Antiviral evaluation of an Hsp90 inhibitor, gedunin, against dengue virus

Purpose: To evaluate the antiviral potential of a tetranortriterpenoid, gedunin, against dengue virus (DENV) replication by targeting the host chaperone, Hsp90.Methods: The compound, gedunin, was tested against the replication of DENV in vitro using BHK-15 cells transfected with DENV-2 subgenomic replicon. Molecular docking of gedunin with Hsp90 protein was performed for evaluation of mode of action, using the program, Autodock vina.Results: In vitro antiviral data showed that gedunin significantly (p < 0.05) reduced DENV replication with EC50 of 10 μM. Further, in silico molecular docking data revealed strong interaction of gedunin with the ATP/ADP binding site of the host protein, Hsp90, with an estimated average free binding energy of -8.9 kcal/mol.Conclusion: The results validate gedunin as a potential antiviral candidate. Further in vitro assays and in vivo viral challenge studies are required to confirm the exact mode of action and pharmacological profile of gedunin in DENV infections.Keywords: Dengue virus replication, Hsp90, Gedunin, Antiviral, Molecular dockin

Frequency of hepatitis E and Hepatitis A virus in water sample collected from Faisalabad, Pakistan

Hepatitis E and Hepatitis A virus both are highly prevalent in Pakistan mainly present as a sporadic disease. The aim of the current study is to isolate and characterized the specific genotype of Hepatitis E virus from water bodies of Faisalabad, Pakistan. Drinking and sewage samples were qualitatively analyzed by using RT-PCR. HEV Genotype 1 strain was recovered from sewage water of Faisalabad. Prevalence of HEV and HAV in sewage water propose the possibility of gradual decline in the protection level of the circulated vaccine in the Pakistani population

Large-Scale in Vitro Transcription, RNA Purification and Chemical Probing Analysis

Background/Aims: RNA elements such as catalytic RNA, riboswitch, microRNA, and long non coding RNA (lncRNA) play central roles in many cellular processes. Studying diverse RNA functions require large quantities of RNA for precise structure analysis. Current RNA structure and function studies can benefit from improved RNA quantity and quality, simpler separation procedure and enhanced accuracy of structural analysis. Methods: Here we present an optimized protocol for analyzing the structure of any RNA, including in vitro transcription, size-exclusion chromatography (SEC) based denaturing purification and improved secondary structure analysis by chemical probing. Results: We observed that higher Mg2+, nucleoside triphosphate (NTP) concentrations and longer reaction duration can improve the RNA yield from in vitro transcription, specifically for longer and more complicated constructs. Our improved SEC-based denaturing RNA purification effectively halved the experiment duration and labor without introducing any contaminant. Finally, this study increased the accuracy and signal-to-noise ratio (SNR) of selective 2′-hydroxyl acylation analyzed by primer extension (SHAPE) chemical probing for analyzing RNA structure. Conclusion: Part or all of our modified method can improve almost any RNA-related study from protein-RNA interaction analysis to crystallography

PCR-Based Molecular Diagnosis of Hepatitis Virus (HBV and HDV) in HCV Infected Patients and Their Biochemical Study

Seroprevalence of HCV indicates that HCV is found in more than 10% of HBV-or HDV-infected patients worldwide leading to liver disease. Here we show HBV and HDV coinfection association with HCV infected Pakistani patients, study of disease severity, and possible interpretation of associated risk factors in coinfected patients. A total of 730 liver diseased patients were included, out of which 501 were found positive for HCV infection via PCR. 5.1% of patients were coinfected with HBV while 1% were coinfected with HBV and HDV both. LFTs were significantly altered in dually and triply infected patients as compared to single HCV infection. Mean bilirubin, AST, and ALT levels were highest (3.25 mg/dL, 174 IU/L, and 348 IU/L) in patients with triple infection while dual infection LFTs (1.6 mg/dL, 61 IU/L, and 74 IU/L) were not high as in single infection (1.9 mg/dL, 76 IU/L, and 91 IU/L). The most prominent risk factor in case of single (22%) and dual infection (27%) group was "reuse of syringes" while in triple infection it was "intravenous drug users" (60%). It is concluded that HBV and HDV coinfections are strongly associated with HCV infected Pakistani patients and in case of severe liver disease the possibility of double and triple coinfection should be kept in consideration

Red Blood Cell Substitutes: Liposome Encapsulated Hemoglobin and Magnetite Nanoparticle Conjugates as Oxygen Carriers

The established blood donation and transfusion system has contributed a lot to human health and welfare, but for this system to function properly, it requires a sufficient number of healthy donors, which is not always possible. Pakistan was a country hit hardest by COVID-19 which additionally reduced the blood donation rates. In order to address such challenges, the present study focused on the development of RBC substitutes that can be transfused to all blood types. This paper reports the development and characterization of RBC substitutes by combining the strategies of conjugated and encapsulated hemoglobin where magnetite nanoparticles would act as the carrier of hemoglobin, and liposomes would separate internal and external environments. The interactions of hemoglobin variants with bare magnetite nanoparticles were studied through molecular docking studies. Moreover, nanoparticles were synthesized, and hemoglobin was purified from blood. These components were then used to make conjugates, and it was observed that only the hemoglobin HbA1 variant was making protein corona. These conjugates were then encapsulated in liposomes to make negatively charged RBC substitutes with a size range of 1–2 μm. Results suggest that these RBC substitutes work potentially in a similar way as natural RBCs work and can be used in the time of emergency

Vaccinomics driven proteome-wide screening of Haemophilus influenzae for the prediction of common putative vaccine candidates

