A Case of Hepatitis B Reactivation due to the Hepatitis B Virus Escape Mutant in a Patient undergoing Chemotherapy

A 62-year-old man had chronic hepatitis B virus （HBV） infection and was diagnosed with liver cirrhosis. At the time of diagnosis the patient＇s virologic markers were positive for hepatitis B surface antigen （HBsAg）, antibody to hepatitis B e antigen （anti-HBe） and antibody to hepatitis B core antigen （anti-HBc）, while antibody to hepatitis B surface antigen （anti-HBs） and HBV DNA were negative. Later the patient received chemotherapy for malignancy. However, this was interrupted due to elevated liver enzymes. At the same time HBV DNA became positive. Lamivudine （LMV） therapy was administered immediately. However, the levels of serum aminotransferase and total bilirubin （TB） were still rising. Finally the patient died of fulminant hepatic failure. A sequence revealed HBV genotype C （HBsAg subtype adw） with immune escape mutations, F8L, $34L, F41S, G44V, F93C, V96G, Lll0I, C149Y and F161Y. The high morbidity and mortality of this complication is one of the major obstacles to completing the standard treatment for malignancy in HBV carriers. Therefore, the relative risk of antiviral prophylactic failure should be further assessed and the optimal strategy for antiviral prophylaxis in HBsAg-positive patients with oncologic and hematologic malignancies undergoing chemotherapy should be revised.

A Case of Hepatitis B Reactivation in an Anti-HBs Positive,Anti-HBc Positive non-Hodgkin’s Lymphoma Patient

Dear Editor,We report a case of HBV reactivation in an anti-HBs positive, anti-HBc positive non-Hodgkin’s lymphoma patient. Hepatitis B virus (HBV) reactivation is a well-recognized complication of patients undergoing chemotherapy or immunosuppressive therapy for lymphomas. The presence of antibodies to the

Different Responses of Two Highly Permissive Cell Lines Upon HCV Infection

The construction of the first infectious clone JFH-1 speeds up the research on hepatitis C virus (HCV). However, Huh7 cell line was the only highly permissive cell line for HCV infection and only a few clones were fully permissive. In this study, two different fully permissive clones of Huh7 cells, Huh7.5.1 and Huh7-Lunet-CD81 (Lunet-CD81) cells were compared for their responses upon HCV infection. The virus replication level was found slightly higher in Huh7.5.1 cells than that in Lunet-CD81 cells. Viability of Huh7.5.1 cells but not of Lunet-CD81 cells was reduced significantly after HCV infection. Further analysis showed that the cell cycle of infected Huh7.5.1 cells was arrested at G1 phase. The G1/S transition was blocked by HCV infection in Huh7.5.1 cells as shown by the cell cycle synchronization analysis. Genes related to cell cycle regulation was modified by HCV infection and gene interaction analysis in GeneSpring GX in Direct Interactions mode highlighted 31 genes. In conclusion, the responses of those two cell lines were different upon HCV infection. HCV infection blocked G1/S transition and cell cycle progress, thus reduced the cell viability in Huh7.5.1 cells but not in Lunet-CD81 cells. Lunet-CD81 cells might be suitable for long term infection studies of HCV.

Regulation of Hepatitis C Virus Replication and Gene Expression by the MAPK-ERK Pathway

The mitogen activated protein kinases-extracellular signal regulated kinases （MAPK-ERK） pathway is involved in regulation of multiple cellular processes including the cell cycle. In the present study using a Huh7 cell line Conl with an HCV replicon, we have shown that the MAPK-ERK pathway plays a significant role in the modulation of HCV replication and protein expression and might influence IFN-a signalling. Epithelial growth factor （EGF） was able to stimulate ERK activation and decreased HCV RNA load while a MAPK-ERK pathway inhibitor U0126 led to an elevated HCV RNA load and higher NS5A protein amounts in Conl cells. It could be further demonstrated that the inhibition of the MAPK-ERK pathway facilitated the translation directed by the HCV internal ribosome entry site. Consistently, a U0126 treatment enhanced activity of the HCV reporter replicon in transient transfeetion assays. Thus, the MAPK-ERK pathway plays an important role in the regulation of HCV gene expression and replication. In addition, cyclin-dependent kinases （CDKs） downstream of ERK may also be involved in the modulation of HCV replication since roscovitine, an inhibitor of CDKs had a similar effect to that of U0126. Modulation of the cell cycle progression by cell cycle inhibitor or RNAi resulted consistently in changes of HCV RNA levels. Further, the replication of HCV replicon in Conl cells was inhibited by IFN-~z. The inhibitory effect of IFN-CZ could be partly reversed by pre-incubation of Con-1 cells with inhibitors of the MAPK-ERK pathway and CDKs. It could be shown that the MAPK-ERK inhibitors are able to partially modulate the expression of interferon-stimulated genes.

