Sustained virologic response to direct-acting antiviral agents predicts better outcomes in hepatitis C virus-infected patients: A retrospective study

BACKGROUND Direct-acting antiviral agents(DAAs)are extremely effective in eradicating hepatitis C virus(HCV)in chronically infected patients.However,the protective role of the sustained virologic response(SVR)achieved by second-and thirdgeneration DAAs against the onset of hepatocellular carcinoma(HCC)and mortality is less well established.AIM To examine the occurrence of HCC or death from any cause in a retrospectiveprospective study of patients treated with DAAs.METHODS Patients were enrolled from a tertiary academic hospital center for liver disease management that collects subject data mainly from northeastern Italy.The study was conducted in 380 patients(age:60±13 years,224 males,32%with cirrhosis)treated with DAAs with or without SVR(95/5%),with a median follow up of 58 wk(interquartile range:38-117).The baseline anthropometric features,HCV viral load,severity of liver disease,presence of extra-hepatic complications,coinfection with HIV and/or HBV,alcohol consumption,previous interferon use,alphafetoprotein levels,and renal function were considered to be confounders.RESULTS The incidence rate of HCC in patients with and without SVR was 1.3 and 59 per 100 person-years,respectively(incidence rate ratio:44,95%CI:15-136,P<0.001).Considering the combined endpoint of HCC or death from any cause,the hazard ratio(HR)for the SVR patients was 0.070(95%CI:0.025-0.194,P<0.001).Other independent predictors of HCC or death were low HCV viremia(HR:0.808,P=0.030),low platelet count(HR:0.910,P=0.041),and presence of mixed cryoglobulinemia(HR:3.460,P=0.044).Considering SVR in a multi-state model,the independent predictors of SVR achievement were absence of cirrhosis(HR:0.521,P<0.001)and high platelet count(HR:1.019,P=0.026).Mixed cryoglobulinemia predicted the combined endpoint in patients with and without SVR(HR:5.982,P=0.028 and HR:5.633,P=0.047,respectively).CONCLUSION DAA treatment is effective in inducing SVR and protecting against HCC or death.A residual risk of HCC persists in patients with advanced liver disease or with complications,such as mixed cryoglobulinemia or renal failure.

Hepatitis C virus reinfection after liver transplantation: Is there a role for direct antiviral agents?

Recurrence of hepatitis C virus(HCV)infection following liver transplantation(LT)is almost universal and can accelerate graft cirrhosis in up to 30%of patients.The development of effective strategies to treat or prevent HCV recurrence after LT remains a major challenge,considering the shortage of donor organs and the accelerated progression of HCV in LT recipients.Standard antiviral therapy with pegylated-interferon plus ribavirin is the current treatment of choice for HCV LT recipients,even though the combination is not as effective as it is in immunocompetent patients.A sustained virological response in the setting of LT improves patient and graft survival,but this is only achieved in 30%-45%of patients and the treatment is poorly tolerated.To improve the efficacy of pre-and post-transplant antiviral therapy,a new class of potent direct-acting antiviral agents (DAAs)has been developed.The aim of this review is to summarize the use of DAAs in LT HCV patients.PubMed,Cochrane Library,MEDLINE,EMBASE,Web of Science and clinical trial databases were searched for this purpose.To date,only three clinical studies on the topic have been published and most of the available data are in abstract form.Although a moderately successful early virological response has been reported,DAA treatment regimens were associated with severe toxicity mitigating their potential usefulness.Moreover,the ongoing nature of data,the lack of randomized studies,the small number of enrolled patients and the heterogeneity of these studies make the results largely anecdotal and questionable.In conclusion,large welldesigned clinical studies on DAAs in HCV LT patients are required before these drugs can be recommended after transplantation.

