Animal models of hepatitis E infection: Advances and challenges

Hepatitis E virus(HEV)is one of the leading causes of acute viral hepatitis worldwide.Although most of HEV infections are asymptomatic,some patients will develop the symptoms,especially pregnant women,the elderly,and patients with preexisting liver diseases,who often experience anorexia,nausea,vom-iting,malaise,abdominal pain,and jaundice.HEV infection may become chronic in immunosuppressed individuals.In addition,HEV infection can also cause several extrahepatic manifestations.HEV exists in a wide range of hosts in nature and can be transmitted across species.Hence,animals susceptible to HEV can be used as models.The establishment of animal models is of great significance for studying HEV transmission,clinical symptoms,extrahepatic manifestations,and therapeutic strategies,which will help us understand the pathogenesis,prevention,and treatment of hepatitis E.This review summarized the animal models of HEV,including pigs,monkeys,rabbits,mice,rats,and other animals.For each animal species,we provided a concise summary of the HEV genotypes that they can be infected with,the cross-species transmission pathways,as well as their role in studying extrahepatic manifestations,prevention,and treatment of HEV infection.The advantages and disadvantages of these animal models were also emphasized.This review offers new perspectives to enhance the current understanding of the research landscape surrounding HEV animal models.

Hepatitis E virus: Epidemiology, diagnosis, clinical manifestations, and treatment

The hepatitis E virus(HEV)is the fifth known form of viral hepatitis and was first recognized as the cause of an epidemic of unexplained acute hepatitis in the early 1980s.Globally,it is one of the most frequent causes of acute viral hepatitis.The majority of HEV infections are asymptomatic and lead to the spontaneous clearance of the virus.Among the eight different genotypes identified to date,HEV genotype 1(HEV1),HEV2,HEV3,and HEV4 are the most frequent genotypes causing infections in humans.HEV1 and HEV2 are prevalent in developing regions and able to result in large-scale outbreaks originating from contaminated water supplies.They are also responsible for severe hepatitis in pregnant patients and infants.In contrast,HEV3 and HEV4 are zoonotic,and the transmission of these genotypes to humans occurs mainly through the fecal contamination of water and consumption of contaminated meat from infected animals.Their main reservoir is the pig,and they are mostly encountered in developed countries.The major risk groups for HEV infection and its ensuing adverse consequences are pregnant women,infants,older people,immunocompromised individuals,patients with underlying chronic liver diseases,and workers that come into close contact with HEV-infected animals.In the clinical perspective,HEV infections have diverse clinical manifestations including acute and self-limiting hepatitis,acute-on-chronic liver disease,chronic hepatitis,cirrhosis,and liver failure.Although HEV mainly results in acute selflimiting infection,chronic HEV infection may occur among immunocompromised patients(e.g.,solid-organ transplant recipients).Additionally,HEV-associated extrahepatic manifestations involving various organs have been reported in the last decade,although the causal link for many of them still needs to be proven.Ribavirin and interferon-alpha are the most widely used agents for the treatment of HEV infections with a certain level of success.However,ribavirin is contraindicated in pregnant patients,and interferon-alpha cannot be used in most transplant recipients.Therefore,there is an urgent need for novel antiviral compounds that are safe and effective particularly for patients having contraindications for ribavirin or interferon-alpha and infected by the ribavirinresistant HEV.In this review article,a literature search using PubMed and MEDLINE databases was performed,up to March 2020.Only the articles published in English were reviewed.

Hepatitis B virus pre S1 deletion is related to viral replication increase and disease progression

