Glycyrrhizic acid conjugates with amino acid methyl esters target the main protease, exhibiting antiviral activity against wild-type and nirmatrelvir-resistant SARS-CoV-2 variants.

COVID-19 pandemic is predominantly caused by SARS-CoV-2, with its main protease, Mpro, playing a pivotal role in viral replication and serving as a potential target for inhibiting different variants. In this study, potent Mpro inhibitors were identified from glycyrrhizic acid (GL) derivatives with amino acid methyl/ethyl esters. Out of the 17 derivatives semisynthesized, Compounds 2, 6, 9, and 15, with methionine methyl esters, D-tyrosine methyl esters, glutamic acid methyl esters, and methionines in the carbohydrate moiety, respectively, significantly inhibited wild-type SARS-CoV-2 Mpro-mediated proteolysis, with IC50 values ranging from 0.06 µM to 0.84 µM. They also demonstrated efficacy in inhibiting trans-cleavage by mutant Mpro variants (Mpro_P132H, Mpro_E166V, Mpro_P168A, Mpro_Q189I), with IC50 values ranging from 0.05 to 0.92 µM, surpassing nirmatrelvir (IC50: 1.17-152.9 µM). Molecular modeling revealed stronger interactions with Valine166 in the structural complex of Mpro_E166V with the compounds compared to nirmatrelvir. Moreover, these compounds efficiently inhibited the post-entry viral processes of wild-type SARS-CoV-2 single-round infectious particles (SRIPs), mitigating viral cytopathic effects and reducing replicon-driven GFP reporter signals, as well as in vitro infectivity of wild-type, Mpro_E166V, and Mpro_Q189I SRIPs, with EC50 values ranging from 0.02 to 0.53 µM. However, nirmatrelvir showed a significant decrease in inhibiting the replication of mutant SARS-CoV-2 SRIPs carrying Mpro_E166V (EC50: >20 µM) and Mpro_Q189I (EC50: 13.2 µM) compared to wild-type SRIPs (EC50: 0.06 µM). Overall, this study identifies four GL derivatives as promising lead compounds for developing treatments against various SARS-CoV-2 strains, including Omicron, and nirmatrelvir-resistant variants.

Endovascular Parent Vessel Occlusion Versus Flow Diversion in the Treatment of Large and Giant Aneurysms: A Randomized Comparison.

OBJECTIVE: Parent vessel occlusion (PVO) is a time-honored treatment for unclippable or uncoilable intracranial aneurysms. Flow diversion (FD) is a recent endovascular alternative that can occlude the aneurysm and spare the parent blood vessel. Our aim was to compare outcomes of FD with endovascular PVO. METHODS: This is a prespecified treatment subgroup analysis of the Flow diversion in Intracranial Aneurysms trial (FIAT). FIAT was an investigator-led parallel-group all-inclusive pragmatic randomized trial. For each patient, clinicians had to prespecify an alternative management option to FD before stratified randomization. We report all patients for whom PVO was selected as the best alternative treatment to FD. The primary outcome was a composite of core-lab determined angiographic occlusion or near-occlusion at 3-12 months combined with an independent clinical outcome (mRS<3). Primary analyses were intent-to-treat. There was no blinding. RESULTS: There were 45 patients (16.2% of the 278 FIAT patients randomized between 2011 and 2020 in 3 centers): 22 were randomly allocated to FD and 23 to PVO. Aneurysms were mainly large or giant (mean 22 mm) anterior circulation (mainly carotid) aneurysms. A poor primary outcome was reached in 11/22 FD (50.0%) compared to 9/23 PVO patients (39.1%) (RR: 1.28, 95% CI [0.66-2.47]; P = 0.466). Morbidity (mRS >2) at 1 year occurred in 4/22 FD and 6/23 PVO patients. Angiographic results and serious adverse events were similar. CONCLUSIONS: The comparison between PVO and FD was inconclusive. More randomized trials are needed to better determine the role of FD in large aneurysms eligible for PVO.

Dual Effects of 3-epi-betulin from Daphniphyllum glaucescens in Suppressing SARS-CoV-2-Induced Inflammation and Inhibiting Virus Entry.

