Replication of hepatitis B virus in primary duck hepatocytes transfected with linear viral DNA

AIM: To explore the expression and replication of hepatitis B virus (HBV) DNA in primary duck hepatocytes (PDHs).METHODS: Complete HBV genome was transfected into PDHs by electroporation (transfected group, 1.19×1012copies of linear HBV DNA/1×107 PDHs). After 1-5 d of transfection, HBsAg and HBeAg in the supernatant and lysate of PDHs were measured with the IMX System.Meanwhile, replicative intermediates of HBV DNA were analyzed by Southern blotting and Dot blotting. PDHs electroporated were used as control group.RESULTS: HBsAg in the hepatocyte lysates of transfected group was 15.24 (1 d), 14.55 (3 d) and 5.13 (5 d; P/N values, positive≥2.1) respectively. HBeAg was negative (＜2.1). Both HBsAg and HBeAg were negative in the supernatant of transfected group. Dot blotting revealed that HBV DNA was strongly positive in the transfected group and negative in the control group. Southern blot analysis of intracellular total DNA indicated that there were relaxed circular (rc DNA), covalently closed circular (ccc DNA), and single-stranded (ss DNA) HBV DNA replicative intermediates in the transfected group, there was no integrated HBV DNA in the cellular genome. These parameters were negative in control group.CONCLUSION: Expression and replication of HBV genes can occur in hepatocytes from non-mammalian species.HBV replication has no critical species-specificity, and yet hepatic-specific regulating factors in hepatocytes may be essential for viral replication.

The antiviral treatment should be initiated before the development of cirrhosis

Hepatitis B virus(HBV)infection increases the risk of liver fibrosis,cirrhosis and hepatocellular carcinoma(HCC)(1).International guidelines recommend antiviral treatment for eligible patients to prevent the development of HCC and the progression of liver disease.However,there is discrepancy in these guidelines regarding treatment recommendations for patients with compensated cirrhosis and low-level viremia(LLV),defined as serum HBV-DNA level of 20-2,000 IU/mL(2-4).And data on the natural history of untreated compensated cirrhotic patients with LLV is limited.

A model of five genes of tumor microenvironment predicts prognosis in Cholangiocarcinoma

Background:Cholangiocarcinoma(CCA)is highly malignant and has a poor prognosis has a high malignant degree and poor prognosis.The purpose of this study is to develop a new prognostic model based on genes related to the tumor microenvironment(TME).Methods:Derived from the discerned differentially expressed genes within The Cancer Genome Atlas(TCGA)dataset,this investigation employed the methodology of weighted gene co-expression network analysis(WGCNA)to ascertain gene co-expressed modules intricately linked to the Tumor Microenvironment(TME)among Cholangiocarcinoma(CCA)patients.The genes associated with prognosis,as identified through Cox regression analysis,were employed in the formulation of a predictive model.This model underwent validation,leading to the development of a risk score formula and nomogram.Concurrently,we validated the model’s reliability using data from CCA patients in the Gene Expression Omnibus(GEO)database(accession:GSE107943).Results:6139 DEGs were divided into 10 co-expressed gene modules using WGCNA.Among these,two modules(blue module with 832 genes and brown module with 1379 genes)showed high correlation with the TME.Five prognostic genes(BNIP3,COL4A3,SPRED3,CEBPB,PLOD2)were identified through Cox regression analysis,and a prognostic model and risk score formula were developed based on these genes.Risk score formula:Risk score=BNIP3×1.70520-COL4A3×2.39815+SPRED3×1.17936+CEBPB×0.40456+PLOD2×0.24785.Kaplan-Meier survival analysis revealed that the survival probabilities of the low-risk group were significantly higher than those of the high-risk group.Furthermore,the related evaluation indexes suggested that the model exhibited strong predictive ability.Conclusion:The prognostic model,based on five TME-related genes(BNIP3,COL4A3,SPRED3,CEBPB,PLOD2),could accurately assess the prognosis of CCA patients to aid in guiding clinical decisions.

