Viral and host factors related to the clinical outcome of COVID-19.

In December 2019, coronavirus disease 2019 (COVID-19), which is caused by the new coronavirus severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), was identified in Wuhan (Hubei province, China)1; it soon spread across the world. In this ongoing pandemic, public health concerns and the urgent need for effective therapeutic measures require a deep understanding of the epidemiology, transmissibility and pathogenesis of COVID-19. Here we analysed clinical, molecular and immunological data from 326 patients with confirmed SARS-CoV-2 infection in Shanghai. The genomic sequences of SARS-CoV-2, assembled from 112 high-quality samples together with sequences in the Global Initiative on Sharing All Influenza Data (GISAID) dataset, showed a stable evolution and suggested that there were two major lineages with differential exposure history during the early phase of the outbreak in Wuhan. Nevertheless, they exhibited similar virulence and clinical outcomes. Lymphocytopenia, especially reduced CD4+ and CD8+ T cell counts upon hospital admission, was predictive of disease progression. High levels of interleukin (IL)-6 and IL-8 during treatment were observed in patients with severe or critical disease and correlated with decreased lymphocyte count. The determinants of disease severity seemed to stem mostly from host factors such as age and lymphocytopenia (and its associated cytokine storm), whereas viral genetic variation did not significantly affect outcomes.

Identification of a membrane-associated element (MAE) in the C-terminal region of SARS-CoV-2 nsp6 that is essential for viral replication.

The coronavirus disease 2019 (COVID-19) pandemic, caused by the novel coronavirus severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2), has rapidly spread worldwide since its emergence in late 2019. Its ongoing evolution poses challenges for antiviral drug development. Coronavirus nsp6, a multiple-spanning transmembrane protein, participates in the biogenesis of the viral replication complex, which accommodates the viral replication-transcription complex. The roles of its structural domains in viral replication are not well studied. Herein, we predicted the structure of the SARS-CoV-2 nsp6 protein using AlphaFold2 and identified a highly folded C-terminal region (nsp6C) downstream of the transmembrane helices. The enhanced green fluorescent protein (EGFP)-fused nsp6C was found to cluster in the cytoplasm and associate with membranes. Functional mapping identified a minimal membrane-associated element (MAE) as the region from amino acids 237 to 276 (LGV-KLL), which is mainly composed of the α-helix H1 and the α-helix H2; the latter exhibits characteristics of an amphipathic helix (AH). Mutagenesis studies and membrane flotation experiments demonstrate that AH-like H2 is required for MAE-mediated membrane association. This MAE was functionally conserved across MERS-CoV, HCoV-OC43, HCoV-229E, HCoV-HKU1, and HCoV-NL63, all capable of mediating membrane association. In a SARS-CoV-2 replicon system, mutagenesis studies of H2 and replacements of H1 and H2 with their homologous counterparts demonstrated requirements of residues on both sides of the H2 and properly paired H1-H2 for MAE-mediated membrane association and viral replication. Notably, mutations I266A and K274A significantly attenuated viral replication without dramatically affecting membrane association, suggesting a dual role of the MAE in viral replication: mediating membrane association as well as participating in protein-protein interactions.IMPORTANCESevere acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) assembles a double-membrane vesicle (DMV) by the viral non-structural proteins for viral replication. Understanding the mechanisms of the DMV assembly is of paramount importance for antiviral development. Nsp6, a multiple-spanning transmembrane protein, plays an important role in the DMV biogenesis. Herein, we predicted the nsp6 structure of SARS-CoV-2 and other human coronaviruses using AlphaFold2 and identified a putative membrane-associated element (MAE) in the highly conserved C-terminal regions of nsp6. Experimentally, we verified a functionally conserved minimal MAE composed of two α-helices, the H1, and the amphipathic helix-like H2. Mutagenesis studies confirmed the requirement of H2 for MAE-mediated membrane association and viral replication and demonstrated a dual role of the MAE in viral replication, by mediating membrane association and participating in residue-specific interactions. This functionally conserved MAE may serve as a novel anti-viral target.

Aqueous Cellulose Solution Adhesive.