AIM:To investigate the clinical implications of hepatitis B virus(HBV) pre S1 deletion.METHODS:We developed a fluorescence resonance energy transfer-based real-time polymerase chain reaction(RT-PCR) that can detect four genotypes(wild type, 15-bp, 18-bp and 21-bp deletion).The PCR method was used in two cohorts of Korean chronic HBV subjects with genotype C infections.Cohort Ⅰ included 292 chronic HBV subjects randomly selected from Cheju National University Hospital(Jeju, South Korea) or Seoul National University Hospital(Seoul, South Korea), and cohort Ⅱ included 90 consecutive chronic HBV carriers recruited from Konkuk University Hospital(Seoul, South Korea); the cohort Ⅱ patients did not have hepatocellular carcinoma or liver cirrhosis.RESULTS:The method proposed in this study identified 341 of 382 samples(89.3%).Deletion variants were identified in 100(29.3%) of the 341 detected samples.In both cohorts, the subjects with deletions had a significantly higher Hepatitis B virus e antigen(HBe Ag)-positive seroprevalence [cohort Ⅰ, wild(51.0%) vs deletion(75.0%), P < 0.001; cohort Ⅱ, wild(69.2%) vs deletion(92.9%), P = 0.002] and higher HBV DNA levels [cohort Ⅰ, wild(797.7 pg/m L) vs deletion(1678.9 pg/m L), P = 0.013; cohort Ⅱ, wild(8.3 × 108 copies/m L) vs deletion(2.2 × 109 copies/m L), P = 0.049], compared to subjects with wild type HBV.CONCLUSION:HBV genotype C pre S1 deletion may affect disease progression in chronic HBV subjects through an extended duration of HBe Ag seropositive status and increased HBV replications.

mi R-29a promotes hepatitis B virus replication and expression by targeting SMARCE1 in hepatoma carcinoma

AIM To investigate the functional role and underlying molecular mechanism of mi R-29 a in hepatitis B virus(HBV) expression and replication.METHODS The levels of mi R-29 a and SMARCE1 in HBV-infected Hep G2.2.15 cells were measured by quantitative real-time PCR and western blot analysis. HBV DNA replication was measured by quantitative PCR and Southern blot analysis. The relative levels of hepatitis B surface antigen and hepatitis B e antigen were detected by enzyme-linked immunosorbent assay. The Cell Counting Kit-8(CCK-8) was used to detect the viability of Hep G2.2.15 cells. The relationship between mi R-29 a and SMARCE1 were identified by target prediction and luciferase reporter analysis.RESULTS mi R-29 a promoted HBV replication and expression, w h i le S MA R C E 1 r e p r e s s e d H B V r e p lic a t io n a n d expression. Cell viability detection indicated that mi R-29 a transfection had no adverse effect on the host cells. Moreover, SMARCE1 was identified and validated to be a functional target of mi R-29 a. Furthermore, restored expression of SMARCE1 could relieve the increased HBV replication and expression caused by mi R-29 a overexpression.CONCLUSION mi R-29 a promotes HBV replication and expression through regulating SMARCE1. As a potential regulator of HBV replication and expression, mi R-29 a could be a promising therapeutic target for patients with HBV infection.

Clinical features of HBs Ag seroclearance in hepatitis B virus carriers in South Korea: A retrospective longitudinal study

AIM To investigate the characteristic features of hepatitis B surface antigen(HBs Ag) seroclearance among Korean hepatitis B virus(HBV) carriers.METHODS Carriers with HBs Ag seroclearance were selected by analyzing longitudinal data collected from 2003 to 2015. The period of time from enrollment to the negative conversion of HBs Ag(HBs Ag-NC) was compared by stratifying various factors, including age, sex, hepatitis B e antigen(HBe Ag), HBV DNA, sequential changes in the signal-to-cutoff ratio of HBs Ag(HBs Ag-SCR), as measured by qualitative HBs Ag assay, and chronic liver disease on ultrasonography(US-CLD). Quantification of HBV DNA and HBs Ag(HBs Ag-QNT) in the serum was performed by commercial assay.RESULTS Among the 1919 carriers, 90(4.7%) exhibited HBs AgNC at 6.2 ± 3.6 years after registration, with no differences observed among the different age groups. Among these carriers, the percentages of those with asymptomatic liver cirrhosis(LC) and hepatocellular carcinoma(HCC) at registration were 31% and 7.8%, respectively. The frequency of HBs Ag-NC significantly differed according to the HBV DNA titer and US-CLD. HBe Ag influenced HBs Ag-NC in the 40-50 and 50-60 year age groups. HBs Ag-SCR < 1000 was correlated with an HBs Ag-QNT < 200 IU/m L. A gradual decrease in the HBs Ag-SCR to < 1000 predicted HBs Ag-NC. Six patients developed HCC after registration, including two before and four after HBs Ag-NC. The rate at which the patients developed new HCC after HBs Ag seroclearance was 4.8%. LC with excessive drinking and vertical infection were found to be risk factors for HCC in the HBs Ag-NC group.CONCLUSION HCC surveillance should be continued after HBs Ag seroclearance. An HBs Ag-SCR < 1000 and its decrease in sequential testing are worth noting as predictive markers of HBs Ag loss.

