Circulating metabolites are associated with persistent elevations of ALT in patients with chronic hepatitis B with complete viral suppression.

Hepatitis B virus (HBV) can be completely suppressed after antiviral treatment; however, some patients with chronic hepatitis B (CHB) exhibit elevated alanine aminotransferase (ALT) levels and sustained disease progression. This study provides novel insights into the mechanism and potential predictive biomarkers of persistently elevated ALT (PeALT) in patients with CHB after complete viral inhibition. Patients having CHB with undetectable HBV DNA at least 12 months after antiviral treatment were enrolled from a prospective, observational cohort. Patients with PeALT and persistently normal ALT (PnALT) were matched 1:1 using propensity score matching. Correlations between plasma metabolites and the risk of elevated ALT were examined using multivariate logistic regression. A mouse model of carbon tetrachloride-induced liver injury was established to validate the effect of key differential metabolites on liver injury. Of the 1238 patients with CHB who achieved complete viral suppression, 40 (3.23%) had PeALT levels during follow-up (median follow-up: 2.42 years). Additionally, 40 patients with PnALT levels were matched as controls. Ser-Phe-Ala, Lys-Ala-Leu-Glu, 3-methylhippuric acid, 3-methylxanthine, and 7-methylxanthine were identified as critical differential metabolites between the two groups and independently associated with PeALT risk. Ser-Phe-Ala and Lys-Ala-Leu-Glu levels could be used to discriminate patients with PeALT from those with PnALT. Furthermore, N-acetyl- l-methionine (NALM) demonstrated the strongest negative correlation with ALT levels. NALM supplementation alleviated liver injury and hepatic necrosis induced by carbon tetrachloride in mice. Changes in circulating metabolites may contribute to PeALT levels in patients with CHB who have achieved complete viral suppression after antiviral treatment.

Construction of an alpaca immune antibody library for the selection of nanobodies against Drosophila melanogaster proteins.

Introduction: Drosophila melanogaster is a model organism for studying developmental biology and human neural disorders. Nanobodies are the variable domains of the heavy chains of camelid heavy-chain antibodies (VHHs) with high affinity to their antigens and have applications in basic research, similar to traditional antibodies. In addition, nanobodies acting as functionalized antibodies or protein binders have become an additional valuable approach in Drosophila. This study aimed to develop a VHH library against Drosophila proteins and confirm its availability by retrieving some Drosophila protein-specific nanobodies from the library. Methods: An alpaca was first immunized with Drosophila embryo lysate and then its lymphocytes were isolated. Total RNA was extracted and cDNA was synthesized. The vhh sequences were amplified by two round PCR, which were then ligated to a phage display vector pADL-10b. The ligation products were transduced into SS320 competent cells to generate a VHH library. From this library, nanobodies against CG7544, Myc, and CyclinE was enriched and screened by phage display technology and ELISA. DNA sequences of identified nanobodies were cloned into pADL-10b-Flag-His for expression and purification in Escherichia coli SS320. Binding ability of purified nanobodies with corresponding antigens were determined by ELISA and surface plasmon resonance in vitro. Results: In this study, an immune VHH library against Drosophila embryo proteins was generated with a capacity of 3 × 107. From this library, eight nanobodies against three Drosophila proteins, Myc, CyclinE, and CG7544, were identified and the DNA sequences of these nanobodies were obtained. These nanobodies were successfully expressed and purified from Escherichia coli SS320, and were demonstrated to bind corresponding antigens with high affinity in vitro. Moreover, the equilibrium constant between the highest enriched nanobodies and corresponding antigens were calculated. Conclusion: In summary, we report the availability of an immune VHH library and a highly efficient panning strategy for nanobodies against proteins in Drosophila.

Rapid detection of Nipah virus using the one-pot RPA-CRISPR/Cas13a assay.

