transformed cell lines and FACS analysis was done. Subsequently, the T98G glioblastoma cell line was used to ectopically express both HPV16 and 48 with the p130 mutants. Findings The p130mE7 and p130mE7/PM22 mutants prevented the binding of 16E7 in CaSki cells, whereas p130PM22 and p130mE7/PM22 mutants prevented the binding of 48E7 in T98G cells. Interpretation HPV16 E7 targets p130 predominantly through direct interactions via the LXCXE motif whereby HPV48 E7 disrupts p130/DREAM via CDK2 phosphorylation.