Oxidative stress modulation in hepatitis C virus infected cells

Hepatitis C virus(HCV) replication is associated with the endoplasmic reticulum, where the virus can induce cellular stress. Oxidative cell damage plays an important role in HCV physiopathology. Oxidative stress is triggered when the concentration of oxygen species in the extracellular or intracellular environment exceeds antioxidant defenses. Cells are protected and modulate oxidative stress through the interplay of intracellular antioxidant agents, mainly glutathione system(GSH) and thioredoxin; and antioxidant enzyme systems such as superoxide dismutase, catalase, GSH peroxidase, and heme oxygenase-1. Also, the use of natural and synthetic antioxidants(vitamin C and E, N-acetylcysteine, glycyrrhizin, polyenylphosphatidyl choline, mitoquinone, quercetin, S-adenosylmethionine and silymarin) has already shown promising results as co-adjuvants in HCV therapy. Despite all the available information, it is not known how different agents with antiviral activity can interfere with the modulation of the cell redox state induced by HCV and decrease viral replication. This review describes an evidence-based consensus on molecular mechanisms involved in HCV replication and their relationship with cell damage induced by oxidative stress generated by the virus itself and cell antiviral machinery. It also describes some molecules that modify the levels of oxidative stress in HCV-infected cells.

Hepatitis E virus is a leading cause of acute-on-chronic liver disease:experience from a tertiary centre in Bangladesh

BACKGROUND:Acute-on-chronic liver failure(ACLF) is common in Bangladesh.Acute viral E hepatitis is sporadically encountered in this country each year,with a rising incidence in the rainy season.This study aimed to identify the etiology of ACLF in Bangladesh. METHODS:In this retrospective study,69 ACLF patients were included.They presented to our department at the Bangabandhu Sheikh Mujib Medical University in Dhaka.History of diseases was recorded and appropriate investigations were conducted in all patients. RESULTS:Acute hepatitis E virus(HEV)infection was positive in 21.7%(15/69)of the patients,while 14.5% (10/69)had septicemia.Upper gastrointestinal tract hemorrhage was seen in 4.3%of the patients(3/69),while another 4.3%(3/69)had a positive history for alcohol or drugs.None of the patients tested positive for hepatitis A virus infection and no evidence of hepatitis B virus flare was found in any patient.No specific cause for ACLF could be identified. CONCLUSIONS:Acute HEV infection is a leading cause of ACLF in Bangladesh.Many patients were thought to have decompensation of cirrhosis,but subsequently were recognized as having ACLF by a retrospective review according to the definition of the Asian Pacific Association for the Study of the Liver Working Party Meeting on ACLF in New Delhi in early 2008.

Detection of hepatitis E virus RNA in sera of patients with hepatitis E by polymerase chain reaction

BACKGROUND: The duration of viremia during hepatitis E virus (HEV) infection has rarely been reported. This study was undertaken to detect HEV RNA in sera of patients with hepatitis E and to understand the process of HEV infection more thoroughly. METHODS: HEV RNA was detected in the serum samples of hospitalized patients with acute hepatitis E by reverse transcriptase-nested polymerase chain reaction (RT-nPCR) using two pairs of primers from open reading frame (ORF) I of the HEV genome. RESULTS: The serum samples from 44 (70%) of 62 patients were positive for HEV RNA. Thirty-two of these patients, with 288 serial serum specimens, were followed up for the whole process, and 24 patients (75%) were positive for HEV RNA. The positive rates declined with the course of the disease, serum HEV RNA persisting for 20.6 days on average after onset of illness. Serum HEV RNA remained positive in 36 (81.8%) of the 44 patients at the time their alanine aminotransferase (ALT) began to decrease. There was no difference in HEV RNA positivity between serum with high levels of HEV antibody (peak P/N ratio >= 4.0) and that with low levels (peak P/N ratio 4.0), with 25 out of 35 and 19 out of 27 (71.4% vs. 70.4%, P>0.05), respectively. CONCLUSIONS: There is a relatively long period of HEV viremia in patients with hepatitis E. The proportion of HEV viremia and its duration are not directly related to serum ALT values or HEV antibody levels.