Nipah virus (NiV) is a zoonotic pathogen with airborne transmission and high case fatality rates in humans. There is currently no treatment or vaccine against NiV infection approved for humans or animals, therefore early diagnosis is the key to control any potential outbreaks. In this study, we developed an optimized one-pot assay using recombinase polymerase amplification (RPA) coupled to CRISPR/Cas13a for the molecular detection of NiV. The one-pot RPA-CRISPR/Cas13a assay for NiV detection was specific and did not cross-react against other selected (re)-emerging pathogens. The sensitivity of the one-pot RPA-CRISPR/Cas13a assay for NiV detection can detect as little as 103 cp/µL of total synthetic NiV cDNA. The assay was then validated with simulated clinical samples. The results for the one-pot RPA-CRISPR/Cas13a assay could be visualized with either fluorescence or lateral flow strips for convenient clinical or field diagnostics, providing a useful supplement to the gold-standard qRT-PCR assay for detecting NiV detections.

Polystyrene microplastics significantly facilitate influenza A virus infection of host cells.

Microplastics (MPs) are emerging pollutants which exist in various environments and pose a potential threat to human health. However, the effect of MP on respiratory pathogens-infected organisms is unknown. In order to explore the effect of MP on respiratory pathogen infection, we studied the effect of polystyrene microplastics (PS) on influenza A virus (IAV)-infected A549 cells. Western blot, qPCR, and viral plaque assay demonstrated that PS could promote IAV infection. Further study by bioluminescence imaging showed that a large number of IAV could be enriched on PS and entered cells through endocytosis. Meanwhile, the expression of IFITM3 in cells was significantly reduced. In addition, our results showed that PS down-regulated IRF3 and its active form P-IRF3 by down-regulating RIG-I and inhibiting TBK1 phosphorylation activation, which then significantly reduced IFN-ß expression and affected the cellular innate antiviral immune system. Taken together, our results indicate the potential threat of MPs to respiratory diseases caused by IAV and provide new insights into human health protection.

Pterostilbene effectively inhibits influenza A virus infection by promoting the type I interferon production.

With the prevalence of novel strains and drug-resistant influenza viruses, there is an urgent need to develop effective and low-toxicity anti-influenza therapeutics. Regulation of the type I interferon antiviral response is considered an attractive therapeutic strategy for viral infection. Pterostilbene, a 3,5-dimethoxy analog of resveratrol, is known for its remarkable pharmacological activity. Here, we found that pterostilbene effectively inhibited influenza A virus infection and mainly affected the late stages of viral replication. A mechanistic study showed that the antiviral activity of pterostilbene might promote the induction of antiviral type I interferon and expression of its downstream interferon-stimulated genes during viral infection. The same effect of pterostilbene was also observed in the condition of polyinosinic-polycytidylic acid (poly I:C) transfection. Further study showed that pterostilbene interacted with influenza non-structural 1 (NS1) protein, inhibited ubiquitination mediated degradation of RIG-I and activated the downstream antiviral pathway, orchestrating an antiviral state against influenza virus in the cell. Taken together, pterostilbene could be a promising anti-influenza agent for future antiviral drug exploitation and compounds with similar structures may provide new options for the development of novel inhibitors against influenza A virus (IAV).

Prevalence and WGS-based characteristics of MRSA isolates in hospitals in Shanghai, China.

Methicillin-resistant Staphylococcus aureus (MRSA) isolates remain a serious threat to global health despite a decrease in MRSA infections since 2005. MRSA isolates exhibit great diversity worldwide, and their lineages show geographic variation. In this study, we used whole genome sequencing (WGS) to analyze antibiotic resistance genes and virulence genes, spa, staphylococcal cassette chromosome mec, sequence types (STs), and core genome multilocus sequence typing (cgMLST) of MRSA isolates from patients and environmental surface in hospitals in China to determine their prevalence and molecular traits. The highest number of infections by MRSA isolates was observed in patients aged ≥60 years (69.8%, P < 0.05). We identified a total of 19 STs from 162 MRSA isolates from patients. A significant increase was observed in the incidence of ST764-t002-II MRSA infection, which is replacing ST5-t002-II MRSA as the predominant ST. Similarly, isolates from environmental surface were predominantly ST764-t002-II (47%). Notably, most ST764 isolates (97.7%) carried seb, but not arginine catabolic mobile element (ACME), which differed from ST764 isolates in Japan and Thailand. The potential danger of spreading requires rigorous surveillance of emerging ST764 MRSA isolates. We also found higher resistance to seven antimicrobials [OXA, cefoxitin (FOX), ERY, CFZ, ciprofloxacin (CIP), levofloxacin (LEV), and moxifloxacin (MXF)]. Resistance to gentamicin (38.3%), tetracycline (55.9%), and minocycline (41.5%) were also common. Phenotypic resistance to antimicrobials was associated with resistance genes to its content, and cgMLST clustering suggested a strong link between these strains. Overall, our findings revealed the prevalence and molecular characteristics of MRSA isolates in Shanghai, China, providing a theoretical reference for preventing and controlling clonal transmission of MRSA isolates in hospitals in China.