Replication of clinical hepatitis B virus isolate and its application for selecting antiviral agents for chronic hepatitis B patients

AIM: To establish a cell model harboring replicative clinical hepatitis B virus (HBV) isolates and evaluate its application in individualized selection of anti-HBV agents for chronic hepatitis B (CHB) patients. METHODS: The full-length HBV genomic DNA from 8 CHB patients was amplified by polymerase chain reaction (PCR). All the patients were treated with lamivudine for at least seven months and finally became resistant to lamivudine. The amplified HBV DNA fragments were inserted into pHY106 vectors by Sap Ⅰ?digestion. The recombinant plasmids containing 1.1 copies of HBV genome were transiently transfected into Huh7 cell line, and the levels of HBsAg, HBeAg and intercellular HBV replicative intermediates were determined by ELISA and Southern blot analysis, respectively, with or without lamivudine and adefovir treatment. The antiviral treatment with adefovir was administered to the patients and analyzed in parallel. RESULTS: A total of 25 independent HBV isolateswere obtained from the sera of 8 patients, each patient had at least two isolates. One isolate from each individual was selected and subcloned into pHY106 vector, including 5 isolates with YVDD mutation and 3 isolates with YIDD mutation. All recombinant plasmids harboring HBV isolates were transfected into Huh7 cells. The results indicated that HBV genome carried in HBV replicons of clinical HBV isolates could effectively replicate and express in Huh7 cells. Adefovir, but not lamivudine, inhibited HBV replication both in vitro and in vivo, and in vitro inhibition was dose-dependent. CONCLUSION: The novel method described herein enables individualized selection of anti-HBV agents in clinic and is useful in future studies of antiviral therapy for CHB.

Outcomes assessment of hepatitis C virus-positive psoriatic patients treated using pegylated interferon in combination with ribavirin compared to new Direct-Acting Antiviral agents

AIM To evaluate the outcomes in biological treatment and quality of life of psoriatic patients with chronic hepatitis C(CHC) treated with new Direct-Acting Antiviral agents(DAAs) compared to pegylated interferon-2α plus ribavirin(P/R) therapy.METHODS This is a retrospective study involving psoriatic patients in biological therapy who underwent anti-hepatitis C virus(HCV) treatment at the Department of Dermatology Galeazzi Orthopaedic Institute Milan, Italy from January 2010 to November 2017. The patients were divided into two groups: patients that underwent therapy with DAAs and patients that underwent HCV treatment with P/R. Patients were assessed by a dermatologist for psoriasis symptoms, collecting Psoriasis Area Severity Index(PASI) scores and the Dermatology Quality of Life Index(DLQI). PASI and DLQI scores were evaluated 24 wk after the end of HCV treatment and were assumed as an outcome of the progression of psoriasis. Switching to a different b DMARD was considered as an inadequate response to biological therapy. The dropout of HCV therapy and sustained virological response(SVR) were considered as outcomes of HCV therapy.RESULTS Fifty-nine psoriatic patients in biological therapy underwent antiviral therapy for CHC. Of this, 27 patients were treated with DAAs and 32 with P/R. After 24 wk post treatment, the DLQI and the PASI scores were significantly lower(P < 0.001 and P < 0.005, respectively) in the DAAs group compared with P/R group. None of the patients in the DAAs group(0/27) compared to 8 patients of the P/R group(8/32) needed a shift in biological treatment.CONCLUSION DAAs seem to be more effective and safe than P/R in HCV-positive psoriatic patients on biological treatment. Fewer dermatological adverse events may be due to interferon-free therapy.

Advances in Zika virus vaccines and therapeutics:A systematic review

Zika virus(ZIKV)is the causative agent of a viral infection that causes neurological complications in newborns and adults worldwide.Its wide transmission route and alarming spread rates are of great concern to the scientific community.Numerous trials have been conducted to develop treatment options for ZIKV infection.This review highlights the latest developments in the fields of vaccinology and pharmaceuticals developments for ZIKV infection.A systematic and comprehensive approach was used to gather relevant and up-to-date data so that inferences could be made about the gaps in therapeutic development.The results indicate that several therapeutic interventions are being tested against ZIKV infection,such as DNA vaccines,subunit vaccines,live-attenuated vaccines,virus-vector-based vaccines,inactivated vaccines,virus-like particles,and mRNA-based vaccines.In addition,approved anti-ZIKV drugs that can reduce the global burden are discussed.Although many vaccine candidates for ZIKV are at different stages of development,none of them have received Food and Drug Authority approval for use up to now.The issue of side effects associated with these drugs in vulnerable newborns and pregnant women is a major obstacle in the therapeutic pathway.