In the context of sustainable development, research on a biomass-based adhesive without chemical modification as a substitute for petroleum-based adhesive is now crucial. It turns out to be challenging to guarantee a simple and sustainable method to produce high-quality adhesives and subsequently manufacture multifunctional composites. Herein, the inherent properties of cellulose were exploited to generate an adhesive based on a cellulose aqueous solution. The adhesion is simple to prepare structurally and functionally complex materials in a single process. Cellulose-based daily necessities including straws, bags, and cups were prepared by adhering cellulose films, and smart devices like actuators and supercapacitors assembled by adhering hydrogels were also demonstrated. In addition, the composite boards bonded with natural biomass wastes, such as wood chips, displayed significantly stronger mechanical properties than the natural wood or commercial composite boards. Cellulose aqueous adhesives provide a straightforward, feasible, renewable, and inventive bonding technique for material shaping and the creation of multipurpose devices.

Prevalence of Neutralizing Autoantibodies Against Type I Interferon in a Multicenter Cohort of Severe or Critical COVID-19 Cases in Shanghai.

OBJECTIVE: We sought to explore the prevalence of type I interferon-neutralizing antibodies in a Chinese cohort and its clinical implications during the Omicron variant wave of SARS-CoV-2. METHODS: Type I interferon (IFN) autoantibodies possessing neutralizing capabilities were identified using luciferase assays. The capacity of the autoantibodies for in vitro interference with antiviral activity of IFN was assessed by using a SARS-CoV-2 replicon system. An analysis of the demographic and clinical profiles of patients exhibiting neutralizing antibodies was also conducted. RESULTS: In this cohort, 11.8% of severe/critical cases exhibited the existence of type I IFN-neutralizing antibodies, specifically targeting IFN-α2, IFN-ω, or both, with an elderly male patient tendency. Notably, these antibodies exerted a pronounced inhibitory effect on the antiviral activity of IFN against SARS-CoV-2 under controlled in vitro conditions. Furthermore, a noteworthy correlation was discerned between the presence of these neutralizing antibodies and critical clinical parameters, including C-reactive protein (CRP) levels, D-dimer levels, and lymphocyte counts. CONCLUSION: The presence of type I IFN-neutralizing antibodies is a pervasive risk factor for severe/critical COVID-19 in the Chinese population.

PAM-free cascaded strand displacement coupled with CRISPR-Cas12a for amplified electrochemical detection of SARS-CoV-2 RNA.

The early diagnosis of coronavirus disease 2019 (COVID-19) is dependent on the specific and sensitive detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA. Herein, we develop a highly sensitive and specific electrochemical biosensor for SARS-CoV-2 target RNA detection based on the integration of protospacer adjacent motif (PAM)-free cascaded toehold-mediated strand displacement reaction (TSDR) and CRISPR-Cas12a (PfTSDR-CRISPR). In this study, each target is transformed into multiple DNA substrates with bubble structure in the seed region by the cascaded TSDR, which can directly hybridize with guide RNA (gRNA) without PAM requirement and then activate CRISPR-Cas12a's trans-cleavage activity. Subsequently, the hairpin DNA modified with methylene blue (MB-HP) is cleaved by activated CRISPR-Cas12a. Therefore, as MB leaves the electrode surface, a decreased current signal is obtained. With the involvement of PAM-free cascaded TSDRs and CRISPR-Cas12a amplification strategy, the PfTSDR-CRISPR-based electrochemical biosensor achieves the detection of target RNA as low as 40 aM. The biosensor has high sequence specificity, reliability and robustness. Thanks to the PAM-free cascaded TSDR, the biosensor can achieve universal detection of different target RNA without redesigning gRNA sequence of CRISPR-Cas12a. In addition, this biosensor successfully detects SARS-CoV-2 target RNA in complex samples, which highlights its potential for diagnosing COVID-19.

FeS and Fe3O4 Co-modified biochar to build a highly resistant advanced oxidation process system for quinclorac degradation in irrigation water.

