Mycothiol is the major low-molecular-mass thiol in M. tuberculosis, which plays an important role in maintaining redox homeostasis. Mycothiol acetyltransferase (MshD) finishes the synthesis process by transferring the acetyl group of Acetyl-CoA to the mycothiol precursor. In order to explore the role of MshD in M. tuberculosis, we constructed a MshD knockout strain with a phage vector-based homologous recombination technology. Compared with the wild strain H37Ra, the knockout had smaller colonies, slower growth rate, decreased biofilm, and was more sensitive to chemical challengers by 5 mmol/L H₂O₂, 0.05% SDS, 50 ℃ heat shock and hypoxic conditions. The above results indicated that the mshD gene played an important role in M. tuberculosis. This study laid the foundation for further revealing the function and mechanism of the gene.

Objective　To investigate the role of Hepatitis B surface antigen (HBsAg) in the immune escape of innate immunity by Hepatitis B virus (HBV).　Methods After treated with PMA, THP-1 was differentiated into macrophage- like cells. Macrophage- like cells were treated further with LPS and the pam3csk4 in the presence or absence of HBsAg. Cytokine IL-10 , IL-12 protein, IL-10, IL-12 mRNA levels , NF-κB p65 protein nuclear translocation as well as IκB-α degradation and ERK protein phosphorylation were detected to monitor the activation of the TLR signaling pathway. Results LPS and the pam3csk4 induced production of cytokine IL-10 , IL-12 protein, NF-κB p65 protein nuclear translocation as well as IκB-α degradation and ERK protein phosphorylation were inhibited by HBsAg in a dose-dependent manner. Conclusion HBsAg inhibit the activation of TLR2 and TLR4 signaling pathway.

Viruses have been classified into enveloped and non-enveloped subtypes according to their surface structures. The membrane proteins of the enveloped viruses are involved in the attachment, penetration, ncoating,
replication, and release of the viruses. The special membrane proteins are essential for the membrane fusion bywhich the enveloped viruses penetrate into host cells. Furthermore, structural data showthat class I and class II viral fusion proteins utilize a similar principle in membrane fusion. In addition, there are some viral membrane proteins, such as M2 of the influenza virus, Vpu of the human immunodeficiency virus type 1 ( HIV-1) , 3a of the severe acute respiratory syndrome coronavirus ( SARS-CoV) , among others, that have ion channel functions. The processes involved in viral membrane fusion and ion channel function provide newinsights into therapeutic design and proteins as potential targets of antivirals. Here we give three typical viral membrane fusion proteins as examples to reviewthe mechanisms of viral membrane fusion and viral ion channel function and the strategies of antiviral drug design.

Tremendous advances have been made in developing oncolytic viruses in the last few years. Viral infection and amplification eventually induce cancer cells into cell death pathways and elicit host antitumor immune responses. Specifically targeted molecules or signaling pathways in cancer cells or in the tumor microenvironment have been studied and a variety of oncolytic viruses such as adenovirus, herpes simplex virus, poxvirus, measles virus, Newcastle disease virus and influenza virus have been identified. In this review, the characteristics of oncolytic viruses for cancer therapy are discussed.

Autophagy is an important form of programmed cell death that regulates the growth and development of cells. It acts as a ‘two-edge sword’. On one side, autophagy eliminates microbes; on the other, many bacteria have developed distinct mechanisms to regulate and interfere with autophagy for their own replication and survival. Autophagy is an important event in the innate immune response. It can initiate a response to bacteria and bacterial toxins through Toll-like receptor mechanisms or mucosal immune system. Effector cells of the cellular immune system can regulate autophagy by secreting different cytokines, allowing the organism to re-tune its adaptive immune response. Autophagy may play a pivotal role in regulating the immune polarization of Th1/Th2 in fighting intracellular bacteria.

Objective To investigate the antimicrobial resistance of enterococcus faecalis and enterococcus faecium in our hospital for the rational use of antibiotic. Methods GPI plate was applied for bacterial identification and susceptibility testing.Whonet5 software was used for data analysis. Results Less than 50% of enterococcus faecalis and enterococcus faecium isolates were resistant to chloramphenicol and nitrofurantoin. Less than 1% of enterococcus faecalis and enterococcus faecium isolates were resistant to vancomycin. The resistance rate of enterococcus faecalis to penicillin G, high concentration gentamicin, ciprofloxacin, rifampicin and erythromycin descend year by year, and that of enterococcus faecium to ciprofloxacin, rifampicin and nitrofurantoin was opposite. In addition, the resistance rate of enterococcus faecium to most antibiotic was higher than enterococcus faecalis. Conclusions The enterococcus faecalis and enterococcus faecium is becoming multi-resistant bacteria. Antimicrobial therapy should be decided according to the results of susceptibility testing.

