Differential effects of lipid biosynthesis inhibitors on Zika and Semliki Forest viruses

The recent outbreak of infection with Zika virus (ZIKV; Flaviviridae) has attracted attention to this previously neglected mosquito-borne pathogen and the need for efficient therapies. Since flavivirus replication is generally known to be dependent on fatty acid biosynthesis, two inhibitors of this pathway, 5-(tetradecyloxyl)-2-furoic acid (TOFA) and cerulenin, were tested for their potentiality to inhibit virus replication. At concentrations previously shown to inhibit the replication of other flaviviruses, neither drug had a significant antiviral affect against ZIKV, but reduced the replication of the non-related mosquito-borne Semliki Forest virus (Togaviridae)

Discovery and characterisation of host-factors involved in Zika virus

Zika virus (ZIKV, Flaviviridae), like other emerging arboviruses, poses a considerable threat to human health. It is estimated that approximately half of the world’s population is at risk from contracting a mosquito-borne arboviral infection, and this was exemplified during the 2015/16 ZIKV outbreak in the Americas. ZIKV infection is thought to be largely asymptomatic, although ZIKV disease has previously been characterised by mild symptoms such as a maculopapular rash, conjunctivitis, and fever. However, recent outbreaks have been associated with an increased incidence of Guillain-Barré syndrome, and a pattern of neurological and developmental symptoms in neonates which is now termed congenital Zika syndrome. Despite intense efforts, no therapeutic or vaccine has been developed. As such, it is vital that further fundamental research is conducted to discover novel host-virus interactions in both vector and host systems, which may allow development of targeted interventions. Here, multiple approaches were used to generate basic tools for ZIKV research, and siRNA screens and data from mass-spectrometry based proteomics were utilised to uncover important host interactors of ZIKV. A study investigating the Aedes aegypti immune response was conducted, and the classical RNAi effector Argonaute 2 (Ago2) was not found to be antiviral, whereas PIWI 4 was. Data from a previous proteomics experiment suggested that glucose-regulated protein 78 kDa (GRP78) may interact with ZIKV E. In this study, co-immunoprecipitation and immunofluorescence was used to verify that GRP78 interacts with ZIKV E in both mammalian and Aedes aegypti cell culture. GRP78 is a key modulator of the unfolded protein response (UPR), and while small-molecule inhibitors (EGCG and HNK) of the GRP78-mediated UPR did not inhibit ZIKV infection, EGCG was able to inhibit ZIKV entry independent of GRP78, likely through direct binding of the virion. Further study of GRP78 revealed that while it is not important for entry, replication, or egress of ZIKV, it did aid viral translation. Depletion of GRP78 with siRNA resulted in a loss of coordination of viral replication factories and relieved a virus-specific inhibition of host translation. Furthermore, STRING analysis of GRP78 host-interactors followed by a targeted siRNA screen revealed that DnaJC1 is also a pro-viral factor. DnaJC1 has previously been shown to coordinate GRP78 localisation to ribosome exit tunnels, and so may contribute to ZIKV infection through GRP78, though this was not assessed in this study. Additionally, by using a circular polymerase extension reaction (CPER) system, a reverse genetics ZIKV was generated. This CPER ZIKV represents a genetically stable source of virus which can be easily modified and can support future research. Collectively, the data herein informs on important ZIKV interactions in both arthropod vector and mammalian systems, and highlights tools and techniques that can be used to conduct future fundamental ZIKV research

Toward adaptive radiotherapy for head and neck patients: Uncertainties in dose warping due to the choice of deformable registration algorithm.

