中国青海地区喜马拉雅旱獭嗜肝病毒自然感染的组织学研究

应用原位杂交技术、免疫组化技术以土拨鼠肝炎病毒(Woodchuck hepatitis virus,WHV)的检测系统检测50份喜马拉雅旱獭肝组织可能存在的嗜肝病毒c基因、s抗原及c抗原的表达,同时检测肝脏组织病理学改变.结果显示旱獭肝组织中嗜肝病毒s抗原、c抗原的阳性率分别为26%(13/50)、36%(18/50);在抗原双阳性的10份肝组织标本中有c基因的阳性表达,阳性率为50%.c抗原定位于肝细胞胞浆和/或胞核,呈散在、片簇状分布,c基因定位于肝细胞的细胞核,阳性细胞散在分布.50份标本中5份出现肝炎的病理改变,与抗原检出问无明显相关性.使用WHV的病毒检测系统证实青海地区喜马拉雅旱獭可能存在类似WHV的嗜肝病毒感染,从组织学的角度为中国青海地区喜马拉雅旱獭嗜肝病毒自然感染提供证据,此种动物有可能用于建立嗜肝病毒感染的动物模型.     

西伯利亚旱獭中类人乙肝病毒的研究

对111份西伯利亚旱獭血清标本进行血清学,形态和组织病理学检测,HBsAg阳性22份,阳性率19.8％;抗-HBs1份,阳性率0.9％;HBV-DNA核酸杂交,阳性斑点16份,阳性率14.4％。IEM观察在3份标本中发现以22—24nm球形颗粒为主,其中有42—45mm的Dane样颗粒。20.7％的肝脏标本有组织病理改变,其中19份为急性肝类,4份为慢性肝炎。超薄切片肝细胞核内有20nm左右的HBeAg颗粒,结果进一步证实我国西伯利亚旱獭中有嗜肝病毒感染。     

啮齿类动物中汉坦病毒感染血清学和病原学检测方法比较研究

致病性汉坦病毒的宿主主要为啮齿类动物,其病毒感染状况是人间疫情发生的关键影响因素,可通过检测宿主动物标本中病毒基因组RNA、蛋白抗原及特异性抗体而进行监测。本研究利用367份鼠肺及鼠血标本,对双抗原夹心ELISA(ELISA)、实时荧光RT-PCR(RT-PCR)和免疫荧光(IFA)等三种分别检测抗体、核酸和抗原的方法进行比较评估。ELISA法检出抗体阳性鼠血标本46份,阳性率为12.53%;RT-PCR法检出病毒RNA阳性鼠肺标本28份,阳性率为7.63%;IFA检出抗原阳性鼠肺标本24份,阳性率为6.54%。宿主动物组织标本中检出汉坦病毒RNA和(或)结构蛋白抗原的标本,对应的血液标本中可检出病毒特异性抗体,100%(24/24)IFA检测阳性标本和89.3%(25/28)RT-PCR检测阳性标本对应血标本ELISA抗体检测阳性,反之亦然,检出抗体的标本基本包含了可检出抗原和RNA的标本。RT-PCR与IFA检测结果差异无显著性(χa2=0.64,P0.05),一致性检验Kappa系数为0.71,一致性高(Z=13.66,P0.05),首先对血标本开展基于ELISA的特异性抗体检测,可显著缩小RT-PCR或IFA法检测病毒RNA或抗原的范围(χb2=12.04,χc2=20.05,P0.05)。本研究为宿主动物汉坦病毒感染实验室监测方案优化提供了有益的依据。     

喜马拉雅旱獭制动性麻醉方法

目的将盐酸氯胺酮用于喜马拉雅旱獭的麻醉,探讨不同剂量麻醉效果,为合理使用提供参考。方法将30只喜马拉雅旱獭随机分为3组,按15、45、90mg/kg体重给予盐酸氯胺酮,行后肢肌肉注射麻醉,观察临床表现和诱导时间及麻醉时间。结果给予盐酸氯胺酮后喜马拉雅旱獭分别在402.0±60.7s、372.0±72.0s、222.0±66.0s进入麻醉状态,维持时间分别为558.0±96.0s、1344.0±264.0s、3366.0±696.0s。随剂量的增加,麻醉时间相应增加。结论盐酸氯胺酮对喜马拉雅旱獭进行肌肉注射麻醉是有效和安全的,在应用中还可以根据实验需要选择3种剂量进行麻醉,为喜马拉雅旱獭得到广泛应用奠定了基础。     