Advanced oxidation processes (AOPs), based on sulfate radical (SO4·-) produced by peroxymonosulfate (PMS), can effectively mineralize refractory organic pollutants. However, the coexistence of anions and natural organic matters in actual wastewater prevents the application of AOPs. A simple one-step method was used to prepare FeS/Fe3O4 co-modified biochar materials (FFB) that could activate PMS to degrade quinclorac (QNC) with a removal rate of 100%, even exhibiting optimum degradation of QNC reached 99.31% in irrigation water, demonstrating excellent anti-interference performance for co-existing anions and natural organic matter. Meanwhile, ecotoxicity analysis showed that the toxicity of degradation intermediates was lower than that of QNC. Characterization results demonstrated the even distribution of FeS and Fe3O4 onto biochar, supplying abundant Fe2+ to activate PMS producing reactive oxygen species (ROS), while the generated Fe3+ after reactive continue to be reduced with sulfur species to promote the cycle of Fe2+/Fe3+. The coexistence of ·OH, SO4·-, 1O2, and O2·- in the FFB/PMS-QNC system suggest the possession of two pathway with free radical and non-free radical pathways to degrade QNC. The density functional theory (DFT) was used to analyze the adsorption sites and adsorption energy of PMS, as well as the differential charge density, which further proved the generation of SO4·-, O2·- and 1O2. In addition, the electrochemical test results showed that electron transfer also played an important role in the degradation of QNC. This study provides a feasible approach for the removal of organic pollutants in actual water.

Molecular epidemiology and clinical characterization of human rhinoviruses circulating in Shanghai, 2012-2020.

Human rhinoviruses (HRVs) cause acute upper and lower respiratory tract infections and aggravation of asthma and chronic obstructive pulmonary disease. The 5' untranslated region (5' UTR) and the VP4/VP2 region are widely used for genotyping of HRVs. Members of the species Rhinovirus A and Rhinovirus C have been reported to be more frequently associated with severe disease than members of the species Rhinovirus B. We report the clinical and molecular epidemiological characteristics of HRVs circulating from 2012 to 2020 in Shanghai. A total of 5832 nasopharyngeal swabs from patients with acute respiratory infections were collected. A real-time reverse transcription polymerase chain reaction assay was used for virus detection. The 5' untranslated region and VP4/VP2 region were amplified and sequenced for genotyping and phylogenetic analysis. The overall rate of rhinovirus detection was 2.74% (160/5832), with members of species A, B, and C accounting for 68.13% (109/160), 20.00% (32/160), and 11.88% (19/160) of the total, respectively. A peak of HRV infection was observed in autumn (5.34%, 58/1087). Patients in the 3- to 14-year-old age group were the most susceptible to HRV infection (χ2 = 23.88, P = 0.017). Influenza virus and Streptococcus pneumoniae were detected more frequently than other pathogens in cases of coinfection. Recombination events were identified in 10 strains, which were successfully genotyped by phylogenetic analysis based on the 5' UTR-VP4/VP2 region but not the 5' UTR region alone. We observed a high degree of variability in the relative distribution of HRV genotypes and the prevalence of HRV infection in Shanghai and found evidence of recombination events in the portion of the genome containing the 5' UTR and the VP4/VP2 region between HRV-C strains and HRV-A-like strains. This study is important for surveillance of the spread of HRVs and the emergence of new variants.

Application of nanopore sequencing in diagnosis of secondary infections in patients with severe COVID-19.

To explore the application value of nanopore sequencing technique in the diagnosis and treatment of secondary infections in patients with severe coronavirus disease 2019 (COVID-19). A total of 77 clinical specimens from 3 patients with severe COVID-19 were collected. After heat inactivation, all samples were subjected to total nucleic acid extraction based on magnetic bead enrichment. The extracted DNA was used for DNA library construction, then nanopore real-time sequencing detection was performed. The sequencing data were subjected to Centrifuge software database species matching and R program differential analysis to obtain potential pathogen identification. Nanopore sequencing results were compared with respiratory pathogen qPCR panel screening and conventional microbiological testing results to verify the effectiveness of nanopore sequencing detection. Nanopore sequencing results showed that positive pathogen were obtained in 44 specimens (57.1%). The potential pathogens identified by nanopore sequencing included , , and , et al. , , were also detected in clinical microbiological culture-based detection; was detected in respiratory pathogen screening qPCR panel; was only detected by the nanopore sequencing technique. Comprehensive considerations with the clinical symptoms, the patient was treated with antibiotics against , and the infection was controlled. Nanopore sequencing may assist the diagnosis and treatment of severe COVID-19 patients through rapid identification of potential pathogens.

Quasispecies dynamics of a hepatitis E virus 4 from the feces and liver biopsy of an acute hepatitis E patient during virus clearance.