The aims of this work were to evaluate the performance of several deformable image registration (DIR) algorithms implemented in our in-house software (NiftyReg) and the uncertainties inherent to using different algorithms for dose warping

La proteína 78 regulada por glucosa (GRP78) interactúa con la envoltura del virus del Zika y es necesaria para una infección productiva

Zika virus (ZIKV) is a member of the Flaviviridae family and was until recently a relatively obscure tropical disease. Subsequently, ZIKV has been shown to be the causative agent of fetal abnormalities and Guillain-Barré syndrome in outbreaks across the Americas and so efforts towards delineating important factors in the viral lifecycle have increased. Combining protein pull-down with mass spectrometry, it was found that ZIKV envelope (Env) interacts with the endoplasmic reticulum (ER) resident chaperone, glucose regulated protein 78 (GRP78) in A549 cells. Flaviviruses such as Japanese encephalitis virus and dengue virus are known to co-opt ER resident proteins and members of the unfolded protein response, including GRP78, to enhance viral infectivity and propagation. The role these proteins play during the ZIKV lifecycle has yet to be elucidated. To determine the importance of this interaction during ZIKV infection, A549 cells were treated with GRP78-specific siRNAs prior to infection with a NanoLuc expressing reporter virus or a wild-type virus. Depletion of GRP78 significantly reduced both virus luciferase readings and viral titres, indicating that GRP78 is necessary for efficient infection of mammalian cell culture. In contrast, inhibition of GRP78 with small molecule inhibitors did not reduce ZIKV infection. Interestingly, immunofluorescence of ZIKV infected cells reveal that GRP78 re-localises following infection and co-localises with Env. Depletion of GRP78 abrogated localisation of viral replication factories. Further experiments have shown that GRP78 is important for infection post entry and replication, and that putative GRP78 interactions partners are also required during infection

Analysis of Zika virus capsid-<i>Aedes aegypti</i> mosquito interactome reveals pro-viral host factors critical for establishing infection

The escalating global prevalence of arboviral diseases emphasizes the need to improve our understanding of their biology. Research in this area has been hindered by the lack of molecular tools for studying virus-mosquito interactions. Here, we develop an Aedes aegypti cell line which stably expresses Zika virus (ZIKV) capsid proteins in order to study virus-vector protein-protein interactions through quantitative label-free proteomics. We identify 157 interactors and show that eight have potentially pro-viral activity during ZIKV infection in mosquito cells. Notably, silencing of transitional endoplasmic reticulum protein TER94 prevents ZIKV capsid degradation and significantly reduces viral replication. Similar results are observed if the TER94 ortholog (VCP) functioning is blocked with inhibitors in human cells. In addition, we show that an E3 ubiquitin-protein ligase, UBR5, mediates the interaction between TER94 and ZIKV capsid. Our study demonstrates a pro-viral function for TER94/VCP during ZIKV infection that is conserved between human and mosquito cells

Glucose-Regulated Protein 78 Interacts with Zika Virus Envelope Protein and Contributes to a Productive Infection.

Zika virus (ZIKV; Flaviviridae) is a mosquito-borne flavivirus shown to cause fetal abnormalities collectively known as congenital Zika syndrome and Guillain-Barre syndrome in recent outbreaks. Currently, there is no specific treatment or vaccine available, and more effort is needed to identify cellular factors in the viral life cycle. Here, we investigated interactors of ZIKV envelope (E) protein by combining protein pull-down with mass spectrometry. We found that E interacts with the endoplasmic reticulum (ER) resident chaperone, glucose regulated protein 78 (GRP78). Although other flaviviruses are known to co-opt ER resident proteins, including GRP78, to enhance viral infectivity, the role ER proteins play during the ZIKV life cycle is yet to be elucidated. We showed that GRP78 levels increased during ZIKV infection and localised to sites coincident with ZIKV E staining. Depletion of GRP78 using specific siRNAs significantly reduced reporter-virus luciferase readings, viral protein synthesis, and viral titres. Additionally, GRP78 depletion reduced the ability of ZIKV to disrupt host cell translation and altered the localisation of viral replication factories, though there was no effect on viral RNA synthesis. In summary, we showed GRP78 is a vital host-factor during ZIKV infection, which may be involved in the coordination of viral replication factories

The Benefits of Temu Mangga (Curcuma Mangga Val) in Cognitive Functions of Elderly