Persistence of Listeria monocytogenes ST5 in Ready-to-Eat Food Processing Environment.

Most human listeriosis is foodborne, and ready-to-eat (RET) foods contaminated by Listeria monocytogenes during processing are found to be common vehicles. In this study, a total of four L. monocytogens STs (ST5, ST121, ST120, and ST2) have been identified in two RTE food plants from 2019 to 2020 in Shanghai, China. The L. monocytogenes ST5 was predominant in one RTE food processing plant, and it persists in the RTE meat processing plant with continued clone transmission. The genetic features of the four STs isolates were different. ST5 and ST121 had the three genes clpL, mdrL, and lde; however, ST120 and ST2 had two genes except for clpL. SSI-1was present in ST5, ST121, and ST120. Additionally, SSI-2 was present only in the ST121 isolates. ST120 had all six biofilm-forming associated genes (actA, prfA, lmo0673, recO, lmo2504 and luxS). The ST2 isolate had only three biofilm-forming associated genes, which were prfA, lmo0673, and recO. The four ST isolates had different biofilm formation abilities at different stages. The biofilm formation ability of ST120 was significantly higher when grown for one day. However, the biofilm formation ability of ST120 reduced significantly after growing for four days. In contrast, the biofilm formation ability of ST5 and ST121 increased significantly. These results suggested that ST5 and ST121 had stronger ability to adapt to stressful environments. Biofilms formed by all four STs grown over four days can be sanitized entirely by a disinfectant concentration of 500 mg/L. Additionally, only ST5 and ST121 biofilm cells survived in sub-lethal concentrations of chlorine-containing disinfectant. These results suggested that ST5 and ST121 were more resistant to chlorine-containing disinfectants. These results indicated that the biofilm formation ability of L. monocytogenes isolates changed at different stages. Additionally, the persistence in food processing environments might be verified by the biofilm formation, stress resistance, etc. Alternatively, these results underlined that disinfectants should be used at lethal concentrations. More attention should be paid to ST5 and ST121, and stronger surveillance should be taken to prevent and control the clonal spread of L. monocytogenes isolates in food processing plants in Shanghai.

Identification and characterization of coiled-coil motifs across Autographa californica multiple nucleopolyhedrovirus genome.

Coiled coils (CCs) are protein structural motifs universally found in proteins and mediate a plethora of biological interactions, and thus their reliable annotation is crucial for studies of protein structure and function. Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is a large double-stranded DNA (dsDNA) virus and encodes 154 proteins. In this study, genome-wide scans of previously uncharacterized CC motifs throughout AcMNPV was conducted using CC prediction software. In total, 24 CC motifs in 19 CC proteins with high confidence were identified. The characteristic of viral CC motifs were analyzed. The CC proteins could be divided into 12 viral structural proteins and 7 non-structural proteins, including viral membrane fusion proteins, enzymes, and transcription factors. Moreover, CC motifs are conserved in the baculoviral orthologs of 14 of the 19 proteins. It is noted that five CC proteins, including Ac51, Ac66, Exon0, Ac13, and GP16, were previously identified to function in the nuclear egress of nucleocapsids, and Ac66 contains multiple CC motifs, the longest of which comprises 252 amino acids, suggesting a role of CC motifs in this process. Taken together, the CC motifs identified in this study are valuable resource for studying protein function and protein interaction networks during virus replication.

Troglitazone inhibits hepatic oval cell proliferation by inducing cell cycle arrest through Hippo/YAP pathway regulation.

