Sofosbuvir and its tri-phosphate metabolite inhibit the RNA-dependent RNA polymerase activity of non-structural protein 5 from the Kyasanur forest disease virus.

Kyasanur forest disease is a neglected zoonotic disease caused by a single-stranded RNA-based flavivirus, the incidence of which was first recorded in 1957 in the Southern part of India. Kyasanur forest disease virus is transmitted to monkeys and humans through the infected tick bite of Haemophysalis spinigera. Kyasanur forest disease is a febrile illness, which in severe cases, results in neurological complications leading to mortality. The current treatment regimens are symptomatic and supportive, and no targeted therapies are available for this disease. In this study, we evaluated the ability of FDA-approved drugs sofosbuvir (and its active metabolite) and Dasabuvir to inhibit the RNA-dependent RNA polymerase activity of NS5 protein from the Kyasanur forest disease virus. NS5 protein containing the N-terminal methyl transferase domain and C-terminal RNA-dependent RNA polymerase domain was expressed in Escherichia coli, and RNA-dependent RNA polymerase activity was demonstrated with the purified protein. The RNA-dependent RNA polymerase assay conditions were optimized, followed by the determination of apparent Km,ATP to validate the enzyme preparation. Half maximal-inhibitory concentrations against RNA-dependent RNA polymerase activity were determined for Sofosbuvir and its active metabolite. Dasabuvir did not show detectable inhibition with the tested conditions. This is the first demonstration of the inhibition of RNA-dependent RNA polymerase activity of NS5 protein from the Kyasanur forest disease virus with small molecule inhibitors. These initial findings can potentially facilitate the discovery and development of targeted therapies for treating Kyasanur forest disease.

An improved enzyme nanoparticles based amperometric pyruvate biosensor for detection of pyruvate in serum.

The nanoparticles of commercially available pyruvate oxidase (POx) from Aerococcus species were prepared by desolvation method, which were then characterized and covalently immobilized onto gold electrode (AuE) to construct an improved model of amperometric pyruvate biosensor. The POxNPs/Au electrode was analyzed morphologically by scanning electron microscopy (SEM). On the other hand, cyclic voltammetry studies (CV) and electrochemical impedance spectroscopy (EIS) helped in deciphering the electrochemical properties of the electrode at different stages of construction. The biosensor showed optimum response within 7.5 s, at a potential of 0.28 V, pH 5.5 and 35 °C. A linear relationship was observed between biosensor response i.e. current (µA) and pyruvate concentration in the range, 0.01 µM - 5000 µM, with a lower detection limit of 0.67 µM. The analytical recovery of added pyruvate in sera was 99.0% and 99.5% within and between batch coefficient of variation (CV) were 0.045% and 0.040% respectively. The working electrode displayed an excellent correlation coefficient (R2 = 0.99%) between levels of pyruvate in sera, as detected by the standard spectrophotometric method and the present biosensor. The biosensor was utilized for detection of total pyruvate level in sera of apparently healthy individuals and patients suffering from cardiogenic stress, more specifically cardiac failure. The activity of the biosensor deteriorated by 25%, after its regular use over a period of 240 days, while being stored dry at 4°C.

Neutralizing Antibody Response and Efficacy of Novel Recombinant Tetravalent Dengue DNA Vaccine Comprising Envelope Domain III in Mice.

BACKGROUND: Dengue is a global arboviral threat to humans; causing 390 million infections per year. The availability of safe and effective tetravalent dengue vaccine is a global requirement to prevent epidemics, morbidity, and mortality associated with it. METHODS: Five experimental groups (6 mice per group) each of 5-week-old BALB/c mice were immunized with vaccine and placebo (empty plasmid) (100 µg, i.m.) on days 0, 14 and 28. Among these, four groups (one group per serotype) of each were subsequently challenged 3 weeks after the last boost with dengue virus (DENV) serotypes 1-4 (100 LD50, 20 µl intracerebrally) to determine vaccine efficacy. The fifth group of each was used as a control. The PBS immunized group was used as mock control. Serum samples were collected before and after subsequent immunizations. EDIII fusion protein expression was determined by Western blot. Total protein concentration was measured by Bradford assay. Neutralizing antibodies were assessed by TCID50-CPE inhibition assay. Statistical analysis was performed using Stata/IC 10.1 software for Windows. One-way repeated measures ANOVA and Mann-Whitney test were used for neutralizing antibody analysis and vaccine efficacy, respectively. RESULTS: The recombinant EDIII fusion protein was expressed adequately in transfected 293T cells. Total protein concentration was almost 3 times more than the control. Vaccine candidate induced neutralizing antibodies against all four DENV serotypes with a notable increase after subsequent boosters. Vaccine efficacy was 83.3% (DENV-1, -3, -4) and 50% (DENV-2). CONCLUSION: Our results suggest that vaccine is immunogenic and protective; however, further studies are required to improve the immunogenicity particularly against DENV-2.