Synthesis and Anti-influenza Virus Activity of Ethyl 6-Bromo-5-hydroxyindole-3-carboxylate Derivatives

A series of ethyl 6-bromo-5-hydroxyindole-3-carboxylate derivatives were synthesized and their in vitro anti-influenza virus activity was evaluated. All the compounds were characterized by 1H NMR and MS.

Research progress of anti-plant virus agents based on marine natural products

Plant virus diseases are a serious threat to food crops,vegetables,and cash crops,their prevention and control are extremely difficult,known as‘plant cancer’,and effective prevention and control agents are extremely short.Environmentally friendly natural antiviral active substances are key resources for the development of anti-plant virus drugs.Marine natural products are the most active field of new drugs and lead compounds discovery.However,there are few studies on their pesticide activities,especially their anti-plant virus activities.In the present manuscript,the latest research on anti-plant virus activity,structure-activity relationship,and mode of action of marine natural products were reviewed.It is hoped that this review will provide a useful reference for the research of anti-plant virus drugs.

Antiviral therapies for chronic hepatitis C virus infection with cirrhosis

Patients who are infected with hepatitis C virus(HCV) and also have advanced fibrosis or cirrhosis have beenrecognized as "difficult-to-treat" patients during an era when peginterferon and ribavirin combination therapy is the standard of care. Recent guidelines have clearly stated that treatment should be prioritized in this population to prevent complications such as decompensation and hepatocellular carcinoma. Recent advances in the treatment of chronic hepatitis C have been achieved through the development of direct-acting antiviral agents(DAAs). Boceprevir and telaprevir are first-generation DAAs that inhibit the HCV NS3/4A protease. Boceprevir or telaprevir, in combination with peginterferon and ribavirin, improved the sustained virological response rates compared with peginterferon and ribavirin alone and were tolerated in patients with HCV genotype 1 infection without cirrhosis or compensated cirrhosis. However, the efficacy is lower especially in prior non-responders with or without cirrhosis. Furthermore, a high incidence of adverse events was observed in patients with advanced liver disease, including cirrhosis, in real-life settings. Current guidelines in the United States and in some European countries no longer recommend these regimens for the treatment of HCV. Next-generation DAAs include second-generation HCV NS3/4A protease inhibitors, HCV NS5 A inhibitors and HCV NS5 B inhibitors, which have a high efficacy and a lower toxicity. These drugs are used in interferon-free or in interferon-based regimens with or without ribavirin in combination with different classes of DAAs. Interferon-based regimens, such as simeprevir in combination with peginterferon and ribavirin, are well tolerated and are highly effective especially in treatmentnave patients and in patients who received treatment but who relapsed. The efficacy is less pronounced in nullresponders and in patients with cirrhosis. Interferonfree regimens in combination with ribavirin and/or two or more DAAs could be used for treatment-nave, treatment-experienced and even for interferon-ineligible or interferon-intolerant patients. Some clinical trials have demonstrated promising results, and have shown that the efficacy and safety were not different between patients with and without cirrhosis. There are also promising regimens for genotypes other than genotype 1. Interferonis contraindicated in patients with decompensated cirrhosis, and further studies are needed to establish the optimal treatment regimen for this population. In the future, interferon-free and ribavirin-free regimens with high efficacy and improved safety are expected for HCVinfected patients with advanced liver diseases.

Hepatitis C virus infection and thyroid autoimmunedisorders:A model of interactions between the host and theenvironment

The hepatitis C virus(HCV) infection is an important public health problem and it is associated with hepatic and extrahepatic manifestations. Autoimmune thyroid diseases are common in HCV infected patients and the standard interferon-based treatment is associated with an increase of the immune-mediated thyroid damage. Recent evidence in the literature analyzed critical points of the mechanisms of thyroid damage, focusing on the balance between the two sides of the interaction: The environment(virus infection with potential crossreaction) and the host(susceptibility genes with consistent immune response). The spectrum of antiviral treatment for chronic HCV infection is rapidly expanding for the development of dual o triple therapy. The availability of interferon-free combined treatment with direct antiviral agents for HCV is very promising, in order to ameliorate the patient compliance and to reduce the development of thyroid autoimmunity.