The continuous emergence of SARS-CoV-2 variants has led to a protracted global COVID-19 pandemic with significant impacts on public health and global economy. While there are currently available SARS-CoV-2 vaccines and therapeutics, most of the FDA-approved antiviral agents directly target viral proteins. However, inflammation is the initial immune pathogenesis induced by SARS-CoV-2 infection, there is still a need to find additional agents that can control the virus in the early stages of infection to alleviate disease progression for the next pandemic. Here, we find that both the spike protein and its receptor CD147 are crucial for inducing inflammation by SARS-CoV-2 in THP-1 monocytic cells. Moreover, we find that 3-epi-betulin, isolated from Daphniphyllum glaucescens, reduces the level of proinflammatory cytokines induced by SARS-CoV-2, consequently resulting in a decreased viral RNA accumulation and plaque formation. In addition, 3-epi-betulin displays a broad-spectrum inhibition of entry of SARS-CoV-2 pseudoviruses, including Alpha (B.1.1.7), Eplison (B.1.429), Gamma (P1), Delta (B.1.617.2) and Omicron (BA.1). Moreover, 3-epi-betulin potently inhibits SARS-CoV-2 infection with an EC50 of <20 µM in Calu-3 lung epithelial cells. Bioinformatic analysis reveals the chemical interaction between the 3-epi-betulin and the spike protein, along with the critical amino acid residues in the spike protein that contribute to the inhibitory activity of 3-epi-betulin against virus entry. Taken together, our results suggest that 3-epi-betulin exhibits dual effect: it reduces SARS-CoV-2-induced inflammation and inhibits virus entry, positioning it as a potential antiviral agent against SARS-CoV-2.

Functional assessments of SARS-CoV-2 single-round infectious particles with variant-specific spike proteins on infectivity, drug sensitivity, and antibody neutralization.

Working with severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is restricted to biosafety level III (BSL-3) laboratory. The study used a trans-complementation system consisting of virus-like particles (VLPs) and DNA-launched replicons to generate SARS-CoV-2 single-round infectious particles (SRIPs) with variant-specific spike (S) proteins. S gene of Wuhan-Hu-1 strain (SWH1) or Omicron BA.1 variant (SBA.1), along with the envelope (E) and membrane (M) genes, were cloned into a tricistronic vector, co-expressed in the cells to produce variant-specific S-VLPs. Additionally, the replicon of the WH1-like strain without S, E, M and accessory genes, was engineered under the control by a CMV promoter to produce self-replicating RNAs within VLP-producing cells, led to create SWH1- and SBA.1-based SARS-CoV-2 SRIPs. The SBA.1-based SRIP showed lower virus yield, replication, N protein expression, fusogenicity, and infectivity compared to SWH1-based SRIPs. SBA.1-based SRIP also exhibited intermediate resistance to neutralizing antibodies produced by SWH1-based vaccines, but were effective at infecting cells with low ACE2 expression. Importantly, both S-based SRIPs responded similarly to remdesivir and GC376, with EC50 values ranging from 0.17 to 1.46 µM, respectively. The study demonstrated that this trans-complementation system is a reliable and efficient tool for generating SARS-CoV-2 SRIPs with variant-specific S proteins. SARS-CoV-2 SRIPs, mimicking authentic live viruses, facilitate comprehensive analysis of variant-specific virological characteristics, including antibody neutralization, and drug sensitivity in non-BSL-3 laboratories.