Objective To explore the characteristic and reason for indeterminate HIV Western Blot(WB),the limitation of WB and the possible amendatory measures.Methods Summing up and analysis the distribution of the indeterminate HIV WB among the tested people,the laboratory test results,the follow-up and the final HIV antibody outcome.Results The relative healthy people including the people of voluntary counseling and testing,the blood donors and pregnant women make up 50% of the indeterminate HIV WB,the follow-up for the indeterminate HIV WB is difficult and the there are few people of the indeterminate HIV WB have HIV antibody test again.There is false-positive in WB test,especially the false-positive of P24 is serious.Conclusion The indeterminate HIV WB is related to the false positive of WB test,measures should be taken to reduce the indeterminate HIV WB and to interpret the result accurately.

Opportunistic pathogens do not cause infectious disease when the immune systemis intact. However, under microdysbiosis or in an immunocompromised host, they can cause clinical infectious disease and mortality. Opportunistic infections are gradually increasing with the prolonged survival of immunocompromised patients with congenital immunodeficiency or cancer by modern medical treatment and as a result of microdysbiosis that may be caused by the abuse of antibiotics and increases in resistant strains ( single - drug or multi - drug resistant strains) . Clinical manifestations of opportunistic infections appear to be more complex and atypical. Great difficulties exist in clinical treatment of this type of infection. Therefore, careful examination of common opportunistic pathogens and their characteristics has great clinical significance in the accurate diagnosis and treatment of opportunistic infections.

Microbial culture, ELISA, nucleic acid testing are the main methods for clinic microbial testing today. An effective method for microbial enrichment not only will be helpful on optimizing the sensitivity of the follow-up testing, but also will determine the availability of some testing methods when the amount of microorganisms are extremely low in the sample. The research progress on microbial enrichment methods and its applications are reviewed and discussed in this report.

Accumulating evidence indicates that the gut microbiota is closely linked with the development of obesity , insulin resistance and other metabolic diseases, with the detailed molecular mechanism being elucidated.The gut microbiota can not only regulate energy metabolism and promote excessive fat accumulation,but also act as one of the predisposing factors for driving the host systemic, low-grade chronic inflammationand subsequent insulin resistance and other metabolic disorders. The gut microbiota may become a new target for the prevention and treatment of obesity.

 

Infection with hepatitis C virus (HCV) is currently treated with interferon-α( IFN-α) , but the molecular mechanismby which IFN inhibits HCV replication still remains elusive. Therefore, in this study, we attempted to elucidate the possible mechanismfor the IFN-inducible protein, Viperin, against HCV. Over-expression of Viper in could significantly down-regulate intracellular levels of HCV protein and RNA in both the replicon and HCVcc systems. Membrane flotation analysis indicated that expression of Viperin could interfere with the association of HCV NS3 and NS5A with the lipid raft, resulting in increased sensitivity of these two proteins to the treatment of non-ionic detergent. Furthermore, Viperinwas able to interact with FPPS because co-transfection of Viperin with FPPS could partially reverse the anti-HCV effects of Viperin in replicon cells. The above results indicate a possible novel mechanismof IFN-induced antiviral activity, that is, over-expression of Viperin can interfere with the microdomain of intracellular lipid raft and, therefore, further disrupt the stability of viral replicase complex to inhibit its functions.

Sequence analysis of human enterovirus (HEV) is now widely applied. The technology of sequencing-based identification and typing of HEV is reviewed in the present paper.

细菌生物膜是指微生物（细菌、真菌等）黏附、聚集形成的一个群体，该群体产生并分泌胞外聚合物（extracellular polymeric substance，EPS），形成一定的三维结构及含有营养物质、氧气等生长必需的物质交换通道，微生物细胞在EPS中增殖、生存^[1]。生物膜结构可阻止抗生素或抗体等大分子有效杀伤微生物细胞，目前尚无有效的预防措施和治疗方法控制细菌生物膜所带来的危害。细菌生物膜形成机制的研究能为预防和解决生物膜引起的各种问题奠定良好的基础。在生物膜研究中，需模拟体内或多种环境以观察生物膜的形态和形成，检测生物膜细菌基因组和蛋白质组表达等。因此......

