山东地区输血传播病毒(TTV)检测和部分基因克隆及序列测定

目的：了解TTV山东分离株感染状况及基因变异的情况。方法：应用逆转录聚合酶链反应（PCR）检测26例来自山东泰安、莱芜、济宁、菏泽、枣庄及临沂、日照地区非甲－庚型肝炎和12例肝细胞癌患者，其中HBV感染者5例，HCV感染者2例，HBV＋HCV＋HGV混合感染2例，HBV＋HCV＋TTV感染者1例，HGV＋TTV感染者1例，HEV＋HGV感染者1例。在这26例非甲－非庚型肝炎中有输血或输血制品、手术、拔牙、针灸、注射以及肝炎密切接触史17例。另12例肝癌中有输血、手术、拔牙、静脉注射史10例。检出血清中TTV DNA阳性扩增的产物利用PCR技术片段直接克隆和测序，分析其基因变异的情况。结果：26例非甲－庚型肝炎中11例TTVDNA阳性（42．3％）。对其中两株（TTVSD4、TTVSD5）部分基因克隆测序，并与日本株（ABOO8394）相比较，其核苷酸序列同源性分别为99．9％和100％。而12例肝细胞癌患者中3例TTVDNA阳性（25．0％），对其中一株（TTVSD6）部分基因克隆与测序，与日本株（ABOO8394）相比较，苷核苷酸序列同源性为99％；TTV山东株三株间核苷酸同源性均为99％。结论：本项研究证实山东地区非甲－庚型肝炎和肝细胞癌患者中存在着较高TTV感染、TTV感染可能具有嗜肝性，而且可能与肝功能损害有关，是引起非甲－庚型肝炎重要病原。     

输血传播病毒母婴传播的研究

目的：探讨深圳市孕产妇输血传播病毒（TTV）感染情况及其在母婴间的传播途径。方法：应用套式－聚合酶链反应（Nest－PCR）对400例正常孕产妇分娩前的血清及其新生儿的脐血血清标本、产妇的乳汁配对进行了TTV DNA检测，对阳性的母亲及其新生儿进行的6个月随访，并对其中13对TTV DNA阳性的母血、母乳及其婴儿的血，共做32株克隆，通过DNA序列测定比较分析母婴TTV感染株之间核苷酸的序列同源性。结果：在400对配对标本中，62例孕妇血清、4例新生儿脐血清、20例母乳检测出TTV DNA，孕母TTV感染率15．5％（62／400），出生时母婴TTV传播率6．5％（4／62），阳性母亲乳TTV DNA检出率32．2％（20／62），配对比较母婴间TTV感染株序列同源性为97．4％－99．8％，经6个月随访母血TTV DNA阳性的婴儿血TTV DNA由阴转阳率为38．1％（16／42）。 结论：TTV可通过围产期和哺乳期母婴传播的途径感染，对胎儿的危害及新生儿生长发育的影响值得重视。     

糖尿病患者庚型肝炎病毒感染及其临床意义

目的　了解糖尿病患者 (I型和II型 )庚型肝炎病毒 (HGV)感染情况及临床意义。方法　应用逆转录套式聚合酶链反应 (RT -NestedPCR)检测 183例糖尿病患者血清标本中的HGVRNA ,部分扩增产物进行核苷酸序列分析 ,所有患者均进行ALT检查。结果　I型和II型糖尿病患者血清标本中HGV检出阳性率分别为 4 6 7% (7/ 15 )和 2 0 2 % (34/ 16 8) ,总阳性率为 2 2 4 % (41/ 183) ;5例HGV 5’ -NCR和NS3区扩增产物与文献报道的核苷酸序列同源性分别为 87 2 %～ 89 4 %和 79 8%～ 83 5 % ;HGV感染的糖尿病患者ALT异常 (5 2～ 88U)的比例 (7 1% ,13/ 183)明显高于未感染HGV的糖尿病患者 (1 1% ,2 / 183)。结论　糖尿病患者 ,尤其是I型糖尿病患者的HGV感染率较高 ;不同地区检出的HGV基因组RNA片段的核苷酸序列存在一定的差异 ;HGV感染可导致部分患者轻微的肝脏损害。     

11株经输血传播病毒（TTV）基因型分析

目的 对11株经输血传播病毒（transfusion transmit virus,TTV）进行基因型分析。方法 用巢式PCR扩增不同地区不明病因的急、慢性肝炎、肝硬化、肝癌和自然健康人群TTV DNA片段,并对此巢式PCR产物进行克隆,测序分析和同源性比较。结果 11例标本的222bp9引物序列除外）的TTV DNA同源性结果为：武汉WH1、WH2、WH33个患者血清中TTV DNA序列与N22同源性分别为97.0％、97.0％和98.0%;广州GZ1、GZ2、GZ33患者血清中TTV DNA序列与N22同源性分别为98.0%、95.0%和95.0%;山东SD2、SD3TTV DNA序列与N22同源性分别为94.6%和95.5%;广州GZ4、山东SD1,新疆XJ1患者TTV DNA序列与TX011同源性分别为98.0%、98.0％和95.0%。结论 根据Okamoto的分类方法,这11株TTV可属于两个亚型,即基因1型的a和b亚型。     

