乙型肝炎与戊型肝炎病毒重叠感染者血清HBV标记物结果分析

目的：观察乙型肝炎病毒（ＨＢＶ）与戊型肝炎病毒（ＨＥＶ）重叠感染对ＨＢＶ复制的影响。方法：以血清ＨＢｓＡｇ和抗ＨＥＶ均阳性患者３６例作为重叠组,以单纯ＨＢＶ感染患者３６例作为对照组。两组病例抗ＨＡＶ－ＩｇＭ和抗ＨＣＶ均为阴性。采用ＥＬＩＳＡ法测定两组的ＨＢＶ－Ｍ。结果：重叠组血清ＨＢｅＡｇ的阳性率明显低于对照组（Ｐ＜０．００１）,抗ＨＢｅ阳性率明显高于对照组（Ｐ＜０．０５）。结论：ＨＢＶ与ＨＥＶ重叠感染后,ＨＢＶ的复制受到一定程度的抑制     

Antibody to hepatitis B virus induced by injecting antibodies to the idiotype

Anti-idiotype reagents that recognize a common idiotype associated with antibody to hepatitis B surface antigen (anti-HBs) were used to induce anti-HBs in mice. The anti-idiotype-induced anti-HBs was found to recognize the group-specific a determinant of hepatitis B surface antigen and to express an interspecies idiotype. These findings suggest that anti-idiotypes may be useful as vaccines or vaccine primers.     

用PCR和ELISA方法分析奶牛血清和奶中类乙型肝炎病毒

用人乙型肝炎病毒（ＨＢＶ）表面抗原（ＨＢｓＡｇ）和ｅ抗原（ＨＢｅＡｇ）ＥＬＩＳＡ试剂盒检测８５份奶牛血清和９３份奶样。结果测出３３份血清为ＨＢｓＡｇ阳性，其中４价亦为ＨＢｅＡｇ阳性；２２份奶样为ＨＢｓＡｇ阳性，其中１１份亦为ＨＢｓＡｇ阳性。７个双阳性的样本，用针对ＨＢＶ表面抗原基因不同部位的两对引物作ＰＣＲ分析，结果未能扩增出特异片段，排除了奶牛ＨＢＶ血清学阳性是由人ＨＢＶ感染所致。     

健康人群乙型肝炎病毒感染血清标志模式及免疫状况的分析

目的:了解健康人群HBV感染及获得性免疫的状况,为更好的防治乙型肝炎提供客观依据.方法:采用ELISA法检测健康体检人群血清中HBsAg、HBsAb、HBeAg、HBeAb、HBcAb五项指标并对结果进行统计学分析.结果:8995例健康人群血清中HBV抗原抗体系统五项指标阴性者为48.4%,抗-HBs阳性者为44.7%,感染指标阳性者为6.9%,出现10种感染模式:五项指标均阴性;抗-HBs阳性;HBsAg阳性;抗-HBe阳性;HBsAg、HBeAg、HBcAb三项阳性;HBsAg、HBeAb、HBcAb三项阳性;HBsAg、HBeAb两项阳性;HBsAb、HBeAb、HBcAb三项阳性;HBeAb、HBcAb两项阳性和HBsAg、HBcAb两项阳性.结论:健康人群HBV感染近年来明显下降而免疫力明显上升,但尚有近1/2的人缺乏免疫力,并存在少数传染性强的乙型肝炎患者;应提倡健康人群普种乙肝疫苗,即使注射了乙肝疫苗也要注意自身一般性预防,常规检测乙肝五项指标,及时发现病情及早治疗.     

Specific expression of hepatitis B surface antigen (HBsAg) in transgenic mice

Two transgenic mice were obtained that contain in their chromosomes the complete hepatitis B virus (HBV) genome except for the core gene. These mice secrete particles of HBV surface antigen (HBsAg) in the serum. In one mouse, HBV DNA sequences that had integrated at two different sites were shown to segregate independently in the first filial generation (F1) and only one of the sequences allowed expression of the surface antigen. Among these animals the males produced five to ten times more HBsAg than the females. A 2.1-kilobase messenger RNA species comigrating with the major surface gene messenger RNA is expressed specifically in the liver in the two original mice. The results suggest that the HBV sequences introduced into the mice are able to confer a tissue-specific expression to the S gene. In addition, the HBV transgenic mice represent a new model for the chronic carrier state of hepatitis B virus infection.     