Significance of serum IgA in patients with acute hepatitis E virus infection

瞄准：学习浆液反肝炎 E 的意义在有肝炎 E 的病人的病毒(HEV ) IgA。方法：一个新方法被建立到试金 anti-HEV IgA，它能在感染的中间的阶段被检测。我们在重量的单位把 anti-HEV IgA 试金与 anti-HEV IgM 和 anti-HEV IgG 试金作比较一 from 有积极 HEV-RNA 的 60 个病人。结果：有积极 HEV-RNA 的 60 个病人与否定 HEV-RNA 让 anti-HEV IgA 和 anti-HEV IgM 和 410 个病人被用作控制。与肝炎 E 从 60 个病人获得的周期的浆液样品为 HEV RNA， anti-HEV IgM， anti-HEV IgA 和 anti-HEV IgG 被测试。他们的 HEV-RNA 在浆液是可检测的直到 20 +/- 11 d。我们使用了 anti-HEV IgM 和 anti-HEV IgA 试金检测 HEV 感染，阳性结果在 90 +/- 被发现 15 d 和 120 +/- 23 d 分别地， anti-HEV IgA 的积极的率比 anti-HEV IgM 和 HEV-RNA 的高(P 【 0.05 ) 。结论：在浆液的 anti-HEV IgA 的持续时间比 anti-HEV IgM 的长，并且 anti-HEV IgA 试金是一个好方法检测 HEV 感染。

Experimental infection of Z:ZCLA Mongolian gerbils with human hepatitis E virus

AIM:To investigate whether Z:ZCLA Mongolian gerbils are readily susceptible to infection by human hepatitis E virus(HEV).METHODS:Z:ZCLA Mongolian gerbils were infected with a clinical HEV strain isolated from an acutehepati tis E patient,and virus pathogenesis was assessed in this host.Non-infected gerbils served as the control group.Feces samples from gerbils were collected weekly for reverse transcription-nested polymerase chain reaction.Serum anti-HEV Ig G and alanine aminotransferase(ALT) were detected by enzyme linked immunosorbent assay.At sacrifice,each animal's liver,spleen and kidney were collected for histopathologic examination.RESULTS:HEV-infected gerbils showed fatigue,with histopathological changes observed in the liver,spleen and kidney.HEV RNA was detected in fecal samples taken at day 7 after inoculation and the detectable levels lasted out to day 42 after inoculation.Interestingly,ALT levels were only moderately increased in the HEV-infected animals compared with the noninfected control group.CONCLUSION:Z:ZCLA Mongolian gerbils are susceptible to human HEV.

Seroprevalence and evolutionary dynamics of genotype 4 hepatitis E virus in Shandong Province,China

AIM:To investigate the seroprevalence and evolutionary dynamics of hepatitis E virus(HEV)and assess the ancestor of HEVs in China’s Shandong Province.METHODS:A total of 2028 serum,60 fecal and 82 bile samples were collected from the general human population,patients and swine,respectively.This seroepidemiological study was conducted using an immunnosorbent assay and HEV RNA was detected by the reverse transcription-nested polymerase chain reaction(RTnPCR)method.Complete genome sequences of the prevalent strains(CH-YT-HEV01,CH-YT-HEV02 and CHYT-sHEV01)were determined,and the sequences were analyzed phylogenetically.In addition,the evolutionary dynamics of three HEV isolates were determined using the framework of coalescent analysis in the program package BEAST,and the time of the most recent common ancestors(TMRCAs)of China-indigenous genotype4 HEV isolates was calculated.RESULTS:The overall viral burden in the general human population was 0.1%,and the positive rates of anti-HEV IgG and IgM in the serum specimens were25.1%(509/2028)and 2.3%(51/2028),respectively.In addition,IgG positivity increased with age.The phylogenetic analysis based on the full-length nucleotide sequences showed that the strain CH-YT-HEV02was directly related to CH-YT-sHEV01 with a 94%identity,suggesting that they were involved in crossspecies transmission.The isolate CH-YT-HEV01 was close to HB-3 and CHN-SD-sHEV with a bootstrap value of 100%,sharing a 96.1%-96.4%identity with each other.Surprisingly,the HB-3 strain was a representative strain prevalent in swine in Hubei,and the isolate CHNSD-sHEV was obtained from swine in Shandong in a previous report.TMRCA for the clade of CH-YT-HEV01and HB-3 was 2003,which was consistent with the TMRCA for the clade of CHN-SD-sHEV and HB-3,and they were both earlier than the TMRCA for the clade of CHYT-HEV01 and CHN-SD-sHEV(2004).CONCLUSION:The strains CH-YT-HEV01,CHN-SDsHEV and HB-3 are involved in trans-regional transmission,and the ancestors of HEVs in Shandong come from Hubei Province.

