Japanese encephalitis virus (JEV), which causes viral encephalitis in humans, is a serious risk to global public health. The JEV envelope protein mediates the viral entry pathway, including receptor-binding and low-pH-triggered membrane fusion. Utilizing mutagenesis of a JEV infectious cDNA clone, mutations were introduced into the potential receptor-binding motif or into residues critical for membrane fusion in the envelope protein to systematically investigate the JEV entry mechanism. We conducted experiments evaluating infectious particle, recombinant viral particle, and virus-like particle production and found that most mutations impaired virus production. Subcellular fractionation confirmed that five mutations-in I 0 , ij, BC, and FG and the R9A substitution-impaired virus assembly, and the assembled virus particles of another five mutations-in kl and the E373A, F407A, L221S, and W217A substitutions-were not released into the secretory pathway. Next, we examined the entry activity of six mutations yielding infectious virus. The results showed N154 and the DE loop are not the only or major receptorbinding motifs for JEV entry into BHK-21 cells; four residues, H144, H319, T410, and Q258, participating in the domain I (DI)-DIII interaction or zippering reaction are important to maintain the efficiency of viral membrane fusion. By continuous passaging of mutants, adaptive mutations from negatively charged amino acids to positively charged or neutral amino acids, such as E138K and D389G, were selected and could restore the viral entry activity. IMPORTANCERecently, there has been much interest in the entry mechanism of flaviviruses into host cells, including the viral entry pathway and membrane fusion mechanism. Our study provides strong evidence for the critical role of several residues in the envelope protein in the assembly, release, and entry of JEV, which also contributes to our understanding of the flaviviral entry mechanism. Furthermore, we demonstrate that the H144A, H319A, T410A, and Q258A mutants exhibit attenuated fusion competence, which may be used to develop novel vaccine candidates for flaviviruses. Japanese encephalitis virus (JEV) is a mosquito-borne zoonotic flavivirus that causes viral encephalitis in most of Asia, Papua New Guinea, and the Torres Strait of northern Australia (1, 2). The recent emergence of JEV in the Torres Strait islands and its spread onto the Cape York Peninsula pose a serious risk to public health in Australia and have elicited growing concern that this virus can spread throughout the world (3). JEV is one of the most important members of the JEV serological complex, which includes West Nile virus (WNV), St. Louis encephalitis virus (SLEV), and Murray Valley encephalitis virus (MVEV), causing approximately 67,900 cases of encephalitis annually in countries of Japanese encephalitis (JE) endemicity and having high morbidity and mortality rates (4, 5). The case fatality rate for JE is 20% to 30%, and 30% to 50% of survivors have severe neurological sequelae even years...

Background: Recent studies have validated and confirmed the great potential of nanoscale metal-organic framework (NMOF) in the biomedical field, especially in improving the efficiency of cancer diagnosis and therapy. However, most previous studies only utilized either the metal cluster or the organic ligand of the NMOF for cancer treatments and merely reported limited theranostic functions, which may not be optimized. As a highly designable and easily functionalized material, prospective rational design offers a powerful way to extract the maximum benefit from NMOF for cancer theranostic applications. Materials and Methods: A NMOF based on hafnium (Hf) cluster and Mn(III)-porphyrin ligand was rational designed and synthesized as a high-performance multifunctional theranostic agent. The folic acid (FA) was modified on the NMOF surface to enhance the cancer targeting efficacy. The proposed "all-in-one" FA-Hf-Mn-NMOF (fHMNM) was characterized and identified using various analytical techniques. Then, in vitro and in vivo studies were performed to further explore the effects of fHMNM both as the magnetic resonance imaging (MRI)/computed tomography (CT)/photoacoustic imaging (PAI) contrast agent and as the photothermal therapy (PTT)/radiotherapy (RT) agent. Results: A tumour targeting multifunctional fHMNM was successfully synthesized with high performance for MRI/CT/PAI enhancements and image-guided PTT/RT synergistic therapy properties. Compared with the current clinical CT and MR contrast agents, the X-ray attenuation and T 1 relaxation rate of this integrated nanosystem increased 1.7-fold and 3-5-fold, respectively. More importantly, the catalase-like Mn(III)-porphyrin ligand can decompose H 2 O 2 into O 2 in tumour microenvironments to improve the synergistic treatment efficiency of PTT and RT. Significant tumour growth inhibition was achieved in mouse cancer models without obvious damage to the other organs. Conclusion: This work highlights the potential of fHMNM as an easily designable material for biomedical applications, could be an effective tool for in vivo detection and subsequent treatment of tumour.

The host-virus interaction during the cellular entry of Japanese encephalitis virus (JEV) is poorly characterized. The ubiquitin-proteasome system (UPS), the major intracellular proteolytic pathway, mediates diverse cellular processes, including endocytosis and signal transduction, which may be involved in the entry of virus. Here, we showed that the proteasome inhibitors, MG132 and lactacystin, impaired the productive entry of JEV by effectively interfering with viral intracellular trafficking at the stage between crossing cell membrane and the initial translation of the viral genome after uncoating. Using confocal microscopy, it was demonstrated that a proportion of the internalized virions were misdirected to lysosomes following treatment with MG132, resulting in non-productive entry. In addition, using specific siRNAs targeting ubiquitin, we verified that protein ubiquitination was involved in the entry of JEV. Overall, our study demonstrated the UPS is essential for the productive entry of JEV and might represent a potential antiviral target for JEV infection.

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