Biological impact of hepatitis B virus X-hepatitis C virus core fusion gene on human hepatocytes

AIM： To investigate the biological impact of hepatitis B virus X- hepatitis C virus core （HBV X-HCV C） fusion gene on hepatoma cells.METHODS： The recombinant adenoviruses Ad- XC, Ad-X and Ad-C expressing HBV X-HCV C fusion gene, HBVX gene and HCV C gene were constructed, respectively. Hepatoma cells were infected with different recombinant adenoviruses. MTT, colony- forming experiment, FCM, TUNEL assay were performed to observe the biological impact of the HBV X-HCV C fusion aene on liver cells.RESULTS： MTT showed that the Ad-XC group cells grew faster than the other group cells. Colony-forming experiment showed that the colony-forming rate for the Ad-XC group cells was significantly higher than that for the other group cells. FCM analysis showed that Ad-XC/Ad-X/Ad-C infection enhanced the progression of G1→S phase in the HepG2 cell cycle. The apoptosis index of the Ad-XC, Ad-X, Ad-C group cells was significantly lower than that of the AdO and control group cells. Semi-quantitative RT-PCR showed that the expression level of c-myc was the highest in Ad- XC infected cells. Tumor formation was found at the injected site of mice inoculated with Ad-XC-infected LO2 cells, but not in control mice.CONCLUSION： Ad-XC, Ad-X and Ad-C facilitate the proliferation activity of HepG2 cells and inhibit their apoptosis in vitro. The effect of Ad-XC is significantly stronger than that of Ad-X and Ad-C. Up-regulation of c-myc may be one of the mechanisms underlying the synergism of HBVX and HCV C genes on hepatocarcinogenesis in athymic nude mice.

Hepatitis B virus and hepatocellular carcinoma at the miRNA level

AIM: To study Hepatitis B virus (HBV) infection and its association with hepatocellular carcinoma (HCC) at the miRNA level.METHODS: Three cellular models were used to investigate miRNA expression changes during HBV infection: human HepG2 hepatoblastoma cell line as a model without HBV infection;HepG2 cell line transfected with a 1.3-fold full-length HBV genome as an acute infection model;and HepG2.2.15 cell line,which is derived from HepG2 and stably transfected with a complete HBV genome,as a chronic infection model.The miRNA levels were examined using microarray technology.To explore the relationship between HBV infection and HCC genesis at the miRNA level,we downloaded from national center for biotechnology information Gene Expression Omnibus an miRNA expression dataset derived from HCC patients,most of whom are HBV carriers.We compared the miRNA expression alterations during HBV infection with those in HCC patients,by analyzing miRNA expression change profiles statistically.RESULTS: Seventy-seven and 48 miRNAs were differentially expressed during acute and chronic HBV infection,respectively.Among these miRNAs,25 were in common,the intersection of which was significant under the hypergeometric test (P = 1.3 × 10-11).Fourteen miRNAs were observed to change coherently in the acute and chronic infections,with one upregulated and 13 downregulated.Eleven showed inverse changes during the two phases of infection;downregulated in the acute infection and upregulated in the chronic infection.The results imply that common and specific mechanisms exist at the miRNA level during acute and chronic HBV infection.Besides,comparative analysis of the miRNA expression changes during HBV infection with those in HCC indicates that,although miRNA expression changes during HBV infection are distinct from those in HCC patients (P < 2.2 × 10-16),they exhibited significant correlations (P = 0.0229 for acute infection;P = 0.0084 for chronic infection).Perturbation of miRNA expression during chronic HBV infection was closer to that in HCC patients than that during acute HBV infection.This observation implies the contribution of miRNAs to HCC genesis from HBV infection.According to their patterns of differential expression in acute and chronic HBV infection,as well as in HCC,miRNAs of potential research interest could be identified,such as miR-18a/miR-18b,miR-106a,miR-221 and miR-101.For instance,the gradient expression alteration of miR-221 in the above three phases,which is downregulated in acute HBV infection,normally expressed in chronic HBV infection,and upregulated in HCC,indicates that it may be a key effector for progression of the disease.CONCLUSION: Our analysis provides insights into HBV infection and related HCC in relation to miRNAs,and reveals some candidate miRNAs for future studies.