Construction of a chimeric hepatitis C virus replicon based on a strain isolated from a chronic hepatitis C patient

Subgenomic replicons of hepatitis C virus （HCV） have been widely used for studying HCV replication.Here,we report a new subgenomic replicon based on a strain isolated from a chronically infected patient.The coding sequence of HCV was recovered from a Chinese chronic hepatitis C patient displaying high serum HCV copy numbers.A consensus sequence designated as CCH strain was constructed based on the sequences of five clones and this was classified by sequence alignment as belonging to genotype 2a.The subgenomic replicon of CCH was replication-deficient in cell culture,due to dysfunctions in NS3 and NS5B.Various JFH1/CCH chimeric replicons were constructed,and specific mutations were introduced.The introduction of mutations could partially restore the replication of chimeric replicons.A replication-competent chimeric construct was finally obtained by the introduction of NS3 from JFH1 into the backbone of the CCH strain.

How the Key Finds its Door-Identification of HBV Receptor

HBV is the predominant pathogen associated with hepatitis cases in China. Although the HBV replication mechanism has been extensively documented in recent years, the virus entry mechanism remains elusive; in particular, the HBV receptor has yet to be

Acknowledgement to all reviewers for Virologica Sinica in 2013

In the fruitful year of 2013,the impact of Virologica Sinica (VS) as been largely extended.The successful publication of VS with high-quality articles is largely benefited from the efforts made by experts listed below,who were committed in managing the editorial process and/or in peer-reviewing.Apology was offered to any reviewer whose name has been inadvertently omitted.The time and expertise of all the experts devoted to Virologica Sinica are greatly appreciated!

Measures of insulin resistance and beta cell function before and after treatment of HCV infection

The association between chronic HCV infection and type 2 diabetes mellitus(T2DM)has been established;however,there is limited research onβ-cell function particularly in the pre-diabetic population.Here,we evaluated indices ofβ-cell function and insulin sensitivity across the spectrum from normal glucose tolerance to T2DMin individuals with and without chronic hepatitis C(CHC),and the effects of antiviral treatments on these variables.A total of 153 noncirrhotic,non-fibrotic CHC patients with a BMI<25 were enrolled in the study.Among them,119 were successfully treated with either direct acting antiviral(DAA)drugs or pegylated interferon/ribavirin(IFN/RBV)anti-HCV therapy.Fasting state-and oral glucose tolerance test(OGTT)-derived indexes were used to evaluateβ-cell function and insulin sensitivity.Among all subjects,19(13%)had T2DM and 21%exhibited pre-diabetes including 8%isolated impaired fasting glucose(IFG)and 13%combined IFG and impaired glucose tolerance(IGT).Early and total insulin secretion adjusted for the degree of insulin resistance were decreased in pre-diabetic CHC patients compared to HCVuninfected individuals.Viral eradication through DAA or IFN/RBV therapy demonstrated positive impacts on insulin sensitivity andβ-cell function in CHC patients who achieved sustained virologic response(SVR),regardless of fasting or OGTT state.These findings emphasize the role of HCV in the development ofβ-cell dysfunction,while also suggesting that viral eradication can improve insulin secretion,reverse insulin resistance,and ameliorate glycemic control.These results have important implications for managing pre-diabetic CHC patients and could prevent diabetes-related clinical manifestations and complications.

Farnesyltransferase inhibitor lonafarnib suppresses respiratory syncytial virus infection by blocking conformational change of fusion glycoprotein

Respiratory syncytial virus(RSV)is the major cause of bronchiolitis and pneumonia in young children and the elderly.There are currently no approved RSV-specific therapeutic small molecules available.Using high-throughput antiviral screening,we identified an oral drug,the prenylation inhibitor lonafarnib,which showed potent inhibition of the RSV fusion process.Lonafarnib exhibited antiviral activity against both the RSV A and B genotypes and showed low cytotoxicity in HEp-2 and human primary bronchial epithelial cells(HBEC).Time-of-addition and pseudovirus assays demonstrated that lonafarnib inhibits RSV entry,but has farnesyltransferase-independent antiviral efficacy.Cryo-electron microscopy revealed that lonafarnib binds to a triple-symmetric pocket within the central cavity of the RSV F metastable pre-fusion conformation.Mutants at the RSV F sites interacting with lonafarnib showed resistance to lonafarnib but remained fully sensitive to the neutralizing monoclonal antibody palivizumab.Furthermore,lonafarnib dose-dependently reduced the replication of RSV in BALB/c mice.Collectively,lonafarnib could be a potential fusion inhibitor for RSV infection.