Inhibition of hepatitis B virus by oxymatrine in vivo

AIM To investigate the anti-HBV effect ofoxymatrine (oxy) in vivo.METHODS HBV transgenic mice were producedby micro-injection of a 4.2kb fragmentcontaining the complete HBV genomes.Expression level of HBsAg and HBcAg in thetransgenic mice liver was determined byimmunohistochemical assay.RESULTS Four groups (6 mice in each group)were injected intraperitoneally with oxy at thedosage of 100,200, and 300 mg/kg or with salineonce a day for 30 days. Both HBsAg and HBcAgwere positive in livers of all the six mice in thecontrol group (injected with saline), and werepositive in livers of two mice in 100 mg/kg groupand 300mg/kg group. In 200mg/kg group,HBsAg and HBcAg were negative in livers of allthe six mice. Based on the results, 200 mg/kg isthe ideal dosage to explore the effect of oxy atdifferent time points. According to the oxytreatment time, mice were divided into fourgroups: 10 d, 20 d, 30 d and 60 d (4 mice in eachgroup). Each mouse underwent liver biopsy twoweeks before the treatment of oxy. Down-regulation of HBsAg and HBcAg appeared aftertreatment of oxymatrine for 10 d and 20 d, Dane-like particles disappeared after the treatment ofoxy for 20d under electron microscopy,however, the expression level of HBsAg andHBcAg returned to normal 60 d later after oxytreatment.CONCLUSION oxymatrine can reduce thecontents of HBsAg and HBcAg in transgenic miceliver, longer treatment time and larger dosagedo not yield better effects.

Impact of new treatment options for hepatitis c virus infection in liver transplantation

Liver transplant candidates and recipients with hepatitis C virus(HCV)-related liver disease greatly benefit from an effective antiviral therapy. The achievement of a sustained virological response before transplantation can prevent the recurrence of post-transplant HCV disease that occurs universally and correlates with enhanced progression to graft cirrhosis. Previous standard-of-care regimens(e.g.,pegylated-interferon plus ribavirin with or without first generation protease inhibitors,boceprevir and telaprevir) displayed suboptimal results and poor tolerance in liver transplant recipients. A new class of potent direct-acting antiviral agents(DAA) characterized by all-oral regimens with minimal side effects has been approved and included in the recent guidelines for the treatment of liver transplant recipients with recurrent HCV disease. Association of sofosbuvir with ribavirin and/or ledipasvir is recommended in liver transplant recipients and patients with decompensated cirrhosis. Other regimens include simeprevir,daclatasvir,and combination of other DAA. Possible interactions should be monitored,especially in coinfected human immunodeficiency virus/HCV patients receiving antiretrovirals.

Real life efficacy and safety of direct-acting antiviral therapy for treatment of patients infected with hepatitis C virus genotypes 1, 2and 3 in northwest China