The natural history of chronic hepatitis B:a retrospective study

OBJECTIVE: To clarify the natural history, of chronic hepatitis B so as to evaluate its long-term therapeutic outcome of the patients and the efficacy of antiviral drugs. METHODS: A cohort of 183 biopsy-proven chronic hepatitis B patients (mean age of 31.75±8.03 years, male/female ratio: 152:31) and 247 controls were followed up retrospectively for 11.81±4.08 years. This study was focused on long-term clinical outcome including the rates of liver cirrhosis, hepatocellular carcinoma and death, apart from the long-term effect of antiviral drugs and prognostic factors. RESULTS: In the 183 chronic hepatitis B patients, 22 (12.02%) developed liver cirrhosis, 12 (6.56%) developed hepatocellular carcinoma, and 20 (10.93%) died. The 5-, 10- and 15-year survival rates were 97. 27%, 91.62%, and 84.47%, respectively. The 5-, 10- and 15-year incidence rates of HCC were O, 3.19%, and 11.56%, respectively. In the 247 controls, 6 (2.43%) died; none of them developed cirrhosis or HCC. The rates of death, liver cirrhosis, and HCC in the hepatitis B patients were markedly different (P<0. 005) compared with the controls. The overall mortality of hepatitis B patients was 4.5-fold higher than the general population. Cox multiple regression analysis showed that old age, severe histological injury, and positive HBeAg were closely related to liver cirrhosis; old age, severe histological injury, and male were major factors leading to death. The independent variable of predicted HCC was not found. CONCLUSION: The long-term outcome of hepatitis B patients is poor and the efficacy of antiviral drugs needs further study.

Single-cell and spatial heterogeneity landscapes of mature epicardial cells

Tbx18,Wt1,and Tcf21 have been identified as epicardial markers during the early embryonic stage.However,the gene markers of mature epicardial cells remain unclear.Single-cell transcriptomic analysis was performed with the Seurat,Monocle,and CellphoneDB packages in R software with standard procedures.Spatial transcriptomics was performed on chilled Visium Tissue Optimization Slides(10x Genomics)and Visium Spatial Gene Expression Slides(10x Genomics).Spatial transcriptomics analysis was performed with Space Ranger software and R software.Immunofluorescence,whole-mount RNA in situ hybridization and X-gal staining were performed to validate the analysis results.Spatial transcriptomics analysis revealed distinct transcriptional profiles and functions between epicardial tissue and non-epicardial tissue.Several gene markers specific to postnatal epicardial tissue were identified,including Msln,C3,Efemp1,and Upk3b.Single-cell transcriptomic analysis revealed that cardiac cells from wildtype mouse hearts(from embryonic day 9.5 to postnatal day 9)could be categorized into six major cell types,which included epicardial cells.Throughout epicardial development,Wt1,Tbx18,and Upk3b were consistently expressed,whereas genes including Msln,C3,and Efemp1 exhibited increased expression during the mature stages of development.Pseudotime analysis further revealed two epicardial cell fates during maturation.Moreover,Upk3b,Msln,Efemp1,and C3 positive epicardial cells were enriched in extracellular matrix signaling.Our results suggested Upk3b,Efemp1,Msln,C3,and other genes were mature epicardium markers.Extracellular matrix signaling was found to play a critical role in the mature epicardium,thus suggesting potential therapeutic targets for heart regeneration in future clinical practice.

