Antiviral activity of silymarin and baicalein against dengue virus.

Dengue is an arthropod-borne viral disease that has become endemic and a global threat in many countries with no effective antiviral drug available currently. This study showed that flavonoids: silymarin and baicalein could inhibit the dengue virus in vitro and were well tolerated in Vero cells with a half-maximum cytotoxic concentration (CC50) of 749.70 µg/mL and 271.03 µg/mL, respectively. Silymarin and baicalein exerted virucidal effects against DENV-3, with a selective index (SI) of 10.87 and 21.34, respectively. Baicalein showed a better inhibition of intracellular DENV-3 progeny with a SI of 7.82 compared to silymarin. Baicalein effectively blocked DENV-3 attachment (95.59%) to the Vero cells, while silymarin prevented the viral entry (72.46%) into the cells, thus reducing viral infectivity. Both flavonoids showed promising antiviral activity against all four dengue serotypes. The in silico molecular docking showed that silymarin could bind to the viral envelope (E) protein with a binding affinity of - 8.5 kcal/mol and form hydrogen bonds with the amino acids GLN120, TRP229, ASN89, and THR223 of the E protein. Overall, this study showed that silymarin and baicalein exhibited potential anti-DENV activity and could serve as promising antiviral agents for further development against dengue infection.

Methods in Screening Antiviral Drugs Against Enterovirus 71.

Enteroviruses 71 (EV71) is a single-stranded, neurotrophic RNA virus responsible for the numerous outbreaks of hand, foot, and mouth disease (HFMD) in the Asia-Pacific regions. HFMD primarily affects children to cause range of infection, from mild symptoms to acute flaccid paralysis, and hemorrhage. Despite increased incidence of EV71 epidemics globally and research against EV71 becoming prioritized, no antiviral agent against EV71 has yet been licensed and approved worldwide. In this chapter, detailed EV71 antiviral screening techniques are described, including plaque assay which determines viral titers through the use of a semisolid overlay, carboxymethyl cellulose to allow even viral spread and infection across the host cellular monolayers as well as a crystal violet, a distinct counterstain to visualize circular regions of infectious zones-plaques. qRT-PCR is used to quantify the viral genomic RNA in the infected samples and MTS cell viability assay to quantify the cell viability after infection or toxicity of the compound on the cells. Furthermore, various antiviral inhibition assays including prophylactic, post infection, and virucidal assays are demonstrated for estimation of the antiviral activity of potential antiviral drugs against EV71. These methods can be effectively utilized in virology laboratories for effective high-throughput screening of antiviral molecules against EV71 that can assist in the future development of antiviral drugs.

High-Throughput Screening Assays for Dengue Antiviral Drug Development.

Dengue is an arthropod-borne viral disease that has become endemic and a global threat in over 100 countries. The increase in prevalence would require a long-term measure to control outbreaks. Sanofi Pasteur has licensed the tetravalent dengue vaccine (Dengvaxia) in certain dengue endemic countries. However, the efficacy of the vaccine is limited against certain dengue serotypes and can only be used for individuals from the age from 9 to 45 years old. Over the years, there has been intense research conducted on the development of antivirals against dengue virus (DENV) through either inhibiting the virus replication or targeting the host cell mechanism to block the virus entry. However, no approved antiviral drug against dengue is yet available. In this chapter, we describe the dengue antiviral development workflow including (i) prophylactic, (ii) virucidal, and (iii) postinfection assays that are employed in the antiviral drug screening process against DENV. Further, we demonstrate different methods that can be used to enumerate the reduction in virus foci number including foci-forming unit reduction assay (FFURA), estimation of viral RNA copy number through quantitative real-time PCR, and a high-throughput enzyme linked immunosorbent assay (ELISA)-based quantification of virus particles.

Enhancement of Tetravalent Immune Responses to Highly Conserved Epitopes of a Dengue Peptide Vaccine Conjugated to Polystyrene Nanoparticles.

Vaccination remains the major approach to the prevention of dengue. Since the only licensed live attenuated vaccine (LAV) lacked efficacy against all four serotypes, other vaccine platforms, such as synthetic peptide vaccines, should be explored. In this study, four multi-epitope peptides (P1-P4) were designed by linking a universal T-helper epitope (PADRE or TpD) to the highly conserved CD8 T cell epitope and B cell epitope (B1 or B2) against all four DENV serotypes. The multi-epitope peptides were conjugated to polystyrene nanoparticles (PSNPs) and four nanovaccines (NP1-NP4) were constructed. Mice immunized with NP1-NP4 elicited significantly higher titers of IgG and neutralizing antibodies when compared to immunization with naked P1-P4. The immune responses in mice immunized with peptide vaccines were compared with nanovaccines using ELISA, ELISPOT, and a neutralization test based on FRNT50. Among the four conjugated peptide nanovaccines, NP3 comprising the TpD T-helper epitope linked to the highly conserved B1 epitope derived from the E protein was able to elicit significant levels of IFN-γ and neutralizing antibodies to all four dengue serotypes. NP3 is a promising tetravalent synthetic peptide vaccine, but the selection of a more effective CD8+ T cell epitope and adjuvants to further improve the immunogenicity is warranted.