BACKGROUND Regimens involving direct-acting antiviral agents(DAAs)are recommended for the treatment of infection with hepatitis C virus(HCV)genotypes 1,2 and 3.But real-world data is still not enough,especially in Asia.AIM To investigate the efficacy and safety of DAA-based regimens in a real-life setting in China.METHODS This study included 366 patients infected with HCV genotypes 1,2 and 3,with or without cirrhosis,who were observed between May 2015 and December 2018.They were treated with ledipasvir and sofosbuvir(SOF)(genotype 1)with or without ribavirin(RBV),SOF and RBV(genotype 2),or SOF and daclatasvir(genotype 3),with or without RBV,for 12 or more wk.The participants’sustained virological responses(SVR)at post-treatment week 12(SVR12)was the primary endpoint.The occurrence of adverse events and drug-drug interactions were recorded.RESULTS In the 366 patients,genotype 1(59.0%)was the most common genotype,followed by genotypes 2(34.4%)and 3(6.6%).Liver cirrhosis was diagnosed in 154(42.1%)patients.Fifty(13.7%)patients were treatment-experienced.Intention-to-treat analysis revealed that SVR12 was 86.3%(316/366).For modified intention-totreat analysis,SVR12 was achieved in 96.6%of overall patients(316/327),96.3%in patients with genotype 1,97.5%in those with genotype 2,and 95.0%in those with genotype 3.Most of the treatment failures were due to lack of follow-up(3cases had non-responses,1 had virological breakthrough,11 relapsed and 36 did not participate in the follow-up).There was no significant difference in SVR between different genotypes and liver statuses(P<0.05).Patients with lower alanine aminotransferase levels at baseline who achieved an end of treatment response were more likely to achieve SVR12(P<0.05).High SVR was observed regardless of age,gender,liver status,alpha-fetoprotein,HCV RNA levels or history of antiviral therapy(P>0.05 for all).The cumulative hepatocellular carcinoma occurrence and recurrence rate after using the DAAs was 0.9%.Most of the adverse events were mild.We found two cases of special adverse events.One case involved facial and bilateral lower extremity edema,and the other case showed an interesting change in lipid levels while on medication.No severe adverse events were noted.CONCLUSION The DAA-based regimens tested in this study have excellent effectiveness and safety in all patients infected with HCV genotypes 1,2 and 3,including those with cirrhosis.

Hepatitis C virus NS5A inhibitors and drug resistance mutations

Some direct-acting antiviral agents for hepatitis C virus (HCV), such as telaprevir and boceprevir have been available since 2011. It was reported that HCV NS5A is associated with interferon signaling related to HCV replication and hepatocarcinogenesis. HCV NS5A inhibitors efficiently inhibited HCV replication in vitro. Human studies showed that dual, triple and quad regimens with HCV NS5A inhibitors, such as daclatasvir and ledipasvir, in combination with other direct-acting antiviral agents against other regions of HCV with or without peginterferon/ribavirin, could efficiently inhibit HCV replication according to HCV genotypes. These combinations might be a powerful tool for &#x0201c;difficult-to-treat&#x0201d; HCV-infected patients. &#x0201c;First generation&#x0201d; HCV NS5A inhibitors such as daclatasvir, ledipasvir and ABT-267, which are now in phase III clinical trials, could result in resistance mutations. &#x0201c;Second generation&#x0201d; NS5A inhibitors such as GS-5816, ACH-3102, and MK-8742, have displayed improvements in the genetic barrier while maintaining potency. HCV NS5A inhibitors are safe at low concentrations, which make them attractive for use despite low genetic barriers, although, in fact, HCV NS5A inhibitors should be used with HCV NS3/4A inhibitors, HCV NS5B inhibitors or peginterferon plus ribavirin. This review article describes HCV NS5A inhibitor resistance mutations and recommends that HCV NS5A inhibitors be used in combination regimens potent enough to prevent the emergence of resistant variants.

Hepatitis C virus protease inhibitor-resistance mutations: Our experience and review

Direct-acting antiviral agents(DAAs)for hepatitis C virus(HCV)infection are one of the major advances in its medical treatment.The HCV protease inhibitors boceprevir and telaprevir were the first approved DAAs in the United States,Europe,and Japan.When combined with peginterferon plus ribavirin,these agents increase sustained virologic response rates to70%-80%in treatment-na?ve patients and previoustreatment relapsers with chronic HCV genotype 1 infection.Without peginterferon plus ribavirin,DAA monotherapies increased DAA-resistance mutations.Several new DAAs for HCV are now in clinical development and are likely to be approved in the near future.However,it has been reported that the use of these drugs also led to the emergence of DAA-resistance mutations in certain cases.Furthermore,these mutations exhibit cross-resistance to multiple drugs.The prevalence of DAA-resistance mutations in HCV-infected patients who were not treated with DAAs is unknown,and it is as yet uncertain whether such variants are sensitive to DAAs.We performed a population sequence analysis to assess the frequency of such variants in the sera of HCV genotype 1-infected patients not treated with HCV protease inhibitors.Here,we reviewed the literature on resistance variants of HCV protease inhibitors in treatment na?ve patients with chronic HCV genotype 1,as well as our experience.