The genes encoding 19kD lipoprotein and early secreting antigenic target-6( ESAT-6) were amplified from genome of the standard Mycobacterium tuberculosis H37Rv using polymerase chain reaction, and then cloned into the vector pMD18-T followed by subcloning into the expression vector pET-21a, respectively. Recombinant 19kD-ESAT6 was expressed in E. coli, and then purified by Ni-NTA. The antigenicity of the fusion protein was analyzed by Western blot analysis. The recombinant 19kD-ESAT6 plasmid was constructed successfully, and could be expressed efficiently in E. coli BL21 ( DE3) . The relative molecular mass of the fusion protein was approximately 29 ×10³ by SDS-PAGE. The recombinant 19kD-ESAT6 protein showed specific antigenicity as determined by Western blot, and can be used for the development of serodiagnostic reagents.

LipL32 is not only the most abundant protein of the outer membrane but also an immunodominant antigen that is highly conserved in pathogenic Leptospira. Recombinant LipL32 (rLipL32) purified under natural conditions was adapted to immunize BALB/c mice for developing monoclonal antibodies (mAbs). Twenty-nine mAbs against eight epitopes were produced and characterized. The capture and detection of antibodies were selected using a one-by-one pairing method with biotin-conjugated mAbs. To evaluate the sensitivity and specificity of the mAb pairs, antigen capture enzyme-linked immunosorbent assay (ELISA) was applied to detect antigens extracted from 15 pathogenic Leptospira strains and from other pathogenic bacteria. One pair of mAbs was selected and the detection capability of rLipL32 by ELISA was found to be approximately 1 ng/ml. mAbs produced with rLipL32 purified under natural conditions may be promising in the further detection of leptospiral antigens.

Campylobacter jejuni ( C. jejuni) is a common causative pathogen of bacterial gastroenteritis in humans. Chemotaxis, adirectional movement of bacteria towards suitable parasitic positions, is the initial key step for C. jejuni to achieve colonization on jejunal mucosa of hosts. The chemotactic migration was controlled and regulated by bacterial chemotaxis-associated two-component signaling system( che-TCS) in a MCPs→Ches→Flis/Mots manner. FliG, amember of Fli protein family, has been confirmed in some other bacterial pathogens to be a flagellar motor protein as well as an essential component of switch complex in bacterial flagellar motor. However, the function of FliG protein in chemotaxis of C. jejuni remains unknown. In the present study we generated a fliG gene knockout ( fliG^- ) mutant from C. jejuni strain NCTC11168 based on homologous recombination, and the motility, chemotaxis and colonization of fliG^- mutant were subsequently determined. The motility test and chemotaxis test in vitro demonstrated that the diameters of colonies on semisolid agar plate and chemotactic rings towards 0. 2 mol/L sodiumdeoxycholate ( SDC) in hard agar plus (HAP) of fliG^- mutant were significantly smaller than those of the wild type strain ( P < 0. 05) . Compared to the wild type strain, the numbers of fliG^- mutant adhering to the surface of jejunal mucosa and existing in jejunal content of BALB/c-ByJ mice were also significantly decreased ( P <0. 05) . All the results of this study lead a conclusion that fliG is an essential gene for flagellar motility and chemotactic movement towards jejunal
mucosa of C. jejuni during infection.