Gene therapy that inhibits NF-κB results in apoptosis of human hepatocarcinoma by recombinant adenovirus

AIM： To investigate whether the recombinant adenovirus induces the TNF-α-mediated apoptosis in vivo. METHODS： Human hepatocarcinoma cell line （HepG2） cells were transfected into BALB/c nude mice, and the tumor growth curve was drawn. We analyzed apoptosis in HepG2 cells by TUNEL, HE staining and electron microscopy. RESULTS： AdIκBαM was expressed stably and efficiently in HepG2 and could not be degraded by induction of TNF-α. Tumor growth in mice could be reduced remarkably if treated by AdIκBαM plus TNF-α. There was apoptosis of 〉 70% of cells treated with AdIκBαM plus TNF-α and about 50% of cells treated with AdIκBαM. In contrast, there was few cell apoptosis in HepG2 cells treated with phosphate buffered saline and AdIκBαM. HepG2 cells in mice also exhibited a high level of apoptosis after in vivo injection with AdIκBαM. The tumor growth curve indicated the tumor transfected with AdIκBαM could be restrained. CONCLUSION： AdIκBαM gene therapy greatly enhances apoptosis due to inhibition of an NF-κB-mediated antiapoptosis signaling pathway.

Specific anti-viral effects of RNA interference on replication and expression of hepatitis B virus in mice

Background RNA interference （RNAi） is a powerful tool to silence gene expression post-transcriptionally. Our previous study has demonstrated that small interfering RNAs （siRNAs） have sufficiently inhibited hepatitis B virus （HBV） replication and expression in vitro. In this study we observed the RNAi-mediated inhibitory effects on HBV replication in mice models and accessed the specificity of these effects. Methods A mutant RNAi vector （pSI-C mut） with two base pairs different from the original target gene sequence at the RNAi vector （pSI-C） was constructed according to the method described in this study, A mouse model of acute hepatitis B virus infection was established by injecting naked plasmid pHBV1.3 via the tail vein with acute circulatory overload, pSI-C, pSI-C mut and the irrelevant RNAi control plasmid for green fluorescent protein （GFP） gene, pSIGFP were respectively delivered with pHBV1.3 by tail vein injection method. Six days post injection, enzyme-linked immunosorbent assay （ELISA） assay was used to measure the concentration of HBV surface antigen （HBsAg） in mouse serum, immunohistochemical straining method was used to visualize the expressin of HBV core protein （HBcAg） in liver tissues, and the transcriptional level of HBV C mRNA in liver tissues was detectedd by reverse transcriptase PCR （RT-PCR） analysis. Results Injection of pSI-C exerted magnificent and specific inhibitory effects on the replication and expression of HBV in the murine model. After 6-day post-injection （ p. i. ）, the OD values were shown to be 5.07 ± 1.07 in infecting group and 0.62 ± 0. 59 in pSI-C group. The concentration of HBsAg in pSI-C group was significantly lower than that in infecting group （ P 〈 0. 01 ）. Liver intracellular synthesis of viral core protein was sharply reduced to 0. 9% ±0. 1%, compared with 5.4% ± 1.2% of positive hepatocytes in infecting group （P 〈0. 01 ）, and the transcriptional level of HBV C mRNA was greatly reduced by 84. 7%. However, the irrelevant RNAi control plasmid （pSIGFP）, and the pSI-C mut did not show the same robust inhibitory effects as pSI-C. Conclusion pSI-C exert efficient and specific inhibitory effects on HBV replication and expression in mice models.

Guideline of Prevention and Treatment for Chronic Hepatitis B (2nd Version)

This guideline is established to standardize the prevention,diagnosis and antiviral therapy of chronic hepatitis B(CHB).For other treatment regimens and methods involving CHB,please refer to relevant guidelines and consensuses.The Chinese Society of Hepatology,Chinese Medical Association(CMA)and the Society of Infectious Diseases,CMA organized relevant native experts to establish this Guideline of Prevention and Treatment for Chronic Hepatitis B(1st version)in 2005,and made the first revision in 2010.In the past 5 years,great progress has been made in the native and foreign fundamental and clinical research with respect to CHB,necessitating additional revision of this guideline.