The objective of this study was to investigate the benefit of Temu Mangga extract (Curcuma Mangga Val) with cognitive function test using Montreal Cognitive Assesment- Indonesian version (MoCA-Ina -Registry MoCA-Ina / stroke registry-INA 2012) in Elderly. This Clinical trial study was conducted using a double-blind, randomized controlled trial, pre and post-test group design in 85 female participants from Panti Wreda Nursing Home in Jakarta. A total of 83 participants who followed the study to the end. 43 participants were administered the Temu Mangga Capsule (TM) group 3x500 mg/day for 30 days.  Results: Mean score of MoCA-Ina in TM group increased by 2.37 from 23.93 ± 3.73 to 26.30 ± 3.92 with Wilcoxon test p-value = 0.000, and in the control group with 40 participants increased by 2.55 from 23.58 ± 4.60 to 26.13 ± 4.56 with Wilcoxon test p-value = 0.000. Mann-Whitney test results in both groups p &gt; 0.05. Conclusions: There was an increase in each treatment groups and control groups with significant Moca-Ina values, but between the two groups did not show significant difference changes. Psychological factors such as attention and hope will become healthier are a strong factor in this study

A plasmid DNA-launched SARS-CoV-2 reverse genetics system and coronavirus toolkit for COVID-19 research

The recent emergence of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), the underlying cause of Coronavirus Disease 2019 (COVID-19), has led to a worldwide pandemic causing substantial morbidity, mortality, and economic devastation. In response, many laboratories have redirected attention to SARS-CoV-2, meaning there is an urgent need for tools that can be used in laboratories unaccustomed to working with coronaviruses. Here we report a range of tools for SARS-CoV-2 research. First, we describe a facile single plasmid SARS-CoV-2 reverse genetics system that is simple to genetically manipulate and can be used to rescue infectious virus through transient transfection (without in vitro transcription or additional expression plasmids). The rescue system is accompanied by our panel of SARS-CoV-2 antibodies (against nearly every viral protein), SARS-CoV-2 clinical isolates, and SARS-CoV-2 permissive cell lines, which are all openly available to the scientific community. Using these tools, we demonstrate here that the controversial ORF10 protein is expressed in infected cells. Furthermore, we show that the promising repurposed antiviral activity of apilimod is dependent on TMPRSS2 expression. Altogether, our SARS-CoV-2 toolkit, which can be directly accessed via our website at https://mrcppu-covid.bio/, constitutes a resource with considerable potential to advance COVID-19 vaccine design, drug testing, and discovery science

Analysis of the Zika virus capsid-Aedes aegypti mosquito interactome reveals pro-viral host factors critical for the establishment of infection

These datasets contain measurements and proteomics data contained in the paper

La proteína 78 regulada por glucosa (GRP78) interactúa con la envoltura del virus del Zika y es necesaria para una infección productiva

Zika virus (ZIKV) is a member of the Flaviviridae family and was until recently a relatively obscure tropical disease. Subsequently, ZIKV has been shown to be the causative agent of fetal abnormalities and Guillain-Barré syndrome in outbreaks across the Americas and so efforts towards delineating important factors in the viral lifecycle have increased. Combining protein pull-down with mass spectrometry, it was found that ZIKV envelope (Env) interacts with the endoplasmic reticulum (ER) resident chaperone, glucose regulated protein 78 (GRP78) in A549 cells. Flaviviruses such as Japanese encephalitis virus and dengue virus are known to co-opt ER resident proteins and members of the unfolded protein response, including GRP78, to enhance viral infectivity and propagation. The role these proteins play during the ZIKV lifecycle has yet to be elucidated. To determine the importance of this interaction during ZIKV infection, A549 cells were treated with GRP78-specific siRNAs prior to infection with a NanoLuc expressing reporter virus or a wild-type virus. Depletion of GRP78 significantly reduced both virus luciferase readings and viral titres, indicating that GRP78 is necessary for efficient infection of mammalian cell culture. In contrast, inhibition of GRP78 with small molecule inhibitors did not reduce ZIKV infection. Interestingly, immunofluorescence of ZIKV infected cells reveal that GRP78 re-localises following infection and co-localises with Env. Depletion of GRP78 abrogated localisation of viral replication factories. Further experiments have shown that GRP78 is important for infection post entry and replication, and that putative GRP78 interactions partners are also required during infection