Inhibition of hepatitis B virus production by Boehmeria nivea root extract in HepG2 2.2.15 cells

AIM: To explore the anti-hepatitis B virus (HBV) effects of Boehmeria nivea (B. nivea) root extract (BNE) by using the HepG2 2.2.15 cell model system. METHODS: Hepatitis B surface antigen (HBsAg), hepatitis B virus e antigen (HBeAg), and HBV DNA were measured by using ELISA and real-time PCR, respectively. Viral DNA replication and RNA expression were determined by using Southern and Northern blot, respectively. RESULTS: In HepG2 2.2.15 cells, HBeAg (60%, P < 0.01) and particle-associated HBV DNA (> 99%, P < 0.01) secretion into supernatant were significantly inhibited by BNE at a dose of 100 mg/L, whereas the HBsAg was not inhibited. With different doses of BNE, the reduced HBeAg was correlated with the inhibition of HBV DNA. The anti-HBV effect of BNE was not caused by its cytotoxicity to cells or inhibition of viral DNA replication and RNA expression. CONCLUSION: BNE could effectively reduce the HBV production and its anti-HBV machinery might differ from the nucleoside analogues.

Hepatitis B and C virus reactivation in immunosuppressed patients with inflammatory bowel disease

In recent years,a number of case reports and clinical studies have highlighted the risk of hepatitis B and C virus reactivation in patients with inflammatory bowel disease who are treated with immunosuppressive drugs.The cases of viral hepatitis reactivation that have been reported are characterized by a wide range of clinical manifestations,from viremia without clinically relevant manifestations to fulminant life-threatening hepatitis.The development and dissemination of biological immunosuppressive drugs have led to a significant increase in the number of reports of interest to physicians in a variety of clinical settings.On this topic,there have been a number of published guidelines and reviews that have collected the available evidence,providing recommendations on prophylactic and therapeutic strategies and methods for monitoring patients at risk.However,it should be noted that,to date,very few clinical studies have been published,and most of the recommendations have been borrowed from otherclinical settings.The published studies are mostly retrospective and are based on very heterogeneous populations,using different therapeutic and prophylactic regimens and obtaining conflicting results.Thus,it seems clear that it is desirable to concentrate our efforts on prospective studies,not conducting further reviews of the literature in the continued absence of new evidence.

Novel approaches towards conquering hepatitis B virus infection

Currently approved treatments for hepatitis B virus （HBV） infection include the immunomodulatory agent, IFN-α, and nucleos（t）ide analogues. Their efficacy is limited by their side effects, as well as the induction of viral mutations that render them less potent. It is thus necessary to develop drugs that target additional viral antigens. Chemicals and biomaterials by unique methods of preventing HBV replication are currently being developed, including novel nucleosides and newly synthesized compounds such as capsid assembling and mRNA transcription inhibitors. Molecular therapies that target different stages of the HBV life cycle will aid current methods to manage chronic hepatitis B （CriB） infection. The use of immunomodulators and gene therapy are also under consideration. This report summarizes the most recent treatment possibilities for CHB infection. Emerging therapies and their potential mechanisms, efficacy, and pitfalls are discussed.

Hepatitis C in human immunodeficiency virus co-infected individuals: Is this still a “special population”?

A substantial proportion of individuals with chronic hepatitis C virus(HCV) are co-infected with human immunodeficiency virus(HIV). Co-infected individuals are traditionally considered as one of the "special populations" amongst those with chronic HCV, mainly because of faster progression to end-stage liver disease and suboptimal responses to treatment with pegylated interferon alpha and ribavirin, the benefits of which are often outweighed by toxicity. The advent of the newer direct acting antivirals(DAAs) has given hope that the majority of co-infected individuals can clear HCV. However the "special population" designation may prove an obstacle for those with co-infection to gain access to the new agents, in terms of requirement for separate pre-licensing clinical trials and extensive drug-drug interaction studies. We review the global epidemiology, natural history and pathogenesis of chronic hepatitis C in HIV co-infection. The accelerated course of chronic hepatitis C in HIV co-infection is not adequately offset by successful combination antiretroviral therapy. We also review the treatment trials of chronic hepatitis C in HIV co-infected individuals with DAAs and compare them to trials in the HCV mono-infected. There is convincing evidence that HIV co-infection no longer diminishes the response to treatment against HCV in the new era of DAA-based therapy. The management of HCV co-infection should therefore become a priority in the care of HIV infected individuals, along with public health efforts to prevent new HCV infections, focusing particularly on specific patient groups at risk, such as men who have sex with men and injecting drug users.