Large Disparity between Prevalence and Treatment Rates for Hepatitis C in Western China

Background and Aims:Recently,the World Health Organization adopted the first-ever global hepatitis strategy with the dream of eliminating viral hepatitis as a public health threat by 2030.However,the epidemiology and treatment rates of hepatitis C virus(HCV)infection in Western China are still unknown.Methods:A total of 111,916 adult individuals(15-96 years)who underwent the HCV-antibody(HCV-Ab)test in the Second Affiliated Hospital of Chongqing Medical University between 2013 and 2015 were included in this study.We retrospectively analyzed the electronic medical records'data for each,and the positivity of HCV-Ab and the treatment of HCV RNA-positive patients were evaluated.Results:During 2013-2015,the crude prevalence of HCVAb was 1.4%(95%CI:1.4-1.5;1,611/111,916)and the adjusted prevalence of HCV-Ab was 1.7%(95%CI:1.6-1.8),which was higher than in the 2006 national study(0.43%).The prevalence was 2-times higher in males than females(2.0%vs.1.1%,p<0.01).Notably,only 46%(434/951)of the HCV RNA-positive patients received standard peginterferon plus ribavirin treatment,with 370(82%)that completed treatment,of whom 272(74%)achieved sustained virologic response(SVR).Particularly,11%(32/292)of HCV RNA-positive patients were HBsAg-positive,and the SVR rate for them was lower than for the HBsAg-negative patients,but no significant difference was observed.Conclusions:HCV infection may have increased since 2006 in Western China.The SVR rate of peg-interferon plus ribavirin treatment was high,but the proportion of untreated HCV patients was large.Thus,more efforts need to be made by the government to create a scientific-based policy for HCV treatment and prevention.

P19 of Tomato Bushy Stunt Virus Suppresses RNA Silencing Induced by Short Hairpin RNA in Mammal Cells

To counteract the immune system in parasitic hosts,some viruses encode proteins to suppress the RNA interference(RNAi)effect.In this report,we established two RNAi systems to be easily observed with strong and obvious effect.The function of the P19 of tomato bushy stunt virus,which suppresses RNAi in mammal cells,was then studied using these two systems.Short hairpin RNAs targeting green fluorescence protein(pshRNA-GFP)and firefly luciferase(pshRNA-luc)were designed and inserted into a eukaryotic transcriptional vector pTZU6+1,respectively.The shRNA expressing vectors were co-transfected with plasmids containing the target gene with or without P19.The GFP expression level was assayed by fluorescence microscopy,Western blotting and RT-PCR.The luciferase expression level was analyzed by the dual-luciferase assay system.pshRNA designed in this study down-regulated the target gene specifically and efficiently,with a decrease of expression of both genes of about 70%,respectively.When P19 was introduced into the RNAi systems,the expression of both GFP and the luciferase were mostly recovered compared with the control groups.The RNAi systems of GFP and luciferase were constructed successfully,demonstrating that P19 of tomato bushy stunt virus has the ability to counteract the RNAi effect induced by shRNA in mammal cells.

Inhibiting HMGCR represses stemness and metastasis of hepatocellular carcinoma via Hedgehog signaling