双探针原位杂交法检测慢性肝病和肝细胞癌患者肝组织TTV DNA的感染状况

目的 探讨慢性肝病和肝细胞癌患者肝组织TIV DNA的感染状况。方法采用PCR扩增法分别合成G1a、G2b两种亚型的双链TTV DNA探针。应用两型探针对45例肝组织标本进行TTV DNA原位杂交检测，巢氏PCR法检测血清TTV DNA。结果31例血清TTTV DNA阳性患者的肝组织TTV DNA均为阳性(100％)。14例血清TTV DNA阴性的患者肝组织中TTV DNA阳性者7例(50%)。慢性肝病患者的肝组织中TTV DNA散在分布在汇管区周围的肝细胞核内，肝癌患者TTV DNA则集中分布在肝癌细胞核内及癌组织周围的肝细胞核内。结论慢性肝病与肝癌患者肝组织中TTV DNA的感染状态存在一定差异。     

高胆红素血症新生儿输血传播病毒的基因检测

目的探讨输血传播病毒（TTV）感染与新生儿高胆红素血症的相关性及肝功能受损情况,并分析TTV（广州株）开放阅读框（ORF）1区基因序列特征.方法对58例高直接胆红素（5例肝病综合征）、92例高间接胆红素血症和85例正常新生儿,应用套式聚合酶链反应（PCR）技术进行外周血TTVDNA扩增、电泳及序列分析,同时检测血清丙氨酸转氨酶（ALT）.结果高直接胆红素患儿中,7例外周血检测出TTV DNA（阳性率为12.1%）,其中3例肝病综合征患儿TTV DNA阳性;高间接胆红素血症和正常新生儿中,分别检出1例TTV DNA阳性（阳性率分别为1.1%和1.2%）.高直接胆红素（包括肝病综合征）患儿的TTV DNA阳性率明显高于高间接胆红素患儿和正常新生儿,差异有统计学意义（P＜0.01）.TTV广州株与日本株N22比较,同源性达87.1%～97.8%,但存在着点突变.结论TTV感染可能是本组引起新生儿直接胆红素升高的原因之一,TTV广州株与日本株具有同源性.     

山东泰安地区部分人群中TTV感染调查及TTV山东株部分基因序列分析

目的 了解山东地区输血后丙型肝炎和非甲～庚型肝炎病例中TTV感染及基因变异的状况。方法应用套式PCR方法检测41例输血后丙型肝炎和16例非甲～庚型肝炎患者血清中TTV DNA,并对其中阳性的2例扩增产物直接克隆测序,分析其基因变异的情况。结果 41例输血后丙型肝炎患者血消中TTVDNA阳性检出率为19.5％(8/41),而16例非甲～庚型肝炎患者血清中阳性检出率为31.3％(5/16)。TTV山东第一株序列和山东第二株序列相应位置核苷酸同源性与日本株和中国第一株相比较分别为94％和91％;山东TTV的两株序列间同源性为91％。结论 本项研究证实山东地区输血后丙型肝炎和非甲～庚型肝炎患者中存在较高TTV感染,输血可能为传播TTV感染途径之一,是否该地区存在着不同的TTV亚型还有待于进一步调查证实。     

徐州地区部分人群TTV感染的检测及分析

TTV为1997年底日本学者在输血后非甲～非庚型肝炎患者血清中发现的单链DNA病毒,基因组全长为3739个核苷酸.由于该病毒的存在与血清ALT升高密切相关,而被认为可能是继庚型肝炎病毒(HGV)之后发现的又一新型肝炎相关病毒.有报道,在供血员、不明原因的暴发性肝炎和急、慢性肝炎患者中均存在TTV感染.为了解徐州地区TTV感染状况,我们以套式聚合酶链反应(nested-PCR)扩增技术对部分人群血清进行了TTV DNA检测,现将结果报道如下.     

TTV在非甲～庚型肝炎患者中的检测及序列分析

目的：研究TTV在非甲～庚型肝炎患者中的感染状况及临床意义。方法：采用套式PCR法检测50例非甲～庚型肝炎患者血清标本中TTV DNA。并对其中1株TTV的部分基因序列进行了测定。结果：在50例肝炎患者中,TTV DNA阳性检出率为28．0（14／50）,TTV序列分析与日本株的同源性为98％。结论：TTV感染可能是非甲～庚型肝炎的病因之一。     