Neurological manifestations of hepatitis E virus infection:An overview

Hepatitis E virus(HEV)is an important cause of repeated waterborne outbreaks of acute hepatitis.Recently,several extrahepatic manifestations(EHMs)have been described in patients with HEV infection.Of these,neurological disorders are the most common EHM associated with HEV.The involvement of both the peripheral nervous system and central nervous system can occur together or in isolation.Patients can present with normal liver function tests,which can often be misleading for physicians.There is a paucity of data on HEV-related neurological manifestations;and these data are mostly described as case reports and case series.In this review,we analyzed data of 163 reported cases of HEV-related neurological disorders.The mechanisms of pathogenesis,clinico-demographic profile,and outcomes of the HEV-related neurological disorders are described in this article.Nerve root and plexus disorder were found to be the most commonly reported disease,followed by meningoencephalitis.

Seroepidemiology and genetic characterization of hepatitis E virus in western Yunnan Province

Objective:To investigate the seroepidemiology and genetic characterization of hepatitis E virus(HEV) in western Yunnan Province.Methods:Questionnaire survey was conducted among1638 residents in western Yunnan Province using stratified sampling method.Enzyme-linked immunosorbent assay was used to detect serum anti-HEV IgG and IgM.HEV RNA was extracted from patients with serum anti-HEV IgM positive.The open reading flame 2(ORF2) of HEV that was amplified by nested RT-PCR was sequenced and compared with standard HEV genotypes 1-4.Results:Serum anti-HEV positive was found in 13.929(228/1638) residents.The HEV infection rate in males was significantly higher than that in females with a ratio of 1.47(P<0.01).20-30 and30-40 years old young men showed the highest incidence.20.57%and 20.78%.respectively.While10-20 and 20-30 years old young women exhibited the highest infection rate,11.85%and 15.60%,respectively.According to occupation,the highest HEV infection rate was observed in farmers(20.35%) and migrants(16.50%).We isolated 10 individual HEV isolates from 31 patients with serum anti-HEV IgM positive.Homology analysis and phylogenetic analysis indicated that these10 HEV isolates belonged to HEV genotype 4 with the homology of 78.65%-94.71%.Conclusions:The HEV infection rate is high in western Yunnan Province.HEV genotype 4 is the leading cause of HEV infection and young farmers and migrants are the main infected population.

Open reading frame 3 protein of hepatitis E virus:Multi-function protein with endless potential

Hepatitis E virus (HEV), a fecal-orally transmitted foodborne viral pathogen,causes acute hepatitis in humans and is responsible for hepatitis E outbreaksworldwide. Since the identification of HEV as a zoonotic agent, this virus has beenisolated from a variety of hosts with an ever-expanding host range. HEV-openreading frame (ORF) 3, the smallest ORF in HEV genomes, initially had beenperceived as an unremarkable HEV accessory protein. However, as novel HEVORF3function has been discovered that is related to the existence of a putativethird virion structural form, referred to as “quasi-enveloped” HEV particles, HEVis challenging the conventional virion structure-based classification scheme,which assigns all viruses to two groups, “enveloped” or “non-enveloped”. In thisreview, we systematically describe recent progress that has identified multiplepathogenic roles of HEV-ORF3, including roles in HEV virion release, biogenesisof quasi-enveloped virus, regulation of the host innate immune response, andinterference with host signaling pathways. In addition, implications of HEVORF3-associated quasi-enveloped virions are discussed to guide futuredevelopment of improved vaccines against zoonotic HEV infection.