Cancer stem cells(CSCs)play a crucial role in tumor initiation,recurrence,metastasis,and drug resistance.However,the current understanding of CSCs in hepatocellular carcinoma(HCC)remains incomplete.Through a comprehensive analysis of the database,it has been observed that 3-hydroxy-3-methylglutaryl-coenzyme A reductase(HMGCR),a critical enzyme involved in cholesterol synthesis,is up-regulated in HCC tissues and liver CSCs.Moreover,high expression of HMGCR is associated with a poor prognosis in patients with HCC.Functionally,HMGCR promotes the stemness and metastasis of HCC both in vitro and in vivo.By screening various signaling pathway inhibitors,we have determined that HMGCR regulates stemness and metastasis by activating the Hedgehog signaling in HCC.Mechanistically,HMGCR positively correlates with the expression of the Smoothened receptor and facilitates the nuclear translocation of the transcriptional activator GLI family zinc finger 1.Inhibition of the Hedgehog pathway can reverse the stimulatory effects of HMGCR on stemness and metastasis in HCC.Notably,simvastatin,an FDA-approved cholesterol-lowering drug,has been shown to inhibit stemness and metastasis of HCC by targeting HMGCR.Taken together,our findings suggest that HMGCR promotes the regeneration and metastasis of HCC through the activation of Hedgehog signaling,and simvastatin holds the potential for clinical suppression of HCC metastasis.

Safety and Immunogenicity After Primary and Booster Inactivated SARS-Cov-2 Vaccination in Patients with Autoimmune Liver Diseases

Background and Aims:SARS-CoV-2 vaccines-associated autoimmune liver diseases have been reported in several case reports.However,the safety and immunogenicity after primary and booster inactivated SARS-CoV-2 vaccination in patients with autoimmune liver diseases(AILD)is still unknown.Methods:Eighty-four patients with AILD were prospectively followed up after the second dose(primary)of inactivated SARS-CoV-2 vaccine.Some of them received the third dose(booster)of inactivated vaccine.Adverse events(AEs),autoimmune activation,and liver inflammation exacerbation after primary and booster vaccination were recorded.Meanwhile,dynamics of antireceptor-binding-domain IgG(anti-RBD-IgG),neutralizing antibodies(NAbs)and RBD-specific B cells responses were evaluated.Results:The overall AEs in AILD patients after primary and booster vaccination were 26.2%and 13.3%,respectively.The decrease of C3 level and increase of immunoglobulin light chain K andλlevels were observed in AILD patients after primary vaccination,however,liver inflammation was not exacerbated,even after booster vaccination.Both the seroprevalence and titers of anti-RBD-IgG and NAbs were decreased over time in AILD patients after primary vaccination.Notably,the antibody titers were significantly elevated after booster vaccination(10-fold in anti-RBD-IgG and 7.4-fold in NAbs,respectively),which was as high as in healthy controls.Unfortunately,the inferior antibody response was not enhanced after booster vaccination in patients with immunosuppressants.Changes of atypical memory B cells were inversely related to antibody levels,which indicate that the impaired immune memory was partially restored partly by the booster vaccination.Conclusions:The well tolerability and enhanced humoral immune response of inactivated vaccine supports an additional booster vaccination in AILD patients without immunosuppressants.

PCK1 attenuates tumor stemness via activating the Hippo signaling pathway in hepatocellular carcinoma

Liver cancer stem cells were found to rely on glycolysis as the preferred metabolic program.Phosphoenolpyruvate carboxylase 1(PCK1),a gluconeogenic metabolic enzyme,is down-regulated in hepatocellular carcinoma and is closely related to poor prognosis.The onco-genesis and progression of tumors are closely related to cancer stem cells.It is not completely clear whether the PCK1 deficiency increases the stemness of hepatoma cells and promotes the oncogenesis of hepatocellular carcinoma.Herein,the results showed that PCK1 inhibited the self-renewal property of hepatoma cells,reduced the mRNA level of cancer stem cell markers,and inhibited tumorigenesis.Moreover,PCK1 increased the sensitivity of hepatocellular carci-noma cells to sorafenib.Furthermore,we found that PCK1 activated the Hippo pathway by enhancing the phosphorylation of YAP and inhibiting its nuclear translocation.Verteporfin reduced the stemness of hepatoma cells and promoted the pro-apoptotic effect of sorafenib.Thus,combined treatment with verteporfin and sorafenib may be a potential anti-tumor strat-egy in hepatocellular carcinoma.