Intranasal adenovirus-vectored Omicron vaccine induced nasal immunoglobulin A has superior neutralizing potency than serum antibodies

The upper respiratory tract is the initial site of SARS-CoV-2 infection.Nasal spike-specific secretory immunoglobulin A(slgA)correlates with protection against Omicron breakthrough infection.We report that intranasal vaccination using human adenovirus serotype 5(Ad5)vectored Omicron spike in people who previously vaccinated with ancestral vaccine could induce robust neutralizing slgA in the nasal passage.Nasal slgA was predominantly present in dimeric and multimeric forms and accounted for nearly 40%of total proteins in nasal mucosal lining fluids(NMLFs).A low-level IgG could also be detected in NMLFs but not IgM,IgD,and IgE.After a complete nasal wash,slgA in the nasal passage could be replenished rapidly within a few hours.A comparison of purified paired serum IgA,serum IgG,and nasal slgA from the same individuals showed that slgA was up to 3-logs more potent than serum antibodies in binding to spikes and in neutralizing Omicron subvariants.Serum IgG and IgA failed to neutralize XBB and BA.2.86,while nasal slgA retained potent neutralization against these newly emerged variants.Further analysis showed that slgA Was more effective than IgG or IgA in blocking spike-mediated cell-to-cell transmission and protecting hACE2 mice from XBB challenge.Using a slgA monoclonal antibody as a reference,we estimated that the total nasal slgA contains about 2.6-3.9%spikespecific slgA in NMLFs collected approximately one month after intranasal vaccination.Our study provided insights for developing intranasal vaccines that can induce slgA to build an effective and mutation-resistant first-line immune barrier against constantly emerging variants.

The role of viral protein Ac34 in nuclear relocation of subunits of the actin-related protein 2/3 complex

The actin nucleator actin-related protein complex(Arp2/3) is composed of seven subunits: Arp2,Arp3, p40/ARPC1(P40), p34/ARPC2(P34), p21/ARPC3(P21), p20/ARPC4(P20), and p16/ARPC5(P16). Arp2/3 plays crucial roles in a variety of cellular activities through regulation of actin polymerization. Autographa californica multiple nucleopolyhedrovirus(Ac MNPV), one of the beststudied alphabaculoviruses, induces Arp2/3 nuclear relocation and mediates nuclear actin polymerization to assist in virus replication. We have demonstrated that Ac34, a viral late-gene product, induces translocation of the P40 subunit of Arp2/3 to the nucleus during Ac MNPV infection. However, it remains unknown whether Ac34 could relocate other Arp2/3 subunits to the nucleus. In this study, the effects of the viral protein Ac34 on the distribution of these subunits were studied by an immunofluorescence assay. Arp2, P34, P21, and P20 cloned from Spodoptera frugiperda(Sf9) cells showed mainly cytoplasmic localization and were relocated to the nucleus in the presence of Ac34. In addition, Arp3 was localized in the cytoplasm in both the presence and absence of Ac34, and P16 showed whole-cell localization. In contrast to Sf9 cells, all subunits of mammalian Arp2/3 showed no nuclear relocation in the presence of Ac34. Co-immunoprecipitation analysis of the interaction between Ac34 and Arp2/3 subunits revealed that Ac34 bound to P40,P34, and P20 of Sf9 cells. However, none of the subunits of mammalian Arp2/3 interacted with Ac34, indicating that protein-protein interaction is essential for Ac34 to relocate Arp2/3 subunits to the nucleus.

HBV cccDNA的表观遗传学修饰及调控

乙型肝炎病毒(hepatitis B virus,HBV)感染是一个重大的世界性公共卫生问题.HBV能够引起急性或慢性病毒性肝炎,最终导致肝硬化甚至肝癌的发生.现行的临床抗HBV药物,如α干扰素和核苷(酸)类似物等,虽可有效抑制病毒复制,但不能彻底清除病毒.其根本原因在于,HBV感染肝细胞后形成共价闭合环状DNA(covalently closed circular DNA,ccc DNA),并以微染色体的形式独立稳定存在于肝细胞核内.表观遗传学修饰可在不改变DNA序列的情况下,影响基因的表达.越来越多的证据表明,ccc DNA的表观遗传学修饰是调节HBV生活周期的重要因素.本文就HBV ccc DNA的表观遗传学修饰和调控作用、表观遗传修饰药物和治疗,以及表观遗传学研究方法和体系等进行综述.