Haemophilus influenzae colonizes the respiratory tract and is associated with life-threatening invasive infections. The recent rise in its global prevalence, even in the presence of multiple vaccines, indicate an urgent need for developing cross-strain effective vaccine strategies. Our work focused on identifying the universally conserved antigenic regions of H. influenzae that can be used for developing new vaccines. A variety of bioinformatics tools were applied for the comprehensive geno-proteomic analysis of H. influenzae type “a” strain, as reference serotype, through which subcellular localization, essentiality, virulence, and non-host homology were determined. B and T-Cell epitope mapping of 3D protein structures were performed. Thereafter, molecular docking with HLA DRB1*0101 and comparative genome analysis established the candidature of identified regions. Based on the established vaccinomics criteria, five target proteins were predicted as novel vaccine candidates. Among these, 9 epitopic regions were identified that could regulate the lymphocyte activity through strong protein-protein interactions. Comparative genomic analysis exhibited that the identified regions were highly conserved among the different strains of H. influenzae. Based on multiple immunogenic factors, the five prioritized proteins and their predicted epitopes were identified as the ideal common putative vaccine candidate against typeable strains.The accepted manuscript in pdf format is listed with the files at the bottom of this page. The presentation of the authors' names and (or) special characters in the title of the manuscript may differ slightly between what is listed on this page and what is listed in the pdf file of the accepted manuscript; that in the pdf file of the accepted manuscript is what was submitted by the author

Development of an immunodiagnostic assay for the detection of Sugarcane mosaic virus

Sugarcane mosaic virus (SCMV) has devastating yield losses in sugarcane cultivars around the World, including Pakistan. The disease losses can be minimized, and specific control measures can be employed if the disease could be diagnosed at an early stage of infection. The present study was aimed at developing an enzyme-linked immunosorbent assay (ELISA) based polyclonal antibodies (pAB) for rapid detection of SCMV. The coat protein (CP) of SCMV was used to determine the physicochemical properties, subcellular localization, and immunogenicity through in-silico analysis. The SCMV CP was synthesized by Macrogen, Inc Korea and cloned into an expression vector, the pET28a+, to produce recombinant fusion coat protein in Escherichia coli (E. coli). The recombinant fusion coat protein (40kDa) was produced in soluble form. The fusion protein was purified through an affinity Ni-NTA resin and was visualized on a 12% SDS-PAGE. The purified protein was used, as an antigen in primary response, followed by four weekly intraperitoneal booster injections in mice to raise pAB antisera, collected by cardiac puncture. Raised antisera was able to detect 100 pg of a purified protein in Indirect ELISA. It also detected the SCMV in an infected sugarcane field plant using raised antisera, which was confirmed by RT_PCR. Thus, the results validate an easy approach to detect and diagnose SCMV infection using immunodiagnostic techniques

In Vivo Validation of Novel Synthetic <i>tbp1</i> Peptide-Based Vaccine Candidates against <i>Haemophilus influenzae</i> Strains in BALB/c Mice

Haemophilus influenzae is a Gram-negative bacterium characterized as a small, nonmotile, facultative anaerobic coccobacillus. It is a common cause of a variety of invasive and non-invasive infections. Among six serotypes (a–f), H. influenzae type b (Hib) is the most familiar and predominant mostly in children and immunocompromised individuals. Following Hib vaccination, infections due to other serotypes have increased in number, and currently, there is no suitable effective vaccine to induce cross-strain protective antibody responses. The current study was aimed to validate the capability of two 20-mer highly conserved synthetic tbp1 (transferrin-binding protein 1) peptide-based vaccine candidates (tbp1-E1 and tbp1-E2) predicted using in silico approaches to induce immune responses against H. influenzae strains. Cytokine induction ability, immune simulations, and molecular dynamics (MD) simulations were performed to confirm the candidacy of epitopic docked complexes. Synthetic peptide vaccine formulations in combination with two different adjuvants, BGs (Bacterial Ghosts) and CFA/IFA (complete/incomplete Freund’s adjuvant), were used in BALB/c mouse groups in three booster shots at two-week intervals. An indirect ELISA was performed to determine endpoint antibody titers using the Student’s t-distribution method. The results revealed that the synergistic use of both peptides in combination with BG adjuvants produced better results. Significant differences in absorbance values were observed in comparison to the rest of the peptide–adjuvant combinations. The findings of this study indicate that these tbp1 peptide-based vaccine candidates may present a preliminary set of peptides for the development of an effective cross-strain vaccine against H. influenzae in the future due to their highly conserved nature

Over expression of a synthetic gene encoding interferon lambda using relative synonymous codon usage bias in Escherichia coli

Interferon Lambda (IFN-λ) is a type III interferon which belongs to a novel family of cytokines and possesses antiviral and antitumor properties. It is unique in its own class of cytokines; because of the specificity towards its heterodimer receptors and its structural similarities with cytokines of other classes. This renders IFN-λ a better choice for the treatment against many diseases including viral hepatitis and human coronavirus (HCoV-EMC). The present study describes a computational approach known as relative synonymous codon usage (RSCU); used to enhance the expression of IFN-λ protein in a eukaryotic expression system. Manually designed and commercially synthesized IFN-λ gene was cloned into pET-22b expression plasmid under the control of inducible T7-lac promoter. Maximum levels of IFN-λ expression was observed with 0.4 mM IPTG in transformed E. coli incubated for 4 hours in LB medium. Higher concentrations of IPTG had no or negative effect on the expression of IFN-λ. This synthetically over expressed IFN-λ can be tested as a targeted treatment option for viral hepatitis after purification