Antibodies to hepatitis B surface antigen potentiate the response of human T lymphocyte clones to the same antigen

Human t-helper lymphocyte clones specific for hepatitis B virus surface antigen (HBsAg) proliferate on stimulation with HBsAg in vitro. Antibodies specific for HBsAg, but no other antibodies, augment this proliferative response. In the presence of antibodies to HBsAg, the maximum response could be achieved at HBsAg concentrations that were 1 percent of those required in the absence of the antibodies. These findings suggest that antigen-specific antibodies exert regulatory controls on T cells that recognize the same antigens.     

A poliovirus neutralization epitope expressed on hybrid hepatitis B surface antigen particles

The hepatitis B virus (HBV) envelope protein carrying the surface antigen (HBsAg) is assembled with cellular lipids in mammalian cells into empty viral envelopes. In a study to evaluate the capacity of such particles to present foreign peptide sequences in a biologically active form, in-phase insertions were created in the S gene encoding the major envelope protein. One of the sequences inserted was a synthetic DNA fragment encoding a poliovirus neutralization epitope. Mammalian cells expressing the modified gene secreted hybrid particles closely resembling authentic 22-nanometer HBsAg particles. These particles reacted with a poliovirus-specific monoclonal antibody and induced neutralizing antibodies against poliovirus. The results indicate that empty viral envelopes of HBV may provide a means for the presentation of peptide sequences and for their export from mammalian cells.     

Enhanced immunogenicity of the pre-S region of hepatitis B surface antigen

The 55 codons upstream of the gene sequence encoding the hepatitis B surface antigen (HBsAg) are called the pre-S(2) region. It has been proposed that polypeptides of high molecular weight that contain the pre-S(2) region should be included in future hepatitis B virus (HBV) vaccines. The pre-S(2) region and the S gene product [25 kilodalton (kD)] together compose a polypeptide of high molecular weight (33 kD). As an initial attempt to determine the relevance of the 33-kD polypeptide to development of an HBV vaccine, the murine immune response to pre-S(2)-encoded determinants as compared to S-encoded determinants on the same polypeptide was examined. The results indicate (i) the pre-S(2) region is significantly more immunogenic than the S region of HBsAg, (ii) the 26 amino acid residues at the NH2-terminus of the 33-kD polypeptide represent a dominant antibody binding site on the pre-S(2) region, (iii) the immune response to the pre-S(2) region is regulated by H-2-linked genes distinct from those that regulate the response to the S region, and (iv) immunization of an S region nonresponder strain with HBV envelope particles that contain both the pre-S(2) and S regions can circumvent nonresponsiveness to the S region.     

An assay for circulating antibodies to a major etiologic virus of human non-A, non-B hepatitis

A specific assay has been developed for a blood-borne non-A, non-B hepatitis (NANBH) virus in which a polypeptide synthesized in recombinant yeast clones of the hepatitis C virus (HCV) is used to capture circulating viral antibodies. HCV antibodies were detected in six of seven human sera that were shown previously to transmit NANBH to chimpanzees. Assays of ten blood transfusions in the United States that resulted in chronic NANBH revealed that there was at least one positive blood donor in nine of these cases and that all ten recipients seroconverted during their illnesses. About 80 percent of chronic, post-transfusion NANBH (PT-NANBH) patients from Italy and Japan had circulating HCV antibody; a much lower frequency (15 percent) was observed in acute, resolving infections. In addition, 58 percent of NANBH patients from the United States with no identifiable source of parenteral exposure to the virus were also positive for HCV antibody. These data indicate that HCV is a major cause of NANBH throughout the world.     