Hepatic oval cells have strong proliferation and differentiation capabilities and are activated when chronic liver injury occurs or when liver function is severely impaired. Peroxisome proliferation-activated receptors (PPARs) are ligand-dependent, sequence-specific nuclear transcription factors. PPARγ is closely related to liver diseases (such as liver cancer, liver fibrosis and non-alcoholic fatty liver disease). As the main effector downstream of the Hippo signaling pathway, YAP can activate the hepatic progenitor cell program, and different expression or activity levels of YAP can determine different liver cell fates. We found that troglitazone (TRO), a classic PPARγ activator, can inhibit the growth of hepatic oval cells, and flow cytometry results showed that TRO inhibited the growth of WB-F344 cells by arresting the cells in the G0/1 phase. Western blot results also confirmed changes in G0/1 phase-related protein expression. Further experiments showed that PPARγ agonists induced hepatic oval cell proliferation inhibition and cell cycle G0/1 phase arrest through the Hippo/YAP pathway. Our experiment demonstrated, for the first time, the relationship between PPARγ and the Hippo/YAP pathway in liver oval cells and revealed that PPARγ acts as a negative regulator of liver regeneration by inhibiting the proliferation of oval cells.

aFGF Targeted Mediated by Novel Nanoparticles-Microbubble Complex Combined With Ultrasound-Targeted Microbubble Destruction attenuates Doxorubicin-Induced Heart Failure via Anti-Apoptosis and Promoting Cardiac Angiogenesis.

The purpose of this study was to evaluate the protective effect of acidic fibroblast growth factor targeted mediated by novel nanoparticles-cationic lipid microbubbles complex (aFGF-NP + CPMBs) combined with ultrasound targeted microbubble destruction (UTMD)on doxorubicin-induced heart failure (HF)and its mechanism. Heart failure rats induced by intraperitoneal injection with doxorubicin (DOX) to achieve cummulative dose of 15mg/kg for continuous 6 weeks showed left ventricular dysfunction, seriously oxidative stress, cardiomyocyte apoptosis, and decrease of myocardial vascular density. In contrast, aFGF-NP + CPMBs combined with UTMD therapy (3ug/kg, caudal vein injection, twice a week, 6weeks)prominently ameliorated left ventricular dysfunction by increased ejection fraction (EF) and fractional shortening (FS), decreased brain natriuretic peptide (BNP); strengthened the ability of antioxidant stress confirmed by increasing the activity of SOD and reducing the production of MDA; exerted the effect of anti-cardiomyocyte apoptosis and promotion angiogenesis by inhibited Bax expression and increased Bcl-2 expression and platelet endothelial cell adhesion molecule (CD31) expression. Taken together, the research suggested that aFGF targeted mediated by novel nanoparticles-cationic lipid microbubbles complex combined with UTMD should be a promising targeted treatment for heart failure.

Ultrasound-targeted microbubble destruction promotes myocardial angiogenesis and functional improvements in rat model of diabetic cardiomyopathy.

BACKGROUND: Microvascular insufficiency plays an important role in the development of diabetic cardiomyopathy (DCM), therapeutic angiogenesis has been mainly used for the treatment of ischemic diseases. This study sought to verify the preclinical performance of SonoVue microbubbles (MB) combined ultrasound (US) treatment on myocardial angiogenesis in the rat model of DCM and investigate the optimal ultrasonic parameters. METHODS: The male Sprague-Dawley (SD) rats were induced DCM by streptozotocin through intraperitoneal injecting and fed with high-fat diet. After the DCM model was established, the rats were divided into the normal group, DCM model group, and US + MB group, while the US + MB group was divided into four subsets according to different pulse lengths (PL) (8 cycles;18 cycle;26 cycle; 36 cycle). After all interventions, all rats underwent conventional echocardiography to examine the cardiac function. The rats were sacrificed and myocardial tissue was examined by histology and morphometry evaluations to detect the myocardial protective effect of SonoVue MBs using US techniques. RESULTS: From morphologic observation and echocardiography, the DCM rats had a series of structural abnormalities of cardiac myocardium compared to the normal rats. The US-MB groups exerted cardioprotective effect in DCM rats, improved reparative neovascularization and increased cardiac perfusion, while the 26 cycle group showed significant therapeutic effects on the cardiac functions in DCM rats. CONCLUSION: This strategy using SonoVue MB and US can improve the efficacy of angiogenesis, even reverse the progress of cardiac dysfunction and pathological abnormalities, especially using the 26 cycle parameters. Under further study, this combined strategy might provide a novel approach for early intervention of DCM in diabetic patients.