The presence of a quasispecies in hepatitis E virus (HEV) infection has been documented, however, the implications of a quasispecies in HEV-host interaction are poorly understood. Here, we analyzed the whole genome sequences of a HEV 4d from the feces and liver biopsy of a patient during the icteric and convalescent phases in an acute hepatitis E infection. Viral RNAs were extracted, reversely transcribed and seven fragments encompassing the entire viral genome were amplified and cloned. By sequencing multiple colonies of each cloned viral genome amplicon with Sanger sequencing, we verified the existence of the HEV quasispecies or intra-host genetic variations within the fecal and liver biopsy samples. There were broader genetic variations in the HEV ORF1 region including the PCP, HPX, and RdRp regions during the convalescent phase whereas more genetic variations in the ORF2 P domain during the icteric phase. The quasispecies dynamics might reflect host immune pressure during viral clearance.

Hepatitis C virus NS5A inhibitor daclatasvir allosterically impairs NS4B-involved protein-protein interactions within the viral replicase and disrupts the replicase quaternary structure in a replicase assembly surrogate system.

Daclatasvir (DCV) is a highly potent direct-acting antiviral that targets the non-structural protein 5A (NS5A) of hepatitis C virus (HCV) and has been used with great clinical success. Previous studies have demonstrated its impact on viral replication complex assembly. However, the precise mechanisms by which DCV impairs the replication complex assembly remains elusive. In this study, by using HCV subgenomic replicons and a viral replicase assembly surrogate system in which the HCV NS3-5B polyprotein is expressed to mimic the viral replicase assembly, we assessed the impact of DCV on the aggregation and tertiary structure of NS5A, the protein-protein interactions within the viral replicase and the quaternary structure of the viral replicase. We found that DCV did not affect aggregation and tertiary structure of NS5A. DCV induced a quaternary structural change of the viral replicase, as evidenced by selective increase of NS4B's sensitivity to proteinase K digestion. Mechanically, DCV impaired the NS4B-involved protein-protein interactions within the viral replicase. These phenotypes were consistent with the phenotypes of several reported NS4B mutants that abolish the viral replicase assembly. The DCV-resistant mutant Y93H was refractory to the DCV-induced reduction of the NS4B-involved protein interactions and the quaternary structural change of the viral replicase. In addition, Y93H reduced NS4B-involved protein-protein interactions within the viral replicase and attenuated viral replication. We propose that DCV may induce a positional change of NS5A, which allosterically affects protein interactions within the replicase components and disrupts replicase assembly.

Replicase-mediated shielding of the poliovirus replicative double-stranded RNA to avoid recognition by MDA5.

Replication of the positive-strand RNA viruses generates double-stranded RNAs (dsRNAs) that are recognized by host pattern recognition receptors (PRRs) to trigger innate immune responses. Formation of the viral replication complex (RC) has been thought to shield dsRNA from being recognized by innate sensors. To elucidate the RC-mediated evasion of innate recognition, we selected poliovirus (PV) as a model. We first found that RNAs generated during PV replication were potent interferon (IFN) inducers upon transfection, while there was no obvious IFN production detected in PV-replicating cells. PV replication did not interfere with IFN production when IFN agonists were synchronously introduced with the replicating PV RNAs, and in PV-infected cells, IFN agonist-induced IFN production was only moderately impaired but not completely abolished. When PV-infected cells were in situ permeabilized by digitonin, viral dsRNAs were readily detected by an anti-dsRNA antibody and were resistant to RNase III digestion. When digitonin-permeabilized cells were further solubilized by 1 % triton X-100, the dsRNAs of PV became sensitive to RNase III digestion. A co-localization study showed that PV dsRNA did not co-localize with MDA5 in virally infected cells. Given that the PV replication complex is protruding single-membrane and tubular in form, viral replicative dsRNAs are probably shielded by the replication complex or the viral replicase to avoid being accessed by RNase III and MDA5. We propose that the replication complex- or replicase-mediated shielding of dsRNA may act as a means for innate evasion.

WNT5A promotes migration and invasion of human osteosarcoma cells via SRC/ERK/MMP-14 pathway.