Human immunodeficiency virus type I （HIV-1） infection leads to severe immune dysfunction. In addition to the progressive depletion and dysfunction of CD4+ T cells, HIV-1 infection also leads to extensive defects in the humoral arm of the immune system. This study aimed to describe the distribution of B cell subpopulations and profiles of activated, apoptosis-associated and costimulatory molecules in each subpopulation. The results showed that the peripheral blood B cell counts in HIV-1 infected patients were significantly lower than that in the healthy controls, but could be restored by antiretroviral therapy (ART). The decline of B cell counts was manifested by the decrease of immature B cells, Naïve B cells, resting memory B cells, and plasmablast. However, tissue-like memory B cells increased significantly. ART could restore the frequencies of naïve B and tissue-like memory B cells, but not the resting memory B cells. CD38 was significantly upregulated in immature B cells, naïve B cells, resting memory B cells and tissue-like memory B cells in HIV-1-infected patients, when compared to healthy controls. All subpopulations showed higher expression of CD95, and naïve B cells, tissue-like memory B cells and plasmablasts exhibited decreased levels of Bcl-2. PD-1 was elevated only in the resting memory cells and plasmablasts. The mean fluorescent intensity of CD40 was diminished in all B cell subpopulations, whereas CD70 decreased in all subpopulations except immature B cells. However, ART could only partially restore the altered expression of the mentioned molecules. These results suggest that HIV-1 infection leads to perturbation of B cell subpopulations, and B cell subpopulations display hyperactivation, susceptibility to apoptosis and impaired interaction with T cells. These alterations could only be restored partially by successful ART, therefore, effective immune intervention strategies should be required.

The purpose of the current study was to evaluate the role of cholesterol of the membrane microdomain in the uptake of Francisella tularensis ( F. tularensis) LVS by mouse macrophages. A shuttle plasmid, pFNLTP6 gro-gfp, was transformed by electroporation to F. tularensis LVS. Cell cholesterol was stained with Filipin Ⅲ, and caveolin-1 was detected with monoclonal antibody and visualized with Alexa 594 conjugated goat anti-mouse antibodies. F. tularensis LVS infection was analyzed using a fluorescencemicroscope equipped with amotorized Z-focus. In order to evaluate the effect of depletion of the membrane microdomain on F. tularensis entery into macrophages, interference with lipid-rich plasmamembrane through the depletion of cholesterol was performed by methyl-β-cyclodextrin. The cholesterol-binding agent, Filipin III, was used to detect the effect of cholesterol depletion. The results showed that cell cholesterol was co-localized with F. tularensis live vaccine strain in the early uptake stage, and both had close contact with the membrane microdomain-associated components, such as caveolin-1. F. tularensis requires cholesterol-rich membrane microdomains for entry into macrophages. These findings suggest that cholesterol-rich membrane microdomains and caveolin-1 play an important role in F. tularensis early entry into macrophages.

Interferon-β plays an important role in innate immunity against viral infection. Hepatocytes, as hepatitis viruses-harboring cells, are reported to possess the potential to inductively express interferon-β. However, a practical in vitro cell model for investigating the interplay between hepatitis B virus and host cells is rarely reported. Here, we determined the inductive expression of interferon-β by interferon-β agonists〔(Newcastle disease virus, NDV) and poly(I∶C)〕in immortalized primary hepatic cell line, PH5CH8,and hepatocarcinoma cell lines, Huh-7 and HepG2. The data demonstrated that inductive expression of interferon-β in PH5CH8 cells was significantly higher than that in Huh7 or HepG2 cells. In addition, the expression level of key molecules critical for interferon-β induction was investigated to clarify the underlying mechanism. The results showed that the background expression level was fairly low in Huh7 and HepG2 cell lines, compared to that in PH5CH8 cells. It is suggested that PH5CH8 cells possess intact potential to produce interferon-β and reconstitution of a selective interferon-deficient hepatic cell line might be achieved via introduction of related molecules critical for interferon induction.

Tuberculosis remains a leading infectious cause of morbidity and mortality worldwide despite the availability of the bacille Calmette Guérin (BCG) vaccine and chemotherapy. Progress has been made in understanding immuopathogenesis and vaccine development in recent years. Mycobacterium tuberculosis (Mtb) may activate innate immunity of macrophage by Toll-like receptors (TLRs) and other pattern recognition receptors (PRRs) , which can eliminate the bacteria and regulate the acquired immune responses. Besides major histocompatibility complex (MHC) restricted CD4 ⁺ and CD8 ⁺ T cells, CD1- restricted T cells and γ δ T cells also take part in immune responses to Mtb infection. Memory T cells and regulatory T cells play a special role in regulating immune responses to mycobacterial infection. In view of poor BCG protective efficacy in adults, improved control of tuberculosis requires development of new and more effective vaccines. Various vaccine candidates including recombinant BCG, live-attenuated Mtb, and booster vaccines , such as recombinant modified vaccinia virus Ankara expressing Mtb antigen, nucleic acid vaccines, and subunit protein vaccines with novel adjuvants , are at different stages of development. One promising vaccine strategy is priming with BCG or BCG replacement vaccine followed by boosting with subunit vaccines. However, the vaccine strategy, optimal dose, route, frequency, and timing of the boost remain to be determined. The challenges facing tuberculosis vaccine development include a lack of immune markers for protection in humans, difficulty in prioritizing which candidates to move to clinical trials, shortage of clinical trial sites, lengthy time required for vaccine evaluation, and high cost.

