Nipah virus (NiV) is a lethal bat-borne zoonotic virus that causes mild to acute respiratory distress and neurological manifestations in humans with a high mortality rate. NiV transmission to humans occurs via consumption of bat-contaminated fruit and date palm sap (DPS), or through direct contact with infected individuals and livestock. Since NiV outbreaks were first reported in pigs from Malaysia and Singapore, non-neutralizing antibodies against NiV attachment Glycoprotein (G) have also been detected in a few domestic mammals. NiV infection is initiated after NiV G binds to the host cell receptors Ephrin-B2 and Ephrin-B3. In this study, we assessed the degree of NiV host tropism in domestic and peridomestic mammals commonly found in Bangladesh that may be crucial in the transmission of NiV by serving as intermediate hosts. We carried out a protein-protein docking analysis of NiV G complexes (n = 52) with Ephrin-B2 and B3 of 13 domestic and peridomestic species using bioinformatics tools. Protein models were generated by homology modelling and the structures were validated for model quality. The different protein-protein complexes in this study were stable, and their binding affinity (ΔG) scores ranged between -8.0 to -19.1 kcal/mol. NiV Bangladesh (NiV-B) strain displayed stronger binding to Ephrin receptors, especially with Ephrin-B3 than the NiV Malaysia (NiV-M) strain, correlating with the observed higher pathogenicity of NiV-B strains. From the docking result, we found that Ephrin receptors of domestic rat (R. norvegicus) had a higher binding affinity for NiV G, suggesting greater susceptibility to NiV infections compared to other study species. Investigations for NiV exposure to domestic/peridomestic animals will help us knowing more the possible role of rats and other animals as intermediate hosts of NiV and would improve future NiV outbreak control and prevention in humans and domestic animals.
Malaysia reported the first human case of Nipah virus (NiV) in late September 1998 with encephalitis and respiratory symptoms. As a result of viral genomic mutations, two main strains (NiV-Malaysia and NiV-Bangladesh) have spread around the world. There are no licensed molecular therapeutics available for this biosafety level 4 pathogen. NiV attachment glycoprotein plays a critical role in viral transmission through its human receptors (Ephrin-B2 and Ephrin-B3), so identifying small molecules that can be repurposed to inhibit them is crucial to developing anti-NiV drugs. Consequently, in this study annealing simulations, pharmacophore modeling, molecular docking, and molecular dynamics were used to evaluate seven potential drugs (Pemirolast, Nitrofurantoin, Isoniazid Pyruvate, Eriodictyol, Cepharanthine, Ergoloid, and Hypericin) against NiV-G, Ephrin-B2, and Ephrin-B3 receptors. Based on the annealing analysis, Pemirolast for efnb2 protein and Isoniazid Pyruvate for efnb3 receptor were repurposed as the most promising small molecule candidates. Furthermore, Hypericin and Cepharanthine, with notable interaction values, are the top Glycoprotein inhibitors in Malaysia and Bangladesh strains, respectively. In addition, docking calculations revealed that their binding affinity scores are related to efnb2-pem (- 7.1 kcal/mol), efnb3-iso (- 5.8 kcal/mol), gm-hyp (- 9.6 kcal/mol), gb-ceph (- 9.2 kcal/mol). Finally, our computational research minimizes the time-consuming aspects and provides options for dealing with any new variants of Nipah virus that might emerge in the future.
Nipah virus (NiV), a paramyxovirus, was first discovered in Malaysia in 1998 in an outbreak of infection in pigs and humans and incurred a high fatality rate in humans. Fruit bats, living in vast areas extending from India to the western Pacific, were identified as the natural reservoir of the virus. However, the mechanisms that resulted in severe pathogenicity in humans (up to 70% mortality) and that enabled crossing the species barrier were not known. In this study, we established a system that enabled the rescue of replicating NiVs from a cloned DNA by cotransfection of a constructed full-length cDNA clone and supporting plasmids coding virus nucleoprotein, phosphoprotein, and polymerase with the infection of the recombinant vaccinia virus, MVAGKT7, expressing T7 RNA polymerase. The rescued NiV (rNiV), by using the newly developed reverse genetics system, showed properties in vitro that were similar to the parent virus and retained the severe pathogenicity in a previously established animal model by experimental infection. A recombinant NiV was also developed, expressing enhanced green fluorescent protein (rNiV-EGFP). Using the virus, permissibility of NiV was compared with the presence of a known cellular receptor, ephrin B2, in a number of cell lines of different origins. Interestingly, two cell lines expressing ephrin B2 were not susceptible for rNiV-EGFP, indicating that additional factors are clearly required for full NiV replication. The reverse genetics for NiV will provide a powerful tool for the analysis of the molecular mechanisms of pathogenicity and cross-species infection.
Dental pulp stem cells (DPSCs) originate from the embryonic neural crest and have neurogenic potential. The present study investigated the roles of the forward and reverse EphrinB2 signalling pathways during DPSC neurogenesis. Treatment of DPSCs with recombinant EphrinB2-Fc protein over 7 days in a neural induction culture resulted in significant downregulation of the following neural markers: βIII-Tubulin, neural cell adhesion molecule (NCAM), nestin, neurogenin 2 (NGN2), neurofilament medium polypeptide and Musashi1. Immunocytochemistry revealed that EphrinB2-Fc-treated DPSCs exhibited more rounded morphologies with fewer neurite outgrowths as well as reduced protein expression of βIII-tubulin and NGN2. Treatment of DPSCs with a peptide inhibitor specific to the EphB4 receptor significantly upregulated expression of the neural markers microtubule-associated protein 2, Musashi1, NGN2 and neuron-specific enolase, whereas treatment with a peptide inhibitor specific to the EphB2 receptor exerted negligible effects on neurogenesis. Transgenic expression of EphrinB2 in DPSCs resulted in significant upregulation of Musashi1 and NCAM gene expression, while treatment of DPSCs with recombinant EphB4-Fc protein led to significant upregulation of only Musashi1. Thus, it may be concluded that stimulation of forward EphrinB2-EphB4 signalling markedly inhibited neurogenesis in DPSCs, whereas suppression of this forward signalling pathway with peptide inhibitor specific to EphB4 promoted neurogenesis. Meanwhile, stimulation of reverse EphB4-EphrinB2 signalling only marginally enhanced the neural differentiation of DPSCs. The present findings indicate the potential application of peptide or small molecule inhibitors of EphrinB2 forward signalling in neural tissue engineering with DPSCs.