Structure-Based Design of Antivirals against Envelope Glycoprotein of Dengue Virus.

Dengue virus (DENV) presents a significant threat to global public health with more than 500,000 hospitalizations and 25,000 deaths annually. Currently, there is no clinically approved antiviral drug to treat DENV infection. The envelope (E) glycoprotein of DENV is a promising target for drug discovery as the E protein is important for viral attachment and fusion. Understanding the structure and function of DENV E protein has led to the exploration of structure-based drug discovery of antiviral compounds and peptides against DENV infections. This review summarizes the structural information of the DENV E protein with regards to DENV attachment and fusion. The information enables the development of antiviral agents through structure-based approaches. In addition, this review compares the potency of antivirals targeting the E protein with the antivirals targeting DENV multifunctional enzymes, repurposed drugs and clinically approved antiviral drugs. None of the current DENV antiviral candidates possess potency similar to the approved antiviral drugs which indicates that more efforts and resources must be invested before an effective DENV drug materializes.

Comparative genomic analysis of ten clinical Streptococcus pneumoniae collected from a Malaysian hospital reveal 31 new unique drug-resistant SNPs using whole genome sequencing.

BACKGROUND: Streptococcus pneumoniae or pneumococcus is a leading cause of morbidity and mortality worldwide, specifically in relation to community-acquired pneumonia. Due to the overuse of antibiotics, S. pneumoniae has developed a high degree of resistance to a wide range of antibacterial drugs. METHODS: In this study, whole genome sequencing (WGS) was performed for 10 clinical strains of S. pneumoniae with different levels of sensitivity to standard antibiotics. The main objective was to investigate genetic changes associated with antibiotic resistance in S. pneumoniae. RESULTS: Our results showed that resistant isolates contain a higher number of non-synonymous single nucleotide polymorphisms (SNPs) as compared to susceptible isolates. We were able to identify SNPs that alter a single amino acid in many genes involved in virulence and capsular polysaccharide synthesis. In addition, 90 SNPs were only presented in the resistant isolates, and 31 SNPs were unique and had not been previously reported, suggesting that these unique SNPs could play a key role in altering the level of resistance to different antibiotics. CONCLUSION: Whole genome sequencing is a powerful tool for comparing the full genome of multiple isolates, especially those closely related, and for analysing the variations found within antibiotic resistance genes that lead to differences in antibiotic sensitivity. We were able to identify specific mutations within virulence genes related to resistant isolates. These findings could provide insights into understanding the role of single nucleotide mutants in conferring drug resistance.

Next generation sequencing reveals the antibiotic resistant variants in the genome of Pseudomonas aeruginosa.

Rapid progress in next generation sequencing and allied computational tools have aided in identification of single nucleotide variants in genomes of several organisms. In the present study, we have investigated single nucleotide polymorphism (SNP) in ten multi-antibiotic resistant Pseudomonas aeruginosa clinical isolates. All the draft genomes were submitted to Rapid Annotations using Subsystems Technology (RAST) web server and the predicted protein sequences were used for comparison. Non-synonymous single nucleotide polymorphism (nsSNP) found in the clinical isolates compared to the reference genome (PAO1), and the comparison of nsSNPs between antibiotic resistant and susceptible clinical isolates revealed insights into the genome variation. These nsSNPs identified in the multi-drug resistant clinical isolates were found to be altering a single amino acid in several antibiotic resistant genes. We found mutations in genes encoding efflux pump systems, cell wall, DNA replication and genes involved in repair mechanism. In addition, nucleotide deletions in the genome and mutations leading to generation of stop codons were also observed in the antibiotic resistant clinical isolates. Next generation sequencing is a powerful tool to compare the whole genomes and analyse the single base pair variations found within the antibiotic resistant genes. We identified specific mutations within antibiotic resistant genes compared to the susceptible strain of the same bacterial species and these findings may provide insights to understand the role of single nucleotide variants in antibiotic resistance.

Synthetic B-Cell Epitopes Eliciting Cross-Neutralizing Antibodies: Strategies for Future Dengue Vaccine.

Dengue virus (DENV) is a major public health threat worldwide. A key element in protection from dengue fever is the neutralising antibody response. Anti-dengue IgG purified from DENV-2 infected human sera showed reactivity against several peptides when evaluated by ELISA and epitope extraction techniques. A multi-step computational approach predicted six antigenic regions within the E protein of DENV-2 that concur with the 6 epitopes identified by the combined ELISA and epitope extraction approach. The selected peptides representing B-cell epitopes were attached to a known dengue T-helper epitope and evaluated for their vaccine potency. Immunization of mice revealed two novel synthetic vaccine constructs that elicited good humoral immune responses and produced cross-reactive neutralising antibodies against DENV-1, 2 and 3. The findings indicate new directions for epitope mapping and contribute towards the future development of multi-epitope based synthetic peptide vaccine.