Effects of hepatitis E virus infection on interferon production via ISG15

AIM To assess the effects of hepatitis E virus(HEV) on the production of type Ⅰ interferons(IFNs) and determine the underlying mechanisms.METHODS We measured the production of interferon(IFN)-alpha and-beta(-α/β) in genotype 3 HEV-infected C3 A cells at different time points(0, 8, 12, 24, 48, 72 and 120 h) by enzyme-linked immunosorbent assay(ELISA). The expression levels of IFN-stimulated gene(ISG)15 in HEVinfected C3A cells at different time points were tested by western blotting. The plasmid-expressing open reading frame 3(ORF3) or control plasmids(green fluorescent protein-expressing) were transfected into C3A cells, and the levels of IFN-α/β and ISG15 were evaluated, respectively. Furthermore, the plasmid-expressing ISG15 or small interfering RNA-inhibiting ISG15 was transfected into infected C3A cells. Then, the production of IFN-α/β was also measured by ELISA.RESULTS We showed that genotype 3 HEV could enhance the production of IFN-α/β and induce elevation of ISG15 in C3A cells. HEV ORF3 protein could enhance the production of IFN-α/β and the expression of ISG15. Additionally, ISG15 silencing enhanced the production of IFN-α/β. Overexpression of ISG15 resulted in the reduction of IFN-α/β.CONCLUSION HEV may promote production of IFN-α/β and expression of ISG15 via ORF3 in the early stages, and increased ISG15 subsequently inhibited the production of IFN-α/β.

Expression and Characterization of a Recombinant Truncated Capsid Protein of Hepatitis E Virus in Pichia pastoris

Hepatitis E is an enterically transmitted viral disease caused by infection with hepatitis E virus（HEV）. HEV is a nonenveloped virus that bas been classified in the family of Caliciviridae. The virus appears to be a polya-denylated, positive-stranded RNA virus with three major open reading frames（ORFs）. The capsid protein of HEV is encoded by the open reading frame 2（ORF2）. We attempted to produce a truncated capsid protein, designed p293, in Pichia pastoris. The p293 gene encoding amino acids（aa） 382-674 of HEV ORF2 was designed based on the full length of HEV ORF2, cloned into the yeast vector pPIC9K, and expressed in P. pastoris strain GS 115. SDS-PAGE and Western blotting demonstrated that the recombinant protein p293 could well be expressed in P pastoris. Under optimized conditions （culture medium pH, 6.0-6.5; methanol concentration added daily, 3.0%; inoculum density, OD600=60; induction time point, 72-96 h）, the yield of soluble p293 was approximately 80 mg/L. We also observed p293 secretory expressed in P. pastoris to be 30 nm viral like particles by using electron microscopy. These results show that the p293 may has utility in the analysis of cell specific factors in the protein processing and assembly of HEV, and serve as a useful antigen for both diagnostic and vaccine purposes.

Review on hepatitis B virus precore/core promoter mutations and their correlation with genotypes and liver disease severity

Of 350 million people worldwide are chronically infected with hepatitis B virus(HBV)and are at risk of developing cirrhosis and hepatocellular carcinoma(HCC)later in life.HBV is the most diverse DNA virus,and its genome is composed of four open reading frames:Presurface antigen/surface antigen gene(preS/S),precore/core gene(preC/C),polymerase gene(P),and theχgene(χ).HBV produces quasispecies naturally or in response to antiviral agents because of the absence of proofreading activity amid reverse transcription and a high replication rate.The virus has 10 genotypes(A to J)with different geographical distributions.There are various HBV mutations in the HBV genome,including preC/C mutations,preS/S mutations,P gene mutations,andχgene mutations.The core promoter region plays a vital part in the replication,morphogenesis and pathogenesis of the virus.The precore region also plays a crucial role in viral replication.Both core promoter and precore mutations rescue the virus from host immune surveillance and result in the formation of mutated strains that may have altered pathogenicity.preC/C mutations are associated with liver disease progression.Precore mutations stop hepatitis B e antigen(HBeAg)production and basal core promoter mutations downregulate HBeAg production.Mutations in the basal core promoter are also associated with increased HBV replication and an increased incidence of advanced liver diseases such as cirrhosis and HCC.The emergence of antiviral-resistant mutations is the main reason for treatment failure.This review focuses mainly on preC/C promoter mutations and their correlation with genotypes and liver disease severity.Thorough perception and knowledge of HBV genetic variety and mutants could be vital to discover techniques for the prognosis and control of HBV infection.