STAT1 participates in the induction of substance P expression in airway epithelial cells by respiratory syncytial virus.

PURPOSE: The regulation effect and mechanism of respiratory syncytial virus (RSV) infection on the expression of tachykinin substance P (SP) in airway epithelial cells was investigated. METHODS: The regulation of SP expression by RSV was investigated in the BEAS-2B airway epithelial cell line. RT-qPCR, immunofluorescence, and ELISA assay were used to examine the expression of the SP encoding gene TAC1, the intracellular SP protein expression, and the extracellular SP secretion. RESULTS: The mRNA expression of TAC1 and the intracellular SP protein level in BEAS-2B cells were significantly enhanced by RSV infection with multiplicity of infection (MOI) values of both 1 and 0.1 at 48 hours post infection. Heat-inactivated and UV-inactivated RSV, but not live RSV, significantly induced SP secretion in both control BEAS-2B cells and CX3CR1 receptor knockout cells without affecting the TAC1 gene expression or cell viability. RSV G protein (2-10 µg/ml) and fractalkine (10-50 ng/ml), both CX3CR1 receptor ligands, did not affect SP secretion in BEAS-2B cells. Inhibition of STAT1 phosphorylation by fludarabine (1 µM) markedly reduced the RSV-induced TAC1 gene expression and antagonized the inhibition of RSV replication by interferon-α in BEAS-2B cells. CONCLUSIONS: STAT1 participates in RSV infection-induced SP expression in airway epithelial cells.

Development and Clinical Application of a Rapid and Sensitive Loop-Mediated Isothermal Amplification Test for SARS-CoV-2 Infection.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) outbreak urgently necessitates sensitive and convenient COVID-19 diagnostics for the containment and timely treatment of patients. We aimed to develop and validate a novel reverse transcription-loop-mediated isothermal amplification (RT-LAMP) assay to detect SARS-CoV-2. Patients with suspected COVID-19 and close contacts were recruited from two hospitals between 26 January and 8 April 2020. Respiratory samples were collected and tested using RT-LAMP, and the results were compared with those obtained by reverse transcription-quantitative PCR (RT-qPCR). Samples yielding inconsistent results between these two methods were subjected to next-generation sequencing for confirmation. RT-LAMP was also applied to an asymptomatic COVID-19 carrier and patients with other respiratory viral infections. Samples were collected from a cohort of 129 cases (329 nasopharyngeal swabs) and an independent cohort of 76 patients (152 nasopharyngeal swabs and sputum samples). The RT-LAMP assay was validated to be accurate (overall sensitivity and specificity of 88.89% and 99.00%, respectively) and diagnostically useful (positive and negative likelihood ratios of 88.89 and 0.11, respectively). RT-LAMP showed increased sensitivity (88.89% versus 81.48%) and high consistency (kappa, 0.92) compared to those of RT-qPCR for SARS-CoV-2 screening while requiring only constant-temperature heating and visual inspection. The time required for RT-LAMP was less than 1 h from sample preparation to the result. In addition, RT-LAMP was feasible for use with asymptomatic patients and did not cross-react with other respiratory pathogens. The developed RT-LAMP assay offers rapid, sensitive, and straightforward detection of SARS-CoV-2 infection and may aid the expansion of COVID-19 testing in the public domain and hospitals.IMPORTANCE We developed a visual and rapid reverse transcription-loop-mediated isothermal amplification (RT-LAMP) assay targeting the S gene for SARS-CoV-2 infection. The strength of our study was that we validated the RT-LAMP assay using 481 clinical respiratory samples from two prospective cohorts of suspected COVID-19 patients and on the serial samples from an asymptomatic carrier. The developed RT-LAMP approach showed an increased sensitivity (88.89%) and high consistency (kappa, 0.92) compared with those of reverse transcription-quantitative PCR (RT-qPCR) for SARS-CoV-2 screening while requiring only constant-temperature heating and visual inspection, facilitating SARS-CoV-2 screening in well-equipped labs as well as in the field. The time required for RT-LAMP was less than 1 h from sample preparation to the result (more than 2 h for RT-qPCR). This study showed that the RT-LAMP assay was a simple, rapid, and sensitive approach for SARS-CoV-2 infection and can facilitate COVID-19 diagnosis, especially in resource-poor settings.