A disintegrin and metalloproteinase domain 9 facilitates SARS-CoV-2 entry into cells with low ACE2 expression.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the etiological agent of the Coronavirus disease-19 (COVID-19) pandemic, utilizes angiotensin-converting enzyme 2 (ACE2) as a receptor for virus infection. However, the expression pattern of ACE2 does not coincide with the tissue tropism of SARS-CoV-2, hinting that other host proteins might be involved in facilitating SARS-CoV-2 entry. To explore potential host factors for SARS-CoV-2 entry, we performed an arrayed shRNA screen in H1650 and HEK293T cells. Here, we identified a disintegrin and a metalloproteinase domain 9 (ADAM9) protein as an important host factor for SARS-CoV-2 entry. Our data showed that silencing ADAM9 reduced virus entry, while its overexpression promoted infection. The knockdown of ADAM9 decreased the infectivity of the variants of concern tested-B.1.1.7 (alpha), B.1.617.2 (delta), and B.1.1.529 (omicron). Furthermore, mechanistic studies indicated that ADAM9 is involved in the binding and endocytosis stages of SARS-CoV-2 entry. Through immunoprecipitation experiments, we demonstrated that ADAM9 binds to the S1 subunit of the SARS-CoV-2 Spike. Additionally, ADAM9 can interact with ACE2, and co-expression of both proteins markedly enhances virus infection. Moreover, the enzymatic activity of ADAM9 facilitates virus entry. Our study reveals an insight into the mechanism of SARS-CoV-2 virus entry and elucidates the role of ADAM9 in virus infection. IMPORTANCE COVID-19, an infectious respiratory disease caused by SARS-CoV-2, has greatly impacted global public health and the economy. Extensive vaccination efforts have been launched worldwide over the last couple of years. However, several variants of concern that reduce the efficacy of vaccines have kept emerging. Thereby, further understanding of the mechanism of SARS-CoV-2 entry is indispensable, which will allow the development of an effective antiviral strategy. Here, we identify a disintegrin and metalloproteinase domain 9 (ADAM9) protein as a co-factor of ACE2 important for SARS-CoV-2 entry, even for the variants of concern, and show that ADAM9 interacts with Spike to aid virus entry. This virus-host interaction could be exploited to develop novel therapeutics against COVID-19.

Upregulation of galectin-3 in influenza A virus infection promotes viral RNA synthesis through its association with viral PA protein.

BACKGROUND: Influenza is one of the most important viral infections globally. Viral RNA-dependent RNA polymerase (RdRp) consists of the PA, PB1, and PB2 subunits, and the amino acid residues of each subunit are highly conserved among influenza A virus (IAV) strains. Due to the high mutation rate and emergence of drug resistance, new antiviral strategies are needed. Host cell factors are involved in the transcription and replication of influenza virus. Here, we investigated the role of galectin-3, a member of the ß-galactoside-binding animal lectin family, in the life cycle of IAV infection in vitro and in mice. METHODS: We used galectin-3 knockout and wild-type mice and cells to study the intracellular role of galectin-3 in influenza pathogenesis. Body weight and survival time of IAV-infected mice were analyzed, and viral production in mouse macrophages and lung fibroblasts was examined. Overexpression and knockdown of galectin-3 in A549 human lung epithelial cells were exploited to assess viral entry, viral ribonucleoprotein (vRNP) import/export, transcription, replication, virion production, as well as interactions between galectin-3 and viral proteins by immunoblotting, immunofluorescence, co-immunoprecipitation, RT-qPCR, minireplicon, and plaque assays. We also employed recombinant galectin-3 proteins to identify specific step(s) of the viral life cycle that was affected by exogenously added galectin-3 in A549 cells. RESULTS: Galectin-3 levels were increased in the bronchoalveolar lavage fluid and lungs of IAV-infected mice. There was a positive correlation between galectin-3 levels and viral loads. Notably, galectin-3 knockout mice were resistant to IAV infection. Knockdown of galectin-3 significantly reduced the production of viral proteins and virions in A549 cells. While intracellular galectin-3 did not affect viral entry, it increased vRNP nuclear import, RdRp activity, and viral transcription and replication, which were associated with the interaction of galectin-3 with viral PA subunit. Galectin-3 enhanced the interaction between viral PA and PB1 proteins. Moreover, exogenously added recombinant galectin-3 proteins also enhanced viral adsorption and promoted IAV infection in A549 cells. CONCLUSION: We demonstrate that galectin-3 enhances viral infection through increases in vRNP nuclear import and RdRp activity, thereby facilitating viral transcription and replication. Our findings also identify galectin-3 as a potential therapeutic target for influenza.