Linezolid is an antibacterial belonging to the oxazolidinone class of antibiotics. The importance of linezolid as an antibiotic is related to its activity against a number of clinically significant Gram-positive cocci, including multidrug-resistant staphylococci and vancomycin-resistant enterococci. Although bacterial resistance to linezolid is not a major issue at present, its development in clinical isolates should be paid more attention. In this article, the mechanisms of bacterial resistance to linezolid, and related detection methods were reviewed. We showed that bacterial resistance is mediated by a single-nucleotide mutation and the production of methyltransferase, however, other potential mechanisms have not yet been determined. The detection methods for linezolid resistance include microbiological and molecular biological methods, some of which would benefit fromtechnological improvements.

The present study was aimed to investigate major histocompatibility complex Ⅱ ( MHC Ⅱ ) antigen presentation induced by interferon-γ ( IFN-γ) in macrophages after exposure to Mycobacterium tuberculosis ( M. uberculosis) . The expressions of MHCⅡ, CD86, and CD80 on macrophages, after exposure to M. tuberculosis for 24 h prior to the addition of IFN-γ, were detected by flow cytometry. The mRNA levels of CⅡTA, PⅠ, PⅢ and PⅣ were assayed by reverse transcriptase-polymerase chain reaction. MHCⅡ antigen presentation was detected by enzyme-linked immunosorbent assay ( ELISA) . The results showed that M. tuberculosis inhibited the expressions of MHCⅡ and CD86 but not CD80 induced by IFN-γon macrophages. The ability of antigen presentation was also inhibited. The mRNA levels of CⅡTA, PⅠ, PⅢ and PⅣ induced by IFN-γwere also decreased. It suggests that M. tuberculosis inhibits MHCⅡ antigen presentation induced by IFN-γon macrophages.

流行性感冒(简称流感)病毒已经与人类“相处”了400多年,可在各年龄段的人群中流行和引起发病,每年在全球引起25万～50万人发病[1]。2009年3月在北美地区暴发了新型H1N1流感,截至2009年6月8日,已有73个国家和地区的25288人被感染,其中139人死亡(http://www.who.int/csr/don/2009_06_08/en/index.html)。世界卫生组织(WorldHealthOrganization,WHO)曾称其为猪流感,原因是其多数基因片段来源于猪流感病毒。猪流感这一名称直接导致了埃及4月30日为预防流感蔓延而扑杀了30万头生猪。随后,WHO将其改名为甲型H1N1流感(下称2009甲型H1N1流感)。因此,甲型H1N1流感病毒的起源毫无疑问成为世界各国关注的热点,本文就2009甲型H1N1流感、1997年人际传播的H5N1禽流感以及人类历史上的几次人流感大流行,对甲型流感病毒的基因组结构特点及其特有的进化机制作一简单介绍......