Development and production of nanobodies specifically against green fluorescence protein.

Variable domains of heavy chains of camelid heavy-chain antibodies (VHHs) are known as nanobodies. Nanobodies are approximately 15 kDa in size with high affinity to their antigens. They can be easily manipulated and produced in microorganisms. In this study, an alpaca was immunized with purified green fluorescence protein (GFP) and a VHH library from lymphocytes of the immunized alpaca was constructed with a capacity of 6.7 × 107. The library was biopanned against GFP by phage display technique and four unique DNA sequences coding for anti-GFP nanobodies were identified by enzyme-linked immunosorbent assay, named a12, e6, d5, and b9. The four DNA sequences were then cloned into pADL-10b-6×His or pBAD24-Flag-6×His for expression in bacteria. Purified A12, E6, D5, and B9 were demonstrated to bind GFP specifically both in vitro by enzyme-linked immunosorbent assay and native-PAGE analysis and in vivo by immunofluorescence and immunoprecipitation. Taken together, our results demonstrate that anti-GFP nanobodies are successfully selected from the immune library, are produced in bacteria, and are available for basic research.Key Points• Four different GFP binders were successfully obtained from an immune VHH library.• The four GFP binders were successfully purified from bacteria. • Purified GFP binders can bind GFP both in vitro and in vivo and are ready for use in basic research.

LAG-3 expression on tumor-infiltrating T cells in soft tissue sarcoma correlates with poor survival.

OBJECTIVE: To elucidate the role and prognostic significance of lymphocyte activation-gene-3 (LAG-3) in soft tissue sarcoma (STS). METHODS: The expression of LAG-3 in patient and matched normal blood samples was analyzed by flow cytometry. The localization and prognostic values of LAG-3+ cells in 163 STS patients were analyzed by immunohistochemistry. In addition, the expression of tumor-infiltrating CD3+ T, CD4+ T, and CD8+ T cells and their role in the prognosis of STS were evaluated by immunohistochemistry. The effect of LAG-3 blockade was evaluated in an immunocompetent MCA205 fibrosarcoma mouse model. RESULTS: Peripheral CD8+ and CD4+ T cells from STS patients expressed higher levels of LAG-3 than those from healthy donors. LAG-3 expression in STS was significantly associated with a poor clinical outcome (P = 0.038 ) and was correlated with high pathological grade (P < 0.001), advanced tumor stage ( P = 0.016). Additionally, LAG-3 expression was highly correlated with CD8+ T-cell infiltration (r = 0.7034, P < 0.001). LAG-3 was expressed in murine tumor-infiltrating lymphocytes, and its blockade decreased tumor growth and enhanced secretion of interferon-gamma by CD8 + and CD4+ T cells. CONCLUSIONS: LAG-3 blockade may be a promising strategy to improve the effects of targeted therapy in STS.

The deletion of the AcMNPV ac124 gene resulted in a decrease in chitinase transcription.

The Autographa californica multiple nucleopolyhedrovirus (AcMNPV) ac124 gene has been previously characterized as a viral pathogenicity factor. In this study, an ac124-knockout virus (vAc124KO) was generated to examine the role of the ac124 gene in the context of the AcMNPV genome during infection. Our results showed that the absence of ac124 does not affect the production of budded virus (BV) and occlusion bodies (OBs) in infected Sf9 cells. Western blotting analysis showed that the deletion of ac124 does not affect the temporal expression and the relative levels of GP64, VP39, P6.9, and polyhedrin. qRT-PCR analysis showed that the transcription level of chitinase but not the adjacent cathepsin in vAc124KO infected cells was significantly lower than that of the vAcWT infected cells from 24 to 96 h p.i. Luciferase assays showed that the overexpression of Ac124 could significantly improve the ability of chitinase promoter to initiate reporter genes. Based on the above data, we hypothesize that Ac124 binds to the promoter of chitinase to regulate the expression of chitinase gene.