Host metabolism dysregulation and cell tropism identification in human airway and alveolar organoids upon SARS-CoV-2 infection

The coronavirus disease 2019(COVID-19)pandemic is caused by infection with the severe acute respiratory syndrome coronavirus 2(SARS-CoV-2),which is spread primary via respiratory droplets and infects the lungs.Currently widely used cell lines and animals are unable to accurately mimic human physiological conditions because of the abnormal status of cell lines(transformed or cancer cells)and species differences between animals and humans.Organoids are stem cell-derived selforganized three-dimensional culture in vitro and model the physiological conditions of natural organs.Here we showed that SARS-CoV-2 infected and extensively replicated in human embryonic stem cells(hESCs)-derived lung organoids,including airway and alveolar organoids which covered the complete infection and spread route for SARS-CoV-2 within lungs.The infected ceils were ciliated,club,and alveolar type 2(AT2)cells,which were sequentially located from the proximal to the distal airway and terminal alveoli,respectively.Additionally,RNA-seq revealed early cell response to virus infection including an unexpected downregulation of the metabolic processes,especially lipid metabolism,in addition to the well-known upregulation of immune response.Further,Remdesivir and a human neutralizing antibody potently inhibited SARS-CoV-2 replication in lung organoids.Therefore,human lung organoids can serve as a pathophysiological model to investigate the underlying mechanism of SARS-CoV-2 infection and to discover and test therapeutic drugs for COVID-19.

Productive HBV infection of well-differentiated, hNTCP-expressing human hepatoma-derived(Huh7) cells

Feasible and effective cell models for hepatitis B virus(HBV) infection are required for investigating the complete lifecycle of this virus, including the early steps of viral entry. Resistance to dimethyl sulfoxide/polyethylene glycol(DMSO/PEG), h NTCP expression, and a differentiated state are the limiting factors for successful HBV infection models. In the present study, we used a hepatoma cell line(Hu7^(hDNTCPh)) to overcome these limiting factors so that it exhibits excellent susceptibility to HBV infection. To achieve this goal, different hepatoma cell lines were tested with 2.5% DMSO/4%PEG8000, and one resistant cell line(Huh7 D) was used to construct a stable h NTCP-expressing cell line(Hu7^(hDNTCPh)) using a recombinant lentivirus system. Then, the morphological characteristics and differentiation molecular markers of Hu7^(hDNTCPh) cells with or without DMSO treatment were characterized. Finally, the susceptibility of Hu7^(hDNTCPh) cells to HBV infection was assessed. Our results showed that Huh7 D cells were resistant to 2.5% DMSO/4% PEG8000, whereas the others were not. Hu7^(hDNTCPh) cells were established to express a high level of h NTCP compared to liver extracts, and Hu7^(hDNTCPh) cells rapidly transformed into a non-dividing, well-differentiated polarized phenotype under DMSO treatment. Hu7^(hDNTCPh) cells fully supported the entire lifecycle of HBV infection. This cell culture system will be useful for the analysis of host-virus interactions, which should facilitate the discovery of antiviral drugs and vaccines.

Complementation of Wild-Type and Drug-Resistant Hepatitis B Virus Genomes to Maintain Viral Replication and Rescue Virion Production under Nucleos(t)ide Analogs

As the open reading frames of hepatitis B virus(HBV)genomes are overlapping,resistance mutations(MTs)in HBV polymerase may result in stop codon MTs in hepatitis B surface proteins,which are usually detected as a mixed population with wild-type(WT)HBV.The question was raised how the coexistence of nucleos(t)ide analogs(NAs)resistance MTs and WT sequences affects HBV replication.In the present study,HBV genomes with frequently detected reverse transcriptase(RT)/surface truncation MTs,rtA181T/sW172^*,rtV191I/sW182^*and rtM204I/sW196^*,were phenotypically characterized alone or together with their WT counterparts in different ratios by transient transfection in the absence or presence of Nas.In the absence of Nas,RT/surface truncation MTs impaired the expression and secretion of HBV surface proteins,and had a dose-dependent negative effect on WT HBV virion secretion.However,in the presence of Nas,coexistence of MTs with WT maintained viral replication,and the presence of WT was able to rescue the production of MT HBV virions.Our findings reveal that complementation of WT and MT HBV genomes is highly effective under drug treatment.