Mitochondrial GRIM19 Loss Induces Liver Fibrosis through NLRP3/IL33 Activation via Reactive Oxygen Species/NF-κB Signaling

Background and Aims:Hepatic fibrosis(HF)is a critical step in the progression of hepatocellular carcinoma(HCC).Gene associated with retinoid-IFN-induced mortality 19(GRIM19),an essential component of mitochondrial respiratory chain complex I,is frequently attenuated in various human cancers,including HCC.Here,we aimed to investigate the potential relationship and underlying mechanism between GRIM19 loss and HF pathogenesis.Methods:GRIM19 expression was evaluated in normal liver tissues,hepatitis,hepatic cirrhosis,and HCC using human liver disease spectrum tissue microarrays.We studied hepatocyte-specific GRIM19 knockout mice and clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR-associated protein-9(Cas9)lentivirus-mediated GRIM19 gene-editing in murine hepatocyte AML12 cells in vitro and in vivo.We performed flow cytometry,immunofluorescence,immunohistochemistry,western blotting,and pharmacological intervention to uncover the potential mechanisms underlying GRIM19 loss-induced HF.Results:Mitochondrial GRIM19 was progressively downregulated in chronic liver disease tissues,including hepatitis,cirrhosis,and HCC tissues.Hepatocyte-specific GRIM19 heterozygous deletion induced spontaneous hepatitis and subsequent liver fibrogenesis in mice.In addition,GRIM19 loss caused chronic liver injury through reactive oxygen species(ROS)-mediated oxidative stress,resulting in aberrant NF-κB activation via an IKK/IKB partner in hepatocytes.Further-more,GRIM19 loss activated NLRP3-mediated IL33 signaling administration of the NLRP3 inhibitor MCC950 dramatically via the ROS/NF-κB pathway in hepatocytes.Intraperitoneal alleviated GRIM19 loss-driven HF in vivo.Conclusions:The mitochondrial GRIM19 loss facilitates liver fibrosis through NLRP3/IL33 activation via ROS/NF-κB signaling,providing potential therapeutic approaches for earlier HF prevention.

Chronic Hepatitis B Infection with Low Level Viremia Correlates with the Progression of the Liver Disease

Background and Aims:Currently,insufficient clinical data are available to address whether low-level viremia(LLV)observed during antiviral treatment will adversely affect the clinical outcome or whether treatment strategies should be altered if LLV occurs.This study compared the clinical out-comes of patients with a maintained virological response(MVR)and patients who experienced LLV and their treatment strategies.Methods:A retrospective cohort of 674 patients with chronic hepatitis B virus(HBV)infection who received antiviral treatment for more than 12 months was analyzed for the development of end-stage liver disease and treatment strategies during the follow-up period.End-stage liver disease included decompensated liver cirrhosis and hepatocellular carcinoma(HCC).Results:During a median 42-month follow-up,end-stage liver disease developed more frequently in patients who experienced LLV than in those who experienced MVR(7.73%and 15.85%vs.0.77%and 5.52%at 5 and 10 years,respectively;p=0.000).The trend was consistent after propensity score matching.In the high-risk group of four HCC risk models,LLV patients had a higher risk of HCC development(p<0.05).By Cox proportional hazard model analysis,LLV was an independent risk factor for end-stage liver disease and HCC(hazard ratio[HR]=6.280,confidence interval[CI]=2.081-18.951,p=0.001;HR=5.108,CI=1.392-18.737,respectively;p=0.014).Patients achieved a lower rate of end-stage liver disease by adjusting treatment compared to continuing the original treatment once LLV occurred(p<0.05).Conclusions:LLV is an independent risk factor for end-stage liver disease and HCC,and treatment adjustments can be considered.