WNT5A, a representative ligand of activating several non-canonical WNT signal pathways, plays significant roles in oncogenesis and tumor inhibition. It has been shown that the non-receptor tyrosine kinase SRC is required for WNT5A-induced invasion of osteosarcoma cells. However, the precise molecular mechanism underlying WNT5A/SRC-mediated osteosarcoma cells invasion remains poorly defined. The study was designed to explore the role of ERK1/2 in WNT5A/SRC-induced osteosarcoma cells invasion and the downstream target of the SRC/ERK1/2 signalings. We found that WNT5A (100 ng/mL) remarkably stimulated migration and invasion of human osteosarcoma MG-63 cells, whereas inhibiting either SRC kinase activity by siRNA-mediated SRC silence or ERK1/2 phosphorylation by PD98059 treatment suppressed these effects, which suggested that the activation of SRC and ERK1/2 is essential for WNT5A-induced MG-63 cells migration and invasion. Furthermore, ERK1/2 phosphorylation induced by WNT5A was dramatically blocked by SRC siRNA. Additionally, our study further demonstrated that MMP-14 was upregulated after exposure to WNT5A in MG-63 cells, and the increased expression was blocked by SRC siRNA or PD98059. Collectively, these results indicate that WNT5A activates SRC/ERK1/2 signal pathway, leading to the upregulation of MMP-14 expression and MG-63 cells migration and invasion.

Pelargonidin induces antitumor effects in human osteosarcoma cells via autophagy induction, loss of mitochondrial membrane potential, G2/M cell cycle arrest and downregulation of PI3K/AKT signalling pathway.

PURPOSE: Osteosarcoma is the most common type of primary malignancy of bone in children and adults. The treatment options for osteosarcoma are limited and are associated with a number of drawbacks. Therefore there is an urgent need to look for more efficient options for the treatment of this disease. Flavonoids have been considered as important anticancer agents due to their efficacy and lower cytotoxicity. In the present study we evaluated the anticancer effects of pelargonidin in U2OS osteosarcoma cell line. METHODS: Cell viability was assessed by MTT assay. Reactive oxygen species (ROS), mitochondrial membrane potential (MMP) and cell cycle analysis was done by flow cytometry. Expression of proteins was examined by western blotting. RESULTS: Pelargonidin exhibited significant anticancer effects on osteosarcoma U2OS cell line with an IC50 of 15 µM. The anticancer effects of pelargonidin were due to induction of autophagy as evidenced from the expression of LC3-I, LC-3II and Beclin-1. Moreover, pelargonidin triggered ROSinduced reduction in MMP and triggered G2/M cell cycle arrest. In addition, pelargonidin inhibited the expression of p-PI3K and p-AKT in a concentration-dependent manner. CONCLUSIONS: Taken together, these results indicated that pelargonidin may prove a potential lead drug for the treatment of osteosarcoma.

Aggregation of a hepatitis C virus replicase module induced by ablation of p97/VCP.

Hijacking host membranes to assemble a membrane-associated viral replicase is a hallmark of almost all positive-strand RNA viruses. However, how the virus co-opts host factors to facilitate this energy-unfavourable process is incompletely understood. In a previous study, using hepatitis C virus (HCV) as a model and employing affinity purification of the viral replicase, we identified a valosin-containing protein (p97/VCP), a member of the ATPases associated with diverse cellular activities (AAA+ ATPase family), as a viral replicase-associated host factor. It is required for viral replication, depending on its ATPase activity. In this study, we used VCP pharmacological inhibitors and short hairpin (sh) RNA-mediated knockdown to ablate VCP function and then dissected the roles of VCP in viral replicase assembly in an HCV subgenomic replicon system and a viral replicase assembly surrogate system. Ablation of VCP specifically resulted in the pronounced formation of an SDS-resistant aggregation of HCV NS5A and the reduction of hyperphosphorylation of NS5A. The NS5A dimerization domain was indispensable for aggregation and the NS5A disordered regions also contributed to a lesser extent. The reduction of the hyperphosphorylation of NS5A coincided with the aggregation of NS5A. We propose that HCV may co-opt VCP to disaggregate an aggregation-prone replicase module to facilitate its replicase assembly.

Chaperone-Assisted Protein Folding Is Critical for Yellow Fever Virus NS3/4A Cleavage and Replication.