Zika virus cleaves GSDMD to disseminate prognosticable and controllable oncolysis in a human glioblastoma cell model.

Glioblastoma (GBM) is the most common aggressive malignant brain cancer and is chemo- and radioresistant, with poor therapeutic outcomes. The "double-edged sword" of virus-induced cell death could be a potential solution if the oncolytic virus specifically kills cancer cells but spares normal ones. Zika virus (ZIKV) has been defined as a prospective oncolytic virus by selectively targeting GBM cells, but unclear understanding of how ZIKV kills GBM and the consequences hinders its application. Here, we found that the cellular gasdermin D (GSDMD) is required for the efficient death of a human GBM cell line caused by ZIKV infection. The ZIKV protease specifically cleaves human GSDMD to activate caspase-independent pyroptosis, harming both viral protease-harboring and naive neighboring cells. Analyzing human GSDMD variants showed that most people were susceptible to ZIKV-induced cytotoxicity, except for those with variants that resisted ZIKV cleavage or were defective in oligomerizing the N terminus GSDMD cleavage product. Consistently, ZIKV-induced secretion of the pro-inflammatory cytokine interleukin-1ß and cytolytic activity were both stopped by a small-molecule inhibitor targeting GSDMD oligomerization. Thus, potential ZIKV oncolytic therapy for GBM would depend on the patient's GSDMD genetic background and could be abolished by GSDMD inhibitors if required.

Small Structural Proteins E and M Render the SARS-CoV-2 Pseudovirus More Infectious and Reveal the Phenotype of Natural Viral Variants.

The SARS-CoV-2 pseudovirus is a commonly used strategy that mimics certain biological functions of the authentic virus by relying on biological legitimacy at the molecular level. Despite the fact that spike (S), envelope (E), and membrane (M) proteins together wrap up the SARS-CoV-2 virion, most of the reported pseudotype viruses consist of only the S protein. Here, we report that the presence of E and M increased the virion infectivity by promoting the S protein priming. The S, E, and M (SEM)-coated pseudovirion is spherical, containing crown-like spikes on the surface. Both S and SEM pseudoviruses packaged the same amounts of viral RNA, but the SEM virus bound more efficiently to cells stably expressing the viral receptor human angiotensin-converting enzyme II (hACE2) and became more infectious. Using this SEM pseudovirus, we examined the infectivity and antigenic properties of the natural SARS-CoV-2 variants. We showed that some variants have higher infectivity than the original virus and that some render the neutralizing plasma with lower potency. These studies thus revealed possible mechanisms of the dissemination advantage of these variants. Hence, the SEM pseudovirion provides a useful tool to evaluate the viral infectivity and capability of convalescent sera in neutralizing specific SARS-CoV-2 S dominant variants.

Clinical Uncertainty and Equipoise in the Management of Recurrent Glioblastoma.

BACKGROUND: A significant proportion of glioblastoma (GBM) patients are considered for repeat resection, but evidence regarding best management remains elusive. Our aim was to measure the degree of clinical uncertainty regarding reoperation for patients with recurrent GBM. METHODS: We first performed a systematic review of agreement studies examining the question of repeat resection for recurrent GBM. An electronic portfolio of 37 pathologically confirmed recurrent GBM patients including pertinent magnetic resonance images and clinical information was assembled. To measure clinical uncertainty, 26 neurosurgeons from various countries, training backgrounds, and years' experience were asked to select best management (repeat surgery, other nonsurgical management, or conservative), confidence in recommended management, and whether they would include the patient in a randomized trial comparing surgery with nonsurgical options. Agreement was evaluated using κ statistics. RESULTS: The literature review did not reveal previous agreement studies examining the question. In our study, agreement regarding best management of recurrent GBM was slight, even when management options were dichotomized (repeat surgery vs. other options; κ=0.198 [95% confidence interval: 0.133-0.276]). Country of practice, years' experience, and training background did not change results. Disagreement and clinical uncertainty were more pronounced within clinicians with (κ=0.167 [0.055-0.314]) than clinicians without neuro-oncology fellowship training (κ=0.601 [0.556-0.646]). A majority (51%) of responders were willing to include the patient in a randomized trial comparing repeat surgery with nonsurgical alternatives in 26/37 (69%) of cases. CONCLUSION: There is sufficient uncertainty and equipoise regarding the question of reoperation for patients with recurrent glioblastoma to support the need for a randomized controlled trial.