Single-cell analysis reveals bronchoalveolar epithelial dysfunction in COVID-19 patients

Dear Editor,In 2019,a zoonotic coronavirus named severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)was identified as the causative agent of Coronavirus Disease 2019(COVID-19).As of 8 June 2020,the World Health Organization(WHO)has reported 6,912,751 globally confirmed cases with 400,469 deaths.Although generally causes mild disease,SARS-CoV-2 infection can result in serious outcomes,including acute lung injury(ALI)and acute respiratory distress syndrome(ARDS),the leading cause of mortality in patients with comorbidities.Recent autopsy studies of COVID-19 patients revealed mononuclear infiltration and excessive production of mucus in the infected lung,especially in the damaged small airways and alveoli(Bian and Team,2020;Liu et al.,2020).

Histone Deacetylase 3 Inhibitor Suppresses Hepatitis C Virus Replication by Regulating Apo-A1 and LEAP-1 Expression

Histone deacetylase (HDAC) inhibitors show clinical promise for the treatment of cancers, including hepatocellular carcinoma (HCC). In this study, we investigated the effect of HDAC inhibitor treatment on hepatitis C virus (HCV)replication in Huh7 human liver cells and in a mouse model of HCV infection. Viral replication was markedly suppressed by the HDAC3 inhibitor at concentrations below 1 mmol/L, with no cellular toxicity. This was accompanied by upregulation of liver-expressed antimicrobial peptide 1 (LEAP-1) and downregulation of apolipoprotein-A1 (Apo-A1), as determined by microarray and quantitative RT-PCR analyses. Moreover, HDAC3 was found to modulate the binding of CCAAT-enhancer-binding protein a (C/EBPa), hypoxia-inducible factor 1 a (HIF1 a), and signal transducer and activator of transcription 3 (STAT3) to the LEAP-1 promoter. HDAC3 inhibitor treatment also blocked HCV replication in a mouse model of HCV infection. These results indicate that epigenetic therapy with HDAC3 inhibitor may be a potential treatment for diseases associated with HCV infection such as HCC.

Hepatitis B virus is degraded by autophagosome-lysosome fusion mediated by Rab7 and related components

Dear Editor,With an estimated 240 million chronically infected people worldwide,hepatitis B virus(HBV)infection is a major public health problem(Schweitzer et al.,2015).Despite more than 30 years of in tense research,many aspects of the HBV life cycle still remain unknown.

Identification of serotonin 2A receptor as a novel HCV entry factor by a chemical biology strategy

Hepatitis C virus(HCV)is a leading cause of liver disease worldwide.Although several HCV protease/polymerase inhibitors were recently approved by U.S.FDA,the combination of antivirals targeting multiple processes of HCV lifecycle would optimize anti-HCV therapy and against potential drug-resistanee.Viral entry is an essential target step for antiviral development,but FDA-approved HCV entry inhibitor remains exclusive.Here we identify serotonin 2A receptor(5-HT2aR)is a HCV entry factor amendable to therapeutic intervention by a chemical biology strategy.The silencing of 5-HT2aR and clinically available 5-HT2aR antagonist suppress cell culture-derived HCV(HCVcc)in different liver cells and primary human hepatocytes at late endocytosis process.The mechanism is related to regulate the correct plasma membrane localization of claudin 1(CLDN1).Moreover,phenoxybenzamine(PBZ),an FDAapproved 5-HT2aR antagonist,inhibits all major HCV genotypes in vitro and displays synergy in combination with clinical used anti-HCV drugs.The impact of PBZ on HCV genotype 2a is documented in immune-competent humanized transgenic mice.Our results not only expand the understanding of HCV entry,but also present a promising target for the invention of HCV entry inhibitor.

Host HDAC4 regulates the antiviral response by inhibiting the phosphorylation of IRF3

Class II HDACs, such as HDAC4, are critical regulators of the immune response in various immune cells;however, its role in innate immunity remains largely unknown.Here, we report that the overexpression of HDAC4 suppresses the production of type I interferons triggered by pattern-recognition receptors (PRRs). HDAC4 repressed the translocation of transcription factor IRF3 to the nucleus, thereby decreasing IRF3-mediated IFN-β expression. In particular, we also determined that HDAC4 can be phosphorylated and simultaneously block the phosphorylation of IRF3 at Ser386 and Ser396 by TBK1 and IKKε, respectively, by interacting with the kinase domain of TBK1 and IKKε. Furthermore, IFN-β may stimulate the expression of HDAC4. Our findings suggest that HDAC4 acts as a regulator of PRR signaling and is a novel mechanism of negative feedback regulation for preventing an overreactive innate immune response.