UNLABELLED: DNAJC14, a heat shock protein 40 (Hsp40) cochaperone, assists with Hsp70-mediated protein folding. Overexpressed DNAJC14 is targeted to sites of yellow fever virus (YFV) replication complex (RC) formation, where it interacts with viral nonstructural (NS) proteins and inhibits viral RNA replication. How RCs are assembled and the roles of chaperones in this coordinated process are largely unknown. We hypothesized that chaperones are diverted from their normal cellular protein quality control function to play similar roles during viral infection. Here, we show that DNAJC14 overexpression affects YFV polyprotein processing and alters RC assembly. We monitored YFV NS2A-5 polyprotein processing by the viral NS2B-3 protease in DNAJC14-overexpressing cells. Notably, DNAJC14 mutants that did not inhibit YFV replication had minimal effects on polyprotein processing, while overexpressed wild-type DNAJC14 affected the NS3/4A and NS4A/2K cleavage sites, resulting in altered NS3-to-NS3-4A ratios. This suggests that DNAJC14's folding activity normally modulates NS3/4A/2K cleavage events to liberate appropriate levels of NS3 and NS4A and promote RC formation. We introduced amino acid substitutions at the NS3/4A site to alter the levels of the NS3 and NS4A products and examined their effects on YFV replication. Residues with reduced cleavage efficiency did not support viral RNA replication, and only revertant viruses with a restored wild-type arginine or lysine residue at the NS3/4A site were obtained. We conclude that DNAJC14 inhibition of RC formation upon DNAJC14 overexpression is likely due to chaperone dysregulation and that YFV probably utilizes DNAJC14's cochaperone function to modulate processing at the NS3/4A site as a mechanism ensuring virus replication. IMPORTANCE: Flaviviruses are single-stranded RNA viruses that cause a wide range of illnesses. Upon host cell entry, the viral genome is translated on endoplasmic reticulum (ER) membranes to produce a single polyprotein, which is cleaved by host and viral proteases to generate viral proteins required for genome replication and virion production. Several studies suggest a role for molecular chaperones during these processes. While the details of chaperone roles have been elusive, in this report we show that overexpression of the ER-resident cochaperone DNAJC14 affects YFV polyprotein processing at the NS3/4A site. This work reveals that DNAJC14 modulation of NS3/4A site processing is an important mechanism to ensure virus replication. Our work highlights the importance of finely regulating flavivirus polyprotein processing. In addition, it suggests future studies to address similarities and/or differences among flaviviruses and to interrogate the precise mechanisms employed for polyprotein processing, a critical step that can ultimately be targeted for novel drug development.

Affinity Purification of the Hepatitis C Virus Replicase Identifies Valosin-Containing Protein, a Member of the ATPases Associated with Diverse Cellular Activities Family, as an Active Virus Replication Modulator.

Like almost all of the positive-strand RNA viruses, hepatitis C virus (HCV) induces host intracellular membrane modification to form the membrane-bound viral replication complex (RC), within which viral replicases amplify the viral RNA genome. Despite accumulated information about how HCV co-opts host factors for viral replication, our knowledge of the molecular mechanisms by which viral proteins hijack host factors for replicase assembly has only begun to emerge. Purification of the viral replicase and identification of the replicase-associated host factors to dissect their roles in RC biogenesis will shed light on the molecular mechanisms of RC assembly. To purify the viral replicase in the context of genuine viral replication, we developed an HCV subgenomic replicon system in which two different affinity tags were simultaneously inserted in frame into HCV NS5A and NS5B. After solubilizing the replicon cells, we purified the viral replicase by two-step affinity purification and identified the associated host factors by mass spectrometry. We identified valosin-containing protein (VCP), a member of the ATPases associated with diverse cellular activities (AAA+ATPase) family, as an active viral replication modulator whose ATPase activity is required for viral replication. A transient replication assay indicated that VCP is involved mainly in viral genome amplification. VCP associated with viral replicase and colocalized with a viral RC marker. Further, in an HCV replicase formation surrogate system, abolishing VCP function resulted in aberrant distribution of HCV NS5A. We propose that HCV may co-opt a host AAA+ATPase for its replicase assembly. IMPORTANCE: Almost all of the positive-strand RNA viruses share a replication strategy in which viral proteins modify host membranes to form the membrane-associated viral replicase. Viruses hijack host factors to facilitate this energy-unfavorable process. Understanding of this fundamental process is hampered by the challenges of purifying the replicase because of the technical difficulties involved. In this study, we developed an HCV subgenomic replicon system in which two different affinity tags were simultaneously inserted in frame into two replicase components. Using this dual-affinity-tagged replicon system, we purified the viral replicase and identified valosin-containing protein (VCP) AAA+ATPase as a pivotal viral replicase-associated host factor that is required for viral genome replication. Abolishing VCP function resulted in aberrant viral protein distribution. We propose that HCV hijacks a host AAA+ATPase for its replicase assembly. Understanding the molecular mechanism of VCP regulates viral replicase assembly may lead to novel antiviral strategies targeting the most conserved viral replication step.