肝癌中HBV和HCV基因和抗原的分布及意义

采用原位分子杂交方法检测HCV RNA及HBV X基因;采用免疫组织化学方法研究HCV核心抗原,非结构区C33c抗原及HBxAg在肝细胞肝癌中的定位及分布。结果表明（1）HCV RNA、HBV X基因在肝细胞肝癌组织检出率分别为40％（55／136）和82％（112／136）。HCV RNA定位于癌细胞的胞浆内,阳性细胞呈散在、灶状及弥漫分布三种形式;HBV X基因在肝癌细胞中的分布呈胞浆型、核型及核浆型,阳性细胞也呈上述三种分布形式;（2）HCV C33c抗原、核心抗原在肝细胞肝癌中的阳性率为81％（133／164）及86％（141／164）。C33c抗原定位于癌细胞及肝细胞的浆内;核心抗原毁定位于癌细胞核中,又可定位于胞浆中。C33c抗原阳性细胞以灶状分布为主;而核心抗原阳性细胞在癌组织以弥漫核阳性常见,在癌旁肝组织以胸浆阳性为主;（3）HBxAg在肝细胞肝 癌中的检出率为75％（123／164）,C33c和HBxAg二者同时阳性占63％（103／164）。HCV感染在我国肝细胞肝癌中比较普遍,HCV和HBV重叠感染占相当比例,可能在肝细胞肝癌的发生中起着重要作用。     

一种HBV研究的新动物模型-喜马拉雅旱獭β-actin基因的克隆及序列分析

目的 喜马拉雅旱獭对土拨鼠肝炎病毒（woodchuck hepatitis virus,WHV）高度易感,可作为HBV感染的新动物模型.本研究对喜马拉雅旱獭β肌动蛋白（β-actin）的部分cDNA序列进行了克隆和序列分析,为喜马拉雅旱獭在HBV感染研究中的应用奠定基础.方法 根据Genbank的土拨鼠β-actin cDNA的序列设计特异性引物,提取旱獭脾组织总RNA作为模板,RT-PCR扩增旱獭β-actin cDNA序列;PCR产物纯化后连接至T载体（pMD18-T）,构建重组质粒pMD18-T-mhActin.对重组质粒进行PCR初筛及酶切鉴定,选择阳性克隆测序;对所获得的序列进行同源性和种系进化分析.结果 获得的旱獭β-actin序列为349 bp（nt887～1 235）,其中编码序列为323 bp（nt-887～1209）,编码106个氨基酸,包含形成二硫键的2个丝氨酸位点（氨基酸16和氨基酸105）及与β-actin功能相关的2个ATP结合位点、6个凝溶胶蛋白结合位点和6个profilin结合位点.同源性分析发现上述序列与其它哺乳动物β-actin的同源性均高达88 %以上,与土拨鼠β-actin的同源性最高（99.69 %）,其氨基酸序列的同源性为100 %.种系进化树分析提示喜马拉雅旱獭β-actin与土拨鼠β-actin的亲缘关系最近,其次为其它啮齿类动物.结论 成功克隆了喜马拉雅旱獭β-actin的部分序列.序列分析发现喜马拉雅旱獭β-actin与土拨鼠β-actin的同源性最高.     

喜马拉雅旱獭血液生化指标测定

本文首次测定了人工饲养条件下喜马拉雅旱獭Marmota himalayana的血液生化值。结果显示雄性喜马拉雅旱獭谷丙转氨酶(ALT)和谷草转氨酶(AST)水平显著高于雌性(P<0.05);人工喂养1年后,喜马拉雅旱獭血清谷丙转氨酶(ALT)、总蛋白(TP)、白蛋白(ALB)和肌酐(CREA)水平降低,提示饲料中可适当增加蛋白的含量;冬眠后血清总蛋白(TP)含量明显下降,白蛋白水平略低于冬眠前。不同性别、年龄、不同饲养时间喜马拉雅旱獭其他指标差异均无统计学意义,说明建立的喜马拉雅旱獭人工饲养管理方法可保证动物血液生化指标的稳定性,所测血液生化指标值也可作为喜马拉雅旱獭饲养管理和疫病检测判断的参考依据。     