A truncated hepatitis E virus ORF2 protein expressed in tobacco plastids is immunogenic in mice

瞄准：表示 23-ku pE2 到花费有效地，肝炎 E 病毒(HEV ) 的 3'-portion 包括的 E2 碎片编码的最有希望的子单元疫苗打开读物框架 2 (ORF2 ) 在烟草的质体(Nicotiana 烟 cv。SR1 ) ，在质体调查 transgene 表示和 pE2 累积，并且评估反在老鼠的导出质体的 pE2 的遗传因子的效果。方法：包含米饭 psbA 倡导者驾驶的 E2 碎片的指向质体的向量 pRB94-E2 被构造。在进烟草质体的交货之上，这构造能在质体染色体在 psaB-trnfM 和 trnG-psbC 碎片开始相应再结合，并且导致在二碎片之间插入的 transgene。pRB94-E2 与一个 biolistic 粒子轰炸方法被交付，并且转变质体的工厂被获得在补充 spectinomycin 的媒介上跟随炮轰的叶纸巾的新生。改革工厂的 Transplastomic 地位被 PCR 和南部的污点分析证实， transgene 表示被北污点分析调查，并且 pE2 的累积被 ELISA 测量。而且，蛋白浸出物被用来使老鼠免疫，并且在使免疫的老鼠的浆液样品的 pE2 反应的抗体的存在被 ELISA 学习。结果：PCR 和南部的污点分析证实的 Transplastomic 线能活跃地抄录 E2 mRNA。pE2 多肽象 13.27 microg/g 一样高被积累到水平新鲜叶子。pE2 能刺激使免疫的老鼠产生 pE2 特定的抗体。结论：HEV-E2 碎片能被插入到质体染色体，导出的 recombinant pE2 抗原是在老鼠遗传因子的反。因此，质体可以是为 HEV 疫苗的划算的生产的新奇来源。

High-dose hepatitis B immunoglobulin therapy in hepatocellular carcinoma with hepatitis B virus-DNA/hepatitis B e antigen-positive patients after living donor liver transplantation

AIM: To investigate the impact of high-dose hepatitis B immunoglobulin(HBIG) on hepatocellular carcinoma(HCC) and hepatitis B virus(HBV) recurrence and overall survival after living donor liver transplantation(LDLT).METHODS: We investigated 168 patients who underwent LDLT due to HCC, and who were HBV-DNA/hepatitis B e antigen(HBe Ag)-positive, from January 2008 to December 2013. After assessing whether the patients met the Milan criteria, they were assigned to the low-dose HBIG group and high-dose HBIG group. Using the propensity score 1:1 matching method, 38 and 18 pairs were defined as adhering to and not adhering to the Milan criteria. For each pair, HCC recurrence, HBV recurrence and overall survival were analyzed by the Kaplan-Meier method and the log rank test according to the HBIG dose. RESULTS: Among those who met the Milan criteria, the 6-mo, 1-year, and 3-year HCC recurrence-free survival rates were 88.9%, 83.2%, and 83.2% in the low-dose HBIG group and 97.2%, 97.2%, and 97.2% in the high-dose HBIG group, respectively(P = 0.042).In contrast, among those who did not meet the Milan criteria, HCC recurrence did not differ according to the HBIG dose(P = 0.937). Moreover, HBV recurrence and overall survival did not differ according to the HBIG dose among those who met(P = 0.317 and 0.190, respectively) and did not meet(P = 0.350 and 0.987, respectively) the Milan criteria. CONCLUSION: High-dose HBIG therapy can reduce HCC recurrence in HBV-DNA/HBe Ag-positive patients after LDLT.