Hepatitis C Virus-Associated Cancers.

Most hepatitis C virus (HCV) infection results in persistent infection. Significant portion of chronic HCV-infected patients develop hepatocellular carcinoma (HCC). Chronic hepatitis C is also associated with extrahepatic manifestations, including cryoglobulinemia, lymphoma, insulin resistance, type 2 diabetes, and neurological disorders. The molecular mechanisms of how HCV infection causes liver cancer are largely unknown. HCV replication or viral proteins may perturb cellular hemostasis and induce the generation of reactive oxygen species (ROS); viral components or viral replication products act as agonist to trigger innate immune response and cause chronic inflammation. Within the liver, non-hepatocytes such as hepatic stellate cell (HSC) are activated upon HCV infection to provide the major source of extracellular proteins and play important roles in fibrogenesis. With the great achievements of HCV treatment, especially the direct-acting antivirals (DAAs) against HCV, HCV eradication is possible. However, until now there are only very limited data on the effect of DAA-based anti-HCV treatment on HCC patients.

Hepatitis C virus replicative double-stranded RNA is a potent interferon inducer that triggers interferon production through MDA5.

The cytoplasmic RNA sensors, retinoic acid-inducible gene I and melanoma differentiation-associated gene 5, play crucial roles in innate sensing of hepatitis C virus (HCV). However, the exact identity of the IFN inducer generated during HCV infection is poorly understood. To identify the IFN inducer, we extracted the RNAs from HCV-replicating cells and introduced these into IFN signalling-competent cells to examine IFN production. RNAs isolated from HCV-replicating cells triggered robust IFN-ß and IFN-λ production in Huh7 cells in a viral replication-dependent manner, preferentially through the melanoma differentiation-associated gene 5 but not through the retinoic acid-inducible gene I-mediated pathway. The IFN-inducing capacity of HCV RNA survived after calf intestinal alkaline phosphatase and ssRNA-specific S1 nuclease treatment, but was completely eliminated by dsRNA-specific RNase III digestion, suggesting that viral replicative dsRNA is an IFN inducer. Furthermore, HCV viral RNA extracted from replicating cells was sensitive to 5'-monophosphate-dependent 5'→3' exonuclease (TER) digestion, suggesting that the HCV genome lacks a 5'-triphosphate or -diphosphate. In semi-permeabilized cells, the HCV IFN inducer primarily resided in an enclosed membranous structure that protects the IFN inducer from RNase digestion. Taken together, we identified HCV replicative dsRNA as a viral IFN inducer enclosed within the viral replication factory.

Innate detection of hepatitis B and C virus and viral inhibition of the response.

Viral hepatitis caused by hepatitis B virus (HBV) and hepatitis C virus (HCV) infections poses a significant burden to the public health system. Although HBV and HCV differ in structure and life cycles, they share unique characteristics, such as tropism to infect hepatocytes and association with hepatic and extrahepatic disorders that are of innate immunity nature. In response to HBV and HCV infection, the liver innate immune cells eradicate pathogens by recognizing specific molecules expressed by pathogens via distinct cellular pattern recognition receptors whose triggering activates intracellular signalling pathways inducing cytokines, interferons and anti-viral response genes that collectively function to clear infections. However, HBV and HCV evolve strategies to inactivate innate signalling factors and as such establish persistent infections without being recognized by the innate immunity. We review recent insights into how HBV and HCV are sensed and how they evade innate immunity to establish chronicity. Understanding the mechanisms of viral hepatitis is mandatory to develop effective and safe therapies for eradication of viral hepatitis.

Occurrence of decabromodiphenyl ethane in captive Chinese alligators (Alligator sinensis) from China.

Decabromodiphenyl ethane (DBDPE), a replacement for decabromodiphenyl ether (deca-BDE), was investigated in captive Chinese alligators from China. DBDPE was detected in adult tissues, neonates and eggs of Chinese alligators with concentrations ranging from 4.74-192, 0.24-1.94, and 0.01-0.51 ng g(-1) lipid weight, respectively. Compared to PBDEs and PCBs, DBDPE contamination was limited in Chinese alligators. Additionally, DBDPE concentrations in adult muscles were one to three orders of magnitude higher than those in neonates and eggs, suggesting the limited maternal transfer potential of DBDPE in Chinese alligators. This is the first study to report the occurrence of DBDPE in Chinese alligators.