喜马拉雅旱獭种群微卫星变异及遗传多样性

为了解不同地理种群喜马拉雅旱獭(Marmota himalayana)的种群数量变化并探讨其内在的遗传学机制,通过构建部分基因组文库的方法筛选出8个高变异微卫星位点,根据微卫星位点的测序结果设计相应引物,PCR扩增检测了青藏高原4个地理种群(德令哈、乌兰、沱沱河、安多)喜马拉雅旱獭的遗传多态性及其种群结构.研究结果显示:8个位点在喜马拉雅旱獭种群中均为高度多态,观察等位基因数、有效等位基因数、多态信息含量分别为4.75、3.033 2、0.610 2;喜马拉雅旱獭种群总的期望杂合度和观察杂合度分别为0.670、0.699;3个喜马拉雅旱獭种群显著(P＜0.05)或极显著(P＜0.01)偏离H-W平衡状态,且这些偏离平衡的位点均由杂合过度导致(FIS＜0);喜马拉雅旱獭种群的部分位点已经偏离了突变-漂移平衡.结论:筛选出的8个微卫星位点适合于喜马拉雅旱獭种群遗传多样性的研究,所研究的喜马拉雅旱獭种群有较高的遗传多样性,安多地理种群在近期可能经历过瓶颈效应,种群数量曾经下降.     

喜马拉雅旱獭血液生理指标测定

首次测定了45只人工饲养条件下喜马拉雅旱獭的血液生理值.结果显示人工饲养条件下除淋巴细胞和中性粒细胞百分率因动物年龄不同而有显著性差异外(P<0.05),不同性别、不同饲养时间喜马拉雅旱獭的白细胞数、白细胞分类计数百分率、红细胞、血小板、血红蛋白和平均血红蛋白浓度等7项指标都没有显著性差异,说明建立的旱獭人工饲养管理方法可保证动物的血液生理指标的稳定性,所测血液生理指标也可作为喜马拉雅旱獭饲养管理和疫病检测判断的参考依据.     

ELISA法检测流行性腮腺炎病毒感染特异性IgM抗体的研究

用流行性腮腺炎(流腮)病毒Enders株接种鸡胚尿囊腔培养,尿囊液经聚乙二醇6000处理制备流腮病毒抗原,用ELISA法检测流腮患者血清中特异性IgM抗体,其敏感性,特异性、重复性和稳定性都很高。 79份流腮患者血清,检出特异性IgM72份,阳性率为91％,32例非流腮患者IgM全部阴性、两者有极显著差异(P<0.01)。 10份血清作血清倍比稀释至1∶3200测IgM仍全部阳性,1∶6400稀释仅1例阴性,1∶12800稀释5例中仍有2例阳性。 10份血清作流腮抗原特异性抗体阻断试验,光密度抑制率均大于50％,平均为87％,10份标本作2-ME和SPA阻断后检测IgM抗体,结果2-ME阻断标本全部阴转,而SPA阻断标本仍阳性,证实所检测为流腮特异性抗体。 24份标本2次重复检测流腮IgM,其阴、阳性结果一致,这期间抗原放4℃ 1个月,提示抗原的稳定性和方法的重复性都很好。本方法敏感性明显高于血凝抑制试验,其阳性率分别为91％和61％,两者有显著差异。而且所用试剂简单经济,操作简便,快速,适用于临床早期诊断,易于广泛推广应用。     

A new hepadnavirus endemic in arctic ground squirrels in Alaska.

We present evidence for a novel member of the hepadnavirus family that is endemic in wild arctic ground squirrels (Spermophylus parryi kennicotti) in Alaska. This virus, designated arctic squirrel hepatitis virus (ASHV), was initially detected in the livers of animals bearing large hepatic nodules by nucleic acid hybridization with hepadnavirus probes and in plasma by cross-reactivity with antibodies to hepadnavirus surface and core antigens. The complete nucleotide sequence of the 3,302-bp-long ASHV genome was determined and compared with those of ground squirrel hepatitis virus (GSHV) and woodchuck hepatitis virus (WHV); all sequences were organized into four open reading frames, designated pre-C/C, pre-S/S, pol, and X. Despite roughly equivalent variability among the three rodent hepadnaviruses (around 16% base and 19% amino acid exchanges), ASHV appeared to be more closely related to GSHV than to WHV in phylogenetic analysis. Accordingly, preliminary studies of the pathology of ASHV infection suggested that ASHV may be a less efficient oncogenic agent than WHV. About one-third of aged animals maintained in captivity, including virus-infected as well as uninfected squirrels, developed large liver nodules, consisting of hepatocellular adenomas or carcinomas or nonmalignant lesions characterized by drastic microvesicular steatosis. ASHV-infected arctic ground squirrels may serve as a new model with which to analyze the contribution of hepadnavirus- and host-specific determinants to liver pathology and tumorigenesis.     