Dual Effects of Let-7b in the Early Stage of Hepatitis C Virus Infection.

MicroRNA let-7b expression is induced by infection of hepatitis C virus (HCV) and is involved in the regulation of HCV replication by directly targeting the HCV genome. The current study demonstrated that let-7b directly targets negative regulators of type I interferon (IFN) signaling thereby limiting HCV replication in the early stage of HCV infection. Let-7b-regulated genes which are involved in host cellular responses to HCV infection were unveiled by microarray profiling and bioinformatic analyses, followed by various molecular and cellular assays using Huh7 cells expressing wild-type (WT) or the seed region-mutated let-7b. Let-7b targeted the cytokine signaling 1 (SOCS1) protein, a negative regulator of JAK/STAT signaling, which then enhanced STAT1-Y701 phosphorylation leading to increased expression of the downstream interferon-stimulated genes (ISGs). Let-7b augmented retinoic acid-inducible gene I (RIG-I) signaling, but not MDA5, to phosphorylate and nuclear translocate IRF3 leading to increased expression of IFN-ß. Let-7b directly targeted the ATG12 and IκB kinase alpha (IKKα) transcripts and reduced the interaction of the ATG5-ATG12 conjugate and RIG-I leading to increased expression of IFN, which may further stimulate JAK/STAT signaling. Let-7b induced by HCV infection elicits dual effects on IFN expression and signaling, along with targeting the coding sequences of NS5B and 5' UTR of the HCV genome, and limits HCV RNA accumulation in the early stage of HCV infection. Controlling let-7b expression is thereby crucial in the intervention of HCV infection.IMPORTANCE HCV is a leading cause of liver disease, with an estimated 71 million people infected worldwide. During HCV infection, type I interferon (IFN) signaling displays potent antiviral and immunomodulatory effects. Host factors, including microRNAs (miRNAs), play a role in upregulating IFN signaling to limit HCV replication. Let-7b is a liver-abundant miRNA that is induced by HCV infection and targets the HCV genome to suppress HCV RNA accumulation. In this study, we demonstrated that let-7b, as a positive regulator of type I IFN signaling, plays dual roles against HCV replication by increasing the expression of IFN and interferon-sensitive response element (ISRE)-driven interferon-stimulated genes (ISGs) in the early stage of HCV infection. This study sheds new insight into understanding the role of let-7b in combatting HCV infection. Clarifying IFN signaling regulated by miRNA during the early phase of HCV infection may help researchers understand the initial defense mechanisms to other RNA viruses.

miR-1975 serves as an indicator of clinical severity upon influenza infection.

Emerging evidence highlights the role of non-coding small RNAs in host-influenza interaction. We have identified a Y RNA-derived small RNA, miR-1975, which is upregulated upon influenza A virus infection in A549 cells. The aim of this study is to investigate whether miR-1975 serves as an indicator of clinical severity upon influenza infection. We investigate the abundance of miR-1975 in sera from clinical patients and its correlation with hypoxemia status. We quantified its amounts in sera from influenza virus-infected patients and healthy volunteers by means of stem-loop RT-PCR. Median values of miR-1975 were significantly higher in influenza virus-infected patients, especially in hypoxemic patients. miR-1975 levels at the acute stage of the disease were highly correlated with the fraction of inspired oxygen used by the patients and total ventilator days. Receiver operator characteristic curve analysis revealed that miR-1975 levels in combination with days of fever before presenting to hospital had significant predictive value for hypoxemia and respiratory failure for patients infected with influenza virus. Our results reveal that circulating miR-1975 has great potential to serve as a biomarker for predicting prognosis in patients infected with influenza virus.

Consensus statements on the clinical usage and characteristics of aripiprazole for Hong Kong.