To understand the relationship between variation of enterovirus 71 ( EV 71) and its virulence, the sequence of the VP4 gene of EV71 virus was analyzed. Eight strains of EV71 isolated from clinical samples collected from infants and children with hand, foot and mouth disease in the Children’s Hospital Affiliated to Capital Institute of Pediatrics in Beijing during 2007 to 2008 were used for comparison. Full length VP4 genes from these eight EV71 strains were amplified by reverse-transcriptase PCR ( RT-PCR) and sequenced after the amplicons were cloned into pUCm-T. The sequences were compared with VP4 genes from EV71 in GenBank. Sequence analysis and type identification were performed by bioinformatics ( DNAStar) . The full length of VP4 gene has 207 bp coding for a protein of 69 amino acids with estimated molecular weight of 7 ×10³ . The nucleotide homology of VP4 genes among the eight strains were 94% ～100% and homology of the deduced amino acids were 100% . The nucleotide sequence homology between these eight strains isolated in Beijing and those isolated in Fuyang, Shenzhen and Taiwan was higher than those isolated elsewhere. The homology of deduced amino acid sequences encoded by VP4 between these eight isolates in Beijing and those in GenBank was 100% except one strain isolated from India. The amino acids at the 7th and 54th position of VP4 of the strain from India were different from others. There were many differences between the nucleotide sequences of VP4 from the eight Beijing strains and those from severe cases ( BrCr, MS and NCKU9822) , as reported in the literature, whereas only a few nucleotide differences were observed between severe
cases ( BJ97, BJ110B, BJ110Y and BJ4243) and mild cases ( BJ25, BJ47, BJ65 and BJ67) among these eight Beijing strains. Phylogenetic analysis of the VP4 sequences from these eight strains indicates that the EV71 viruses that circulated in Beijing during 2007 to 2008 should be classified as subtype C4. The VP4 genes from the isolates in Beijing from2007 and 2008 were highly conserved. There was no consistent divergence in the sequences of VP4 between strains isolated fromsevere cases and those frommild cases, suggesting that the virulence of the EV71 virus does not seem to be related to the sequence of VP4.

Objective To establish a molecular biology method for detecting sequences of unknown viruses. Methods The hepatitis B and hepatitis C viruses were used as hypothetically unrecognized DNA, RNA viruses respectively to test the efficacy of this random PCR-based method. Virus-containing serum was first filtered through a 0.22-μm disk filter followed by DNase I treatment to eliminate the host genomic DNA. After the 26mer 5’ end anchored random primer was annealed to the extracted nucleic acids either by Klenow polymerase extension (DNA as template) or by reverse transcription (RNA as template), the virus genomes were randomly amplified with the 20mer anchor primer. Purified PCR products were cloned, sequenced and subjected to BLAST search for further analysis. Results HBV and HCV genomes were amplified by random PCR and their fragments could be detected in the picked clones. The detective rate was about 15% at 1&#61620;106 copies/ml viral load. Positive clones could still be obtained with as few as 1&#61620;104 copies/ml viral load. Conclusion This culture-independent random PCR-based method can be used to investigate unknown viruses and will be useful for the early diagnosis of emerging infectious disease.

Objective To establish the HHV-6-specific CD4⁺ T cell clones and further study the character of HHV-6-specific CD4⁺ T cells. Methods We acquired CD4⁺ T cell clones by the liquid microwell limiting dilution technique, and selected HHV-6-specific CD4⁺ T cell clones by means of 3H-thymidine uptake. The phenotype of HHV-6-specific T cell clones were analyzed with FACS. We tested the IL-10, IL-4 or IFN-γ secretion from HHV-6-specific CD4⁺ T cell clones by ELISA. Results Of the five HHV-6-specific CD4⁺ T cell clones, four showed proliferative responses to the stimulation with HHV-6 infected JJHAN lysates. Moreover, proliferation responses of HHV-6-specific CD4⁺T cell clones depended on the concentration of the HHV-6-infected JJHAN lysates. The phenotypic analysis with FACS demonstrated that HHV-6-specific T cell clones consisted of CD3⁺ CD4⁺ T cells. CD4⁺ T cell clones-W-2 and W-4 possessed the feature of high levels of IL-10 production, while CD4⁺ T cell clones-W-1 possessed the feature of high levels of IL-10 and IFN-γ production and W-3 possessed the feature of high levels of IFN-γ production. Conclusions The HHV-6-specific CD4⁺ T cell clones, were established in our study so as to further study their pneotype, the character of cytokine secrection, and to provide experimental basis for selecting HHV-6-specific CD4⁺ Treg cell clones.

The hepatitis B virus ( HBV) is a retroid virus that contains a small DNA genome with very limited coding capacity. Yet, HBV is extremely successful both atmultiplyingwithin individual host cells and at spreading across populations. In this review, wewill highlight some recent progress in understanding HBV-host cell interactions that are likely to contribute to the extraordinary ability of HBV to replicate and persist within the host cell, focusing primarily on studies carried out inmy own laboratory. The major HBV-cell interactions to be discussed include the role of cellular chaperones for the initiation of HBV reverse transcription and nucleocapsid assembly, host regulation of viral nucleocapsid maturation through the dynamic modulation of its state of phosphorylation, and the formation of the viral nuclear episomal DNA responsible for HBV persistence.