Replication of naturally occurring woodchuck hepatitis virus deletion mutants in primary hepatocyte cultures and after transmission to naive woodchucks

Woodchuck hepatitis virus (WHV) mutants with core internal deletions (CID) occur naturally in chronically WHV-infected woodchucks, as do hepatitis B virus mutants in humans. We studied the replication of WHV deletion mutants in primary woodchuck hepatocyte cultures and in vivo after transmission to naive woodchucks. By screening 14 wild-caught, chronically WHV-infected woodchucks, two woodchucks, WH69 and WH70, were found to harbor WHV CID mutants. Consistent with previous results, WHV CID mutants from both animals had deletions of variable lengths (90 to 135 bp) within the middle of the WHV core gene. In woodchuck WH69, WHV CID mutants represented a predominant fraction of the viral population in sera, normal liver tissues, and to a lesser extent, in liver tumor tissues. In primary hepatocytes of WH69, the replication of wild-type WHV and CID mutants was maintained at least for 7 days. Although WHV CID mutants were predominant in fractions of cellular WHV replicative intermediates, mutant covalently closed circular DNAs (cccDNAs) appeared to be a small part of cccDNA-enriched fractions. Analysis of cccDNA-enriched fractions from liver tissues of other woodchucks confirmed that mutant cccDNA represents only a small fraction of the total cccDNA pool. Four naive woodchucks were inoculated with sera from woodchuck WH69 or WH70 containing WHV CID mutants. All four woodchucks developed viremia after 3 to 4 weeks postinoculation (p.i.). They developed anti-WHV core antigen (WHcAg) antibody, lymphoproliferative response to WHcAg, and anti-WHV surface antigen. Only wild-type WHV, but no CID mutant, was found in sera from these woodchucks. The WHV CID mutant was also not identified in liver tissue from one woodchuck sacrificed in week 7 p.i. Three remaining woodchucks cleared WHV. Thus, the presence of WHV CID mutants in the inocula did not significantly change the course of acute self-limiting WHV infection. Our results indicate that the replication of WHV CID mutants might require some specific selective conditions. Further investigations on WHV CID mutants will allow us to have more insight into hepadnavirus replication.     

In vitro model for the nuclear transport of the hepadnavirus genome.

Hepadnaviruses contain a DNA genome, but they replicate via an RNA intermediate, synthesized by the cellular RNA polymerase II in the nucleus of the infected cell. Thus, nuclear transport of the viral DNA is required in the viral life cycle. Protein-free DNA is only poorly imported into the nucleus, so one or more of the viral proteins must be involved in the transport of the viral genome. In order to identify these viral proteins, we purified woodchuck hepadnavirus (WHV) core particles from infected woodchuck liver, isolated WHV DNA, and extracted the covalent complex of viral polymerase from the particles using urea. Intact core particles, the polymerase-DNA complex, or protein-free WHV DNA from core particles was added to digitonin-permeabilized HuH-7 cells, in which the cytosol was substituted by rabbit reticulocyte lysate (RRL) and an ATP-generating system. The distribution of the viral genome was analyzed by semiquantitative PCR or by hybridization in total nuclei, RRL, nuclear membranes, and nucleoplasm. The polymerase-DNA complex was efficiently transported into the nucleus, as indicated by the resistance of the nucleus-associated DNA to a short-term treatment with DNase I of the intact nuclei. The DNA within core particles stayed mainly in the cytosol and remained protected against DNase I. A minor part of the encapsidated DNA was bound to nuclei. It was protected against DNase I but became accessible after disruption of the nuclei. Deproteinized viral DNA completely remained in the cytosol. These data show that the viral polymerase is probably sufficient for mediating the transport of a hepadnavirus genome into the nucleus and that the viral core particles may release the genome at the nuclear membrane.     