Low-level viremia in nucleoside analog-treated chronic hepatitis B patients

Low-level viremia(LLV)was defined as persistent or intermittent episodes of detectable hepatitis B virus(HBV)DNA(<2000 IU/mL,detection limit of 10 IU/mL)after 48 weeks of antiviral treatment.Effective antiviral therapies for chronic hepatitis B(CHB)patients,such as entecavir(ETV),tenofovir disoproxil fumarate(TDF),and tenofovir alafenamide(TAF),have been shown to inhibit the replication of HBV DNA and prevent liver-related complications.However,even with long-term antiviral therapy,there are still a number of patients with persistent or intermittent LLV.At present,the research on LLV to address whether adversely affect the clinical outcome is limited,and the follow-up treatment for these patients is open to question.At the same time,the mechanism of LLV is not clear.In this review,we summarize the incidence of LLV,the association between LLV and long-term outcomes,possible mechanisms,and management strategies in these patient populations.

Development of cell-based pseudovirus entry assay to identify potential viral entry inhibitors and neutralizing antibodies against SARS-CoV-2

Severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)is the causative virus of the coronavirus disease 2019(COVID-19)pandemic.To establish a safe and convenient assay system for studying entry inhibitors and neutralizing antibodies against SARS-CoV-2,we constructed a codon-optimized,full-length C-terminal mutant spike(S)gene of SARS-CoV-2.We generated a luciferase(Luc)-expressing pseudovirus containing the wild-type or mutant S protein of SARS-CoV-2 in the envelope-defective HIV-1 backbone.The key parameters for this pseudovirus-based assay,including the S mutants and virus incubation time,were optimized.This pseudovirus contains a Luc reporter gene that enabled us to easily quantify virus entry into angiotensin-converting enzyme 2(ACE2)-expressing 293T cells.Cathepsin(Cat)B/L inhibitor E64d could significantly block SARS-CoV-2 pseudovirus infection in 293T-ACE2 cells.Furthermore,the SARS-CoV-2 spike pseudotyped virus could be neutralized by sera from convalescent COVID-19 patients or recombinant ACE2 with the fused Fc region of human IgG1.Thus,we developed a pseudovirus-based assay for SARS-CoV-2,which will be valuable for evaluating viral entry inhibitors and neutralizing antibodies against this highly pathogenic virus.

Liver-infiltrating CD11b−CD27− NK subsets account for NK-cell dysfunction in patients with hepatocellular carcinoma and are associated with tumor progression

Natural killer(NK)cells have a vital role in killing hepatocellular carcinoma(HCC)cells;however,the mechanism underlying tumor-infiltrating NK(TINK)-cell dysfunction remains poorly understood.Using flow cytometry staining,we precisely characterized the frequency,phenotype and function of NK subsets distinguished by CD27 and CD11b in 30 patients with HCC in comparison to 30 healthy controls.Interestingly,we found a substantial proportion of liver-infiltrating CD11b−CD27−(DN)NK subsets in tumor tissue from HCC patients.Remarkably,these relatively expanded DN NK subsets exhibited an inactive and immature phenotype.By detecting the expression of CD107a and interferon-gamma(IFN-γ)on NK subsets and NK cells,we demonstrated that DN NK subsets exhibited a poor cytotoxic capacity and deficient potential to produce IFN-γin comparison to the other three subsets,which contributed to the dysfunction of TINK cells in HCC patients.In addition,we found that the presence of DN NK cells was closely associated with the clinical outcomes of HCC patients,as the frequency of DN NK cells among TINK cells was positively correlated with tumor stage and size.A large percentage of DN NK cells among TINK cells was an independent prognostic factor for lower survival in the 60-month follow-up period.In conclusion,a substantial proportion of CD11b−CD27−NK subsets among TINK cells accounts for NK-cell dysfunction in patients with HCC and is associated with tumor progression.Our study may provide a novel therapeutic target for the treatment of patients with HCC.