Viral clearance without destruction of infected cells during acute HBV infection

Viral clearance during hepatitis B virus (HBV) infection has been thought to reflect the destruction of infected hepatocytes by CD8(+) T lymphocytes. However, in this study, HBV DNA was shown to largely disappear from the liver and the blood of acutely infected chimpanzees long before the peak of T cell infiltration and most of the liver disease. These results demonstrate that noncytopathic antiviral mechanisms contribute to viral clearance during acute viral hepatitis by purging HBV replicative intermediates from the cytoplasm and covalently closed circular viral DNA from the nucleus of infected cells.     

人、猪和鸡血清中抗禽戊型肝炎病毒抗体的检测与鉴定

通过对人、猪和鸡血清中的抗禽戊型肝炎病毒(hepatitis E virus, HEV)ORF2抗体的检测以及对阳性血清抗体特异性的鉴定,本研究结果表明,人、猪和禽血清中抗禽HEV ORF2抗体阳性率为0.91%～62.5%.因为禽与哺乳动物HEV ORF2抗原具有共有的抗原表位,而阳性血清中抗禽HEV ORF2特异性抗原表位抗体的阳性率为41.3%～92.1%,提示人、猪和鸡都有感染禽HEV的可能性,这为进一步验证禽HEV可能造成的人兽共感染提供新的科学依据.     

HCV persistence and immune evasion in the absence of memory T cell help

Spontaneous resolution of hepatitis C virus (HCV) infection in humans usually affords long-term immunity to persistent viremia and associated liver diseases. Here, we report that memory CD4+ Tcells are essential for this protection. Antibody-mediated depletion of CD4+ Tcells before reinfection of two immune chimpanzees resulted in persistent, low-level viremia despite functional intra-hepatic memory CD8+ Tcell responses. Incomplete control of HCV replication by memory CD8+ Tcells in the absence of adequate CD4+ Tcell help was associated with emergence of viral escape mutations in class I major histocompatibility complex-restricted epitopes and failure to resolve HCV infection.     

Amino acid homology between the encephalitogenic site of myelin basic protein and virus: mechanism for autoimmunity

Amino acid sequence homology was found between viral and host encephalitogenic protein. Immune responses were then generated in rabbits by using the viral peptide that cross-reacts with the self protein. Mononuclear cell infiltration was observed in the central nervous systems of animals immunized with the viral peptide. Myelin basic protein (MBP) is a host protein whose encephalitogenic site of ten amino acids induces experimental allergic encephalomyelitis. By computer analysis, hepatitis B virus polymerase (HBVP) was found to share six consecutive amino acids with the encephalitogenic site of rabbit MBP. Rabbits given injections of a selected eight- or ten-amino acid peptide from HBVP made antibody that reacted with the predetermined sequences of HBVP and also with native MBP. Peripheral blood mononuclear cells from the immunized rabbits proliferated when incubated with either MBP or HBVP. Central nervous system tissue taken from these rabbits had a histologic picture reminiscent of experimental allergic encephalomyelitis. Thus, viral infection may trigger the production of antibodies and mononuclear cells that cross-react with self proteins by a mechanism termed molecular mimicry. Tissue injury from the resultant autoallergic event can take place in the absence of the infectious virus that initiated the immune response.     

鸭病毒性肝炎病原分离鉴定与防治研究

从三门峡某鸭场疑似鸭病毒性肝炎的发病或病死肉雏鸭的肝脏中分离到 1株病毒 ,经电镜观察、雏鸭保护试验、鸭胚中和试验鉴定为血清 型鸭病毒性肝炎病毒。从而证明引起雏鸭大量发病和死亡的主要原因为鸭病毒性肝炎所致。用鸭病毒性肝炎标准毒株多次强化免疫家兔 ,获得兔抗鸭病毒性肝炎高免血清 ,用此高免血清给发病雏鸭紧急注射 ,获得了满意的治疗效果     

猪类乙型肝炎病毒的血清学检测及其S和C基因的克隆与分析

对甘肃省兰州市周边地区采集的300份猪血清的表面抗原(HBsAg)、E抗原(HBeAg)、E抗体(抗-HBe)、核心抗体(抗-HBc)、表面抗体(抗-HBs)5项指标进行检测,并从HBsAg和抗-HBs抗体阳性血清中提取DNA,进行乙型肝炎病毒(HBV)S基因和C基因的巢式PCR扩增、克隆、测序和序列分析.结果表明:血清学检测的上述5项指标阳性率分别为10.7%3、.0%、6.1%、3.3%和88.5%,大三阳(即HBsAg、HBeAg、抗-HBs抗体均为阳性)的猪血清样本6份,阳性率为2%;序列分析表明,猪类HBV的S基因和C基因与GenBank中的人HBV同源性达到99%,说明猪存在类HBV感染,且猪类HBV与人HBV存在相关关系.     