Application of hepatitis B virus replication mouse model

AIM:To evaluate the value of the hepatitis B virus(HBV) replication mouse model with regard to several aspects of the study of HBV biology.METHODS:To evaluate the HBV replication mouse model in detecting the efficacy of anti-HBV agents,the interferon inducer polyinosinic-polytidylin acid(polyIC) and nucleotide analogues adefovir and entecavir were administered to mice injected with wild type pHBV4.1,and the inhibiting effect of these agents on HBV DNA replication was evaluated.To identify the model's value in a replication ability study of HBV drug-resistant mutants and a HBx-minus mutant,telbivudine resistance mutants(rtM204I,ayw subtype),adefovir resistance mutants(rtA181V + rtN236T,ayw subtype) and HBxminus mutants were injected respectively,and their corresponding HBV DNA replication intermediates in mouse liver were assessed.RESULTS:Compared with the wild type HBV replication mouse model without antiviral agent treatment,the HBV DNA replication intermediates of the polyICtreated group were decreased 1-fold;while in the entecavir-and adefovir-treated groups,the levels of HBV DNA replication intermediates were inhibited 13.6-fold and 1.4-fold,respectively.For the mouse models injected with telbivudine resistance mutant,adefovir resistance mutant and HBx-minus mutant,HBV DNA replication intermediates could still be detected,but the levels of HBV DNA replication intermediates of these mutants decreased 4.5-fold,5.6-fold and 2.9-fold respectively,compared with the mouse model with wild type HBV plasmid.CONCLUSION:The HBV replication mouse model we established was a useful and convenient tool to detect the efficacy of antiviral agents and to study the replication ability of HBV mutants in vivo.

Inhibition of hepatitis B virus replication by pokeweed antiviral protein in vitro

AIM: To explore the inhibitory effects of pokeweed antiviral protein seed (PAP-S) and PAP encoded by a eukaryotic expression plasmid on hepatitis B virus (HBV) replication in vitro. METHODS: HepG2 2.2.15 cells in cultured medium were treated with different concentrations of PAP-S. HBsAg, HBeAg and HBV DNA in supernatants were determined by ELISA and fluorescent quantitative PCR respectively. MTT method was used to assay for cytotoxicity. HepG2 were cotransfected with various amounts of PAP encoded by a eukaryotic expression plasmid and replication competent wild-type HBV 1.3 fold over- length plasmid. On d 3 after transfection, HBsAg and HBeAg were determined by using ELISA. Levels of HBV core-associated DNA and RNA were detected by using Southern and Northern blot, respectively. RESULTS: The inhibitory effects of PAP-S on HBsAg, HBeAg and HBV DNA were gradually enhanced with the increase of PAP concentration. When the concentration of PAP-S was 10 μg/mL, the inhibition rates of HBsAg, HBeAg and HBV DNA were 20.9%, 30.2% and 50%, respectively. After transfection of 1.0 μg and 2.0 μg plasmid pXF3H-PAP, the levels of HBV nucleocapside- associated DNA were reduced by 38.0% and 74.0% respectively, the levels of HBsAg in the media by 76.8% and 99.7% respectively, and the levels of HBeAg by 72.7% and 99.3% respectively as compared with controls. Transfection with 2 μg plasmid pXF3H-PAP reduced the levels of HBV nucleocapside-associated RNA by 69.0%.CONCLUSION: Both PAP-S and PAP encoded by a eukaryotic expression plasmid could effectively inhibit HBV replication and antigen expression in vitro, and the inhibitory effects were dose-dependent.