Author Correction: Ephrin receptor A2 is an epithelial cell receptor for Epstein-Barr virus entry.

In the version of this Letter originally published, the authors reported on the use of 2,5-dimethylpyrrolyl benzoic acid to block Ephrin receptors. In 2011, it was reported that newly synthesized 2,5-dimethylpyrrolyl benzoic acid lacked the previously reported EphA2 antagonizing activity1. However, the purchased compound did in fact have the activity initially reported, suggesting that an uncharacterized alteration occurred during storage. The authors therefore wish to clarify that the compound used in their study should be more accurately referred to as a 2,5-dimethylpyrrolyl benzoic acid derivative. All references to 2,5-dimethylpyrrolyl benzoic acid in the Letter have now been changed to reflect this.Although 2,5-dimethylpyrrolyl benzoic acid derivatives have been reported to have off-target effects2, as do most small-molecule inhibitors, the multiple complementary methods and techniques used demonstrate that EphA2 is a key Epstein-Barr virus epithelial cell receptor. The conclusions of the study are therefore unchanged.

Structure of Schlafen13 reveals a new class of tRNA/rRNA- targeting RNase engaged in translational control.

Cleavage of transfer (t)RNA and ribosomal (r)RNA are critical and conserved steps of translational control for cells to overcome varied environmental stresses. However, enzymes that are responsible for this event have not been fully identified in high eukaryotes. Here, we report a mammalian tRNA/rRNA-targeting endoribonuclease: SLFN13, a member of the Schlafen family. Structural study reveals a unique pseudo-dimeric U-pillow-shaped architecture of the SLFN13 N'-domain that may clamp base-paired RNAs. SLFN13 is able to digest tRNAs and rRNAs in vitro, and the endonucleolytic cleavage dissevers 11 nucleotides from the 3'-terminus of tRNA at the acceptor stem. The cytoplasmically localised SLFN13 inhibits protein synthesis in 293T cells. Moreover, SLFN13 restricts HIV replication in a nucleolytic activity-dependent manner. According to these observations, we term SLFN13 RNase S13. Our study provides insights into the modulation of translational machinery in high eukaryotes, and sheds light on the functional mechanisms of the Schlafen family.

Pathogenicity and transmissibility of three avian influenza A (H5N6) viruses isolated from wild birds.

Since 2013, highly pathogenic H5N6 avian influenza viruses (AIVs) have emerged in poultry and caused sporadic human infections in Asia. The recent discovery of three new avian H5N6 viruses - A/oriental magpie-robin/Guangdong/SW8/2014 (H5N6), A/common moorhen/Guangdong/GZ174/2014 (H5N6) and A/Pallas's sandgrouse/Guangdong/ZH283/2015 (H5N6) - isolated from apparently healthy wild birds in Southern China in 2014-2015 raises great concern for the spread of these highly pathogenic AIVs (HPAIVs) and their potential threat to human and animal health. In our study, we conducted animal experiments and tested their pathogenicity in ducks, chickens and mice. Ducks can carry and shed the H5N6 HPAIVs, but show no ill effects. On the other hand, these H5N6 HPAIVs can efficiently infect, transmit and cause death in chickens. Due to the overlap of habitats, domestic ducks play an important role in circulating AIVs between poultry and wild birds. Our results raise the possibility that wild birds disseminate these H5N6 HPAIVs to poultry along their flyways and thus pose a great threat to the poultry industry. These viruses are also highly pathogenic to mice, suggesting they pose a potential threat to mammals and, thus, public health. One virus isolated in 2015 replicates much more efficiently and is more lethal in these animals than the two other viruses isolated in 2014. It seems that the H5N6 viruses tend to be more lethal as time passes. Therefore, it is necessary to vigilantly monitor H5N6 HPAIVs in wild birds and poultry.