Aripiprazole, a dopamine partial agonist, is a second-generation anti-psychotic that is widely used for the treatment of schizophrenia and other psychotic disorders. A group of psychiatric experts in Hong Kong developed a set of consensus statements, aiming to facilitate the understanding of clinical properties and usages of aripiprazole among local physicians. Of note, because aripiprazole long-acting injectable has been available locally not long before the establishment of the consensus panel, which limited the discussion on its use in the local context, the consensus statements were focused primarily on oral aripiprazole. To draft the consensus statements, the panellists discussed the published evidence and their clinical experience regarding aripiprazole in a series of meetings based on several areas. At the final meeting, each drafted statement was voted on anonymously by all panellists based on its practicability of recommendation in Hong Kong. A set of consensus statements on the characteristics and clinical use of aripiprazole was established and accepted by the panel. These statements serve to provide a practical reference for physicians in Hong Kong, and possibly other parts of the Asia-Pacific region, on the use of aripiprazole in people with schizophrenia spectrum disorders and other psychotic problems.

DNA-induced 2'3'-cGAMP enhances haplotype-specific human STING cleavage by dengue protease.

The cytosolic DNA sensor cGMP-AMP synthase (cGAS) synthesizes the noncanonical cyclic dinucleotide 2'3'-cGAMP to activate the adaptor protein stimulator of IFN genes (STING), thus awakening host immunity in response to DNA pathogen infection. However, dengue virus (DENV), an RNA virus without a DNA stage in its life cycle, also manipulates cGAS-STING-mediated innate immunity by proteolytic degradation of STING. Here, we found that the sensitivity of STING to DENV protease varied with different human STING haplotypes. Exogenous DNA further enhanced DENV protease's ability to interact and cleave protease-sensitive STING. DNA-enhanced STING cleavage was reduced in cGAS-knockdown cells and triggered by the cGAS product 2'3'-cGAMP. The source of DNA may not be endogenous mitochondrial DNA but rather exogenous reactivated viral DNA. Cells producing 2'3'-cGAMP by overexpressing cGAS or with DNA virus reactivation enhanced STING cleavage in neighboring cells harboring DENV protease. DENV infection reduced host innate immunity in cells with the protease-sensitive STING haplotype, whose homozygote genotype frequency was found significantly reduced in Taiwanese people with dengue fever. Therefore, the human STING genetic background and DNA pathogen coinfection may be the missing links contributing to DENV pathogenesis.

Exosome-delivered and Y RNA-derived small RNA suppresses influenza virus replication.

BACKGROUND: Multiple interplays between viral and host factors are involved in influenza virus replication and pathogenesis. Several small RNAs have recently emerged as important regulators of host response to viral infections. The aim of this study was to characterize the functional role of hsa-miR-1975, a Y5 RNA-derived small RNA, in defending influenza virus and delineate the mechanisms. METHODS: We performed high throughput sequencing of small RNAs in influenza virus-infected cells to identify up- or down- regulated small RNA species. The expression of the most abundant RNA species (hsa-miR-1975) was validated by stem-loop reverse transcription-polymerase chain reaction (RT-PCR). Antiviral effects of hsa-miR-1975 were confirmed by Western Blot, RT-PCR and plaque assay. In vitro perturbation of hsa-miR-1975 combined with exosomes isolation was used to elucidate the role and mechanism of hsa-miR-1975 in the context of antiviral immunity. RESULTS: Small RNA sequencing revealed that hsa-miR-1975 was the most up-regulated small RNA in influenza virus-infected cells. The amount of intracellular hsa-miR-1975 increased in the late stage of the influenza virus replication cycle. The increased hsa-miR-1975 was at least partially derived from degradation of Y5RNA as a result of cellular apoptosis. Unexpectedly, hsa-miR-1975 mimics inhibited influenza virus replication while hsa-miR-1975 sponges enhanced the virus replication. Moreover, hsa-miR-1975 was secreted in exosomes and taken up by the neighboring cells to induce interferon expression. CONCLUSIONS: Our findings unravel a critical role of Y-class small RNA in host's defense against influenza virus infection and reveal its antiviral mechanism through exosome delivery. This may provide a new candidate for targeting influenza virus.