ELISA法与FQ-PCR对HBV三种血清标志物检测结果的比较

为了探讨与评价联合检测乙型肝炎HBeAg和Pre-S1Ag、HBV-DNA等血清免疫标志物在乙型病毒性肝炎临床诊断、治疗中的意义,利用酶联免疫吸附试验（ELISA）和荧光实时定量聚合酶链式反应（FQ-PCR）,对619例疑似或确诊乙肝患者的血清样本分别进行乙肝免疫标志物HBeAg、Pre-S1Ag和HBV-DNA的联合检测。结果表明,血清乙肝免疫标志物HbeAg检测为阴性时,不能完全表明患者乙肝病毒复制终止或病毒血症的消失;血清Pre-S1Ag检测结果有助于乙型肝炎的早期诊断,也可以作为乙肝病毒DNA复制的指标之一;而FQ—PCR检测血清HBVDNA结果则有助于乙型肝炎病毒的抗原或抗体血清滴度较低时肝炎的诊断。     

硫酸铵浓度对疏水作用层析纯化重组乙肝表面抗原的影响

以表面抗原活性收率和纯度为考察指标,转化乙肝病毒preS2-S基因重组酵母株,经甘油培养基充分增殖,转移到甲醇培养基中诱导表达.破碎细胞收集细胞原液,离心15000 r·min-1除去细胞碎片,超滤浓缩.结果表明,硫酸铵0.12、0.16 mol·L-1时盐浓度低,HBsAg未结合到疏水介质上;硫酸铵0.24 mol·L-1时疏水性强, HBsAg不宜从介质上洗脱.表明0.20 mol·L-1硫酸铵更适合重组HBsAg纯化.     

HLA and NK cell inhibitory receptor genes in resolving hepatitis C virus infection

Natural killer (NK) cells provide a central defense against viral infection by using inhibitory and activation receptors for major histocompatibility complex class I molecules as a means of controlling their activity. We show that genes encoding the inhibitory NK cell receptor KIR2DL3 and its human leukocyte antigen C group 1 (HLA-C1) ligand directly influence resolution of hepatitis C virus (HCV) infection. This effect was observed in Caucasians and African Americans with expected low infectious doses of HCV but not in those with high-dose exposure, in whom the innate immune response is likely overwhelmed. The data strongly suggest that inhibitory NK cell interactions are important in determining antiviral immunity and that diminished inhibitory responses confer protection against HCV.     

兔出血症病毒衣壳蛋白VP60的原核表达及多克隆抗体制备

【目的】构建兔出血症病毒衣壳蛋白VP60基因的原核表达载体,进行原核表达并制备其蛋白抗体。【方法】以保存的VP60基因的质粒(pTeasy-VP60)为模版,用PCR方法获得VP60基因,并将其克隆到pET28a(+)载体上,构建重组表达载体pET28a(+)-VP60,酶切鉴定和测序验证后转入大肠杆菌BL21(DE3)中进行诱导表达,优化诱导表达时间和IPTG浓度;利用镍离子螯合层析纯化重组蛋白,并制备了高效价的抗VP60多克隆抗体。【结果】成功构建了兔出血症病毒衣壳蛋白VP60的原核表达质粒pET28a(+)-VP60,其在大肠杆菌中以包涵体形式高效表达,重组蛋白分子质量为60 ku,制备的多克隆抗体具有较强的免疫结合活性。【结论】成功实现了VP60基因的原核表达,制备了重组蛋白VP60的多克隆抗体,为进一步的VP60转基因研究奠定了基础。