Quantitative detection of hepadnavirus-infected lymphoid cells by in situ PCR combined with flow cytometry: implications for the study of occult virus persistence

The detection of small amounts of viral pathogens in infected cells by classical PCR is hampered by a partial loss of virus nucleic acid due to extraction and by difficulties in discrimination between truly intracellular virus genome material and that possibly adhered to the cell surface. These impediments limit reliable identification of virus traces within infected cells, which are typically encountered in latent and persistent occult infections. In this study, hepadnavirus-specific in situ PCR combined with the enzymatic elimination of extracellular virus and flow cytometry permitted detection of viral genomes in lymphoid cells without nucleic acid isolation and allowed quantification of infected cells during the course of persistent infection with woodchuck hepatitis virus (WHV). The validity of the procedure was confirmed by hybridization analysis of the in situ-amplified viral sequences. The results showed that hepadnavirus can be directly detected within lymphoid cells not only in serologically accountable infection, but also years after recovery from viral hepatitis and in the course of primary occult virus carriage. Percentages of infected peripheral lymphoid cells in symptomatic WHV hepatitis fluctuate between 3.4 and 20.4% (mean +/- standard error of the mean, 9.6% +/- 1.7%), whereas those in persistent, serologically mute WHV infection range from 1.1 to 14.6% (mean +/- standard error of the mean, 4.8% +/- 0.8%) (P = 0.005). The data obtained provide further evidence that WHV infection continues indefinitely in the lymphatic system independently of whether it is symptomatic or concealed. They document that hepadnavirus can be detected in a significant proportion of circulating lymphoid cells in both immunovirologically apparent as well as occult persistent infection.     

Persistence of the Recombinant Genomes of Woodchuck Hepatitis Virus in the Mouse Model

Hydrodynamic injection (HI) with a replication competent hepatitis B virus (HBV) genome may lead to transient or prolonged HBV replication in mice. However, the prolonged HBV persistence after HI depends on the specific backbone of the vector carrying HBV genome and the genetic background of the mouse strain. We asked whether a genetically closely related hepadnavirus, woodchuck hepatitis virus (WHV), may maintain the gene expression and replication in the mouse liver after HI. Interestingly, we found that HI of pBS-WHV1.3 containing a 1.3 fold overlength WHV genome in BALB/c mouse led to the long presence of WHV DNA and WHV proteins expression in the mouse liver. Thus, we asked whether WHV genome carrying foreign DNA sequences could maintain the long term gene expression and persistence. For this purpose, the coding region of HBV surface antigen (HBsAg) was inserted into the WHV genome to replace the corresponding region. Three recombinant WHV-HBV genomes were constructed with the replacement with HBsAg a-determinant, major HBsAg, and middle HBsAg. Serum HBsAg, viral DNA, hepatic WHV protein expression, and viral replication intermediates were detected in mice after HI with recombinant genomes. Similarly, the recombinant genomes could persist for a prolonged period of time up to 45 weeks in mice. WHV and recombinant WHV-HBV genomes did not trigger effective antibody and T-cell responses to viral proteins. The ability of recombinant WHV constructs to persist in mice is an interesting aspect for the future investigation and may be explored for in vivo gene transfer.     

In vitro and in vivo infectivity and pathogenicity of the lymphoid cell-derived woodchuck hepatitis virus

Woodchuck hepatitis virus (WHV) and human hepatitis B virus are closely related, highly hepatotropic mammalian DNA viruses that also replicate in the lymphatic system. The infectivity and pathogenicity of hepadnaviruses propagating in lymphoid cells are under debate. In this study, hepato- and lymphotropism of WHV produced by naturally infected lymphoid cells was examined in specifically established woodchuck hepatocyte and lymphoid cell cultures and coculture systems, and virus pathogenicity was tested in susceptible animals. Applying PCR-based assays discriminating between the total pool of WHV genomes and covalently closed circular DNA (cccDNA), combined with enzymatic elimination of extracellular viral sequences potentially associated with the cell surface, our study documents that virus replicating in woodchuck lymphoid cells is infectious to homologous hepatocytes and lymphoid cells in vitro. The productive replication of WHV from lymphoid cells in cultured hepatocytes was evidenced by the appearance of virus-specific DNA, cccDNA, and antigens, transmissibility of the virus through multiple passages in hepatocyte cultures, and the ability of the passaged virus to infect virus-naive animals. The data also revealed that WHV from lymphoid cells can initiate classical acute viral hepatitis in susceptible animals, albeit small quantities (approximately 10(3) virions) caused immunovirologically undetectable (occult) WHV infection that engaged the lymphatic system but not the liver. Our results provide direct in vitro and in vivo evidence that lymphoid cells in the infected host support propagation of infectious hepadnavirus that has the potential to induce hepatitis. They also emphasize a principal role of the lymphatic system in the maintenance and dissemination of hepadnavirus infection, particularly when infection is induced by low virus doses.