Stress and burden faced by family caregivers of people with schizophrenia and early psychosis in Hong Kong.

BACKGROUND/AIM: Families, especially in Chinese society, play a crucial role in care provision for relatives with schizophrenia, but the burden of caregiving has shown to cause significant distress among caregivers. The aim of the study is to assess the degree of stress and burden among caregivers of relatives with schizophrenia and early psychosis in Hong Kong. METHODS: A cross-sectional survey was conducted in 454 caregivers recruited from two mental health non-governmental organisations and the outpatient clinic of a psychiatric hospital. Data were collected through a questionnaire administered via face-to-face or telephone interview. RESULTS: Caregivers attributed most of their conflicts with the ill relative or other family members to their own lack of knowledge of patient symptoms (56.4%), other family members' lack of knowledge of patient symptoms (46.9%) or the ill relative's refusal to take medications (43.0%). Most of the caregivers had corresponding stress scores of 5 (scale: 1-5; mean = 3.88, 3.85 and 4.19, respectively). Nearly, a third (30.2%) of the caregivers surveyed reported an overall stress score of 5 (mean = 3.56). Regarding psychosocial problems, 78.0%, 49.8% and 45.8% of caregivers experienced anxiety, reduced socialising and insomnia, respectively. CONCLUSIONS: Caregivers of relatives with schizophrenia and early psychosis experience significant stress and psychosocial burden. To help them cope with distress, community support services should be strengthened. Moreover, long-acting injectable antipsychotics are worth considering to alleviate caregiver burden due to ill relatives' medication compliance issues.

How Dengue Virus Circumvents Innate Immunity.

In the battle between a virus and its host, innate immunity serves as the first line of defense protecting the host against pathogens. The antiviral actions start with the recognition of pathogen-associated molecular patterns derived from the virus, then ultimately turning on particular transcription factors to generate antiviral interferons (IFNs) or proinflammatory cytokines via fine-tuned signaling cascades. With dengue virus (DENV) infection, its viral RNA is recognized by the host RNA sensors, mainly retinoic acid inducible gene-I (RIG-I)-like receptors (RLRs) and toll-like receptors. DENV infection also activates the cyclic GMP-AMP synthase-stimulator of interferon genes (cGAS-STING)-mediated DNA-sensing pathway despite the absence of a DNA stage in the DENV lifecycle. In the last decade, DENV has been considered a weak IFN-inducing pathogen with the evidence that DENV has evolved multiple strategies antagonizing the host IFN system. DENV passively escapes from innate immunity surveillance and also actively subverts the innate immune system at multiple steps. DENV targets both RNA-triggered RLR-mitochondrial antiviral signaling protein (RLR-MAVS) and DNA-triggered cGAS-STING signaling to reduce IFN production in infected cells. It also blocks IFN action by inhibiting IFN regulatory factor- and signal transducer and activator of transcription-mediated signaling. This review explores the current understanding of how DENV escapes the control of the innate immune system by modifying viral RNA and viral protein and by post-translational modification of cellular factors. The roles of the DNA-sensing pathway in DENV infection, and how mitochondrial dynamics participates in innate immunity are also discussed.

Quantitative RT-PCR Analysis of Influenza Virus Endocytic Escape.

Although several virus families are internalized into their host cells by direct fusion of the viral envelope with the plasma membrane, most viruses, for example, influenza virus, make use of endocytic pathways for productive entry and infection. After endocytosis, the influenza virus escapes from the endocytic compartment to the cytosol. The distribution of the incoming influenza virus could be traced by detection of the viral RNA in the distinct cellular compartments, including endosome, cytosol, and nucleus. To accomplish this work, we developed a subcellular fractionation method based on density gradient ultracentrifugation and detected the viral RNA using quantitative reverse transcription-polymerase chain reaction analysis. This chapter is devoted to the practical methods and precautions for studying endocytic traffic of virus as well as host cellular factors affecting viral endocytosis.