不同引物检测庚型肝炎病毒及其与丙型肝炎病毒混合感染的研究

目的评价不同引物检测庚型肝炎病毒（HGV）的效果和了解其与丙型肝炎病毒（HCV）混台感染率及相互关系．方法5’-NCR,NSS引物和PCRELISA检测HGV,定量RT－PCR检测HCV及部分标本中的HCV分型,部分扩增产物测定核苷酸序列。结果211例HCV阳性血清标本中HGV5－NCRRNA和HGVNSSRNA检出率分别为47．9％和31．8％,总阳性率高达49．8％。结论HGV和HCV有较高的混合感染率。PCRELISA是一种极为敏感的HGV检测方法,HGV5’－NCR引物的检测效果优于HGVNS5。核苷酸序列分析证实PCR—ELISA检测结果可靠。标本中HCV浓度与HGV感染呈负相关,可能存在相互抑制的机制。     

Hepatitis delta virus propagation enabled by hepatitis C virus—Scientifically intriguing,but is it relevant to clinical practice?

In vitro cell culture experiments and animal models have demonstrated that hepatitis delta virus (HDV) can theoretically propagate being enveloped by human pathogenic viruses other than hepatitis B virus (HBV), namely hepatitis C virus (HCV) and dengue virus. However, the clinical relevance of these findings and whether HDV replication occurs in real‐world hepatitis B surface antigen (HBsAg)–negative HCV patient cohorts remain unknown. To this aim, we analysed 323 HCV‐RNA–positive and HBsAg‐negative sera for the presence of HDV‐RNA and anti‐HDV antibodies (anti‐HDV). All 323 (100%) samples were negative for HDV‐RNA. Interestingly, 8/316 samples tested positive for anti‐HDV. The HBV serology of these eight patients showed a positive result for HBV core antibodies (anti‐HBc) indicating a seroconversion of an acute HBV infection in the past. None of the anti‐HBc–negative patients were positive for anti‐HDV. Our results indicate a distinctly low probability of replicative HDV infection in HCV mono‐infected patients in Germany. Current German clinical guidelines rightly recommend performing HDV screening only in HBsAg‐positive patients. However, larger studies on this subject should be performed in regions that are endemic for chronic HBV/HDV as well as HCV infections.     

Age-specific antibody to hepatitis E virus has remained constant during the past 20 years in Japan

We investigated the presence of antibodies to hepatitis E virus (anti-HEV) and hepatitis A virus (anti-HAV) by enzyme immunoassays in sera from 1015 individuals collected in 1974, 1984 and 1994. Age-specific profiles of anti-HEV remained unchanged with a peak at 40-49 years, while those of anti-HAV started to increase in individuals aged 20-29 years in 1974, 30-39 years in 1984 and 40-49 years in 1994. These results suggest that a silent HEV infection has been taking place in the last 20 years or so in Japan, while HAV infection has been terminated at least since 1974.     

HBV感染者HCV的重叠感染关系研究

目的 研究HBV感染患者中HCV的重叠感染状况及其相互关系。 方法 采用ELISA法对767例HBV感染患者同步检测HBV和HCV血清标志物,对可疑HCV感染但抗HCV阴性和/或抗-HCV阳性患者血清,采用PCR法检测HCV-RNA。 结果 HCV重叠感染率为4.82％,且在各类乙肝患者中存在非常显著差异(P<0.01);HBV/HCV感染组重症肝炎的发生率显著高于非HCV感染组(P<0.01);HBV/HCV感染组HBsAg阳性率显著低于单纯HBV感染组(P<0.05);HBV/HCV感染组HCV-RNA阳性率显著低于单纯HCV感染组(P<0.05)。 结论 HCV重叠感染与乙肝患者的发病、病情加重及重症肝炎的发生相关;HCV可抑制或中止HBsAg携带状态,但这种作用远不如对病情的加重作用重要;同时HBV对HCV的复制亦存在抑制作用。     

Low prevalence of hepatitis C virus infection in patients with auto-immune hepatitis type 1

Hepatitis C virus (HCV) antibodies were measured in 28 patients with auto-immune hepatitis type 1 using six different assay kits, three for C100–3 antibody and three for second generation HCV antibody, and two confirmatory tests to determine the prevalence of HCV infection in auto-immune hepatitis. These patients were confirmed to have human leucocyte antigen DR 4 or 2 which is susceptible to auto-immune hepatitis in Japanese. Of the 28 patients, four (14.3%) were positive for HCV antibody in all assays and reacted positively in at least one of the two confirmatory tests, indicating a true positive finding. Eight were positive for HCV antibody only by the Ortho ELISA kit and were negative in both confirmatory tests. The cut-off level for these results was low and became negative soon after the patients received corticosteroid treatment. Thus, these eight patients are presumed to be false-positive reactors. Hepatitis C virus RNA was detected in the serum of two of the four patients with HCV antibody and in none of 24 patients without HCV antibody. No significant difference was observed between the patients with and without HCV antibody in terms of clinical background, liver function tests and auto-antibodies. Our results showed that the prevalence of a past or present HCV infection in patients with auto-immune hepatitis in Japan is low; thus, auto-immune hepatitis is thought to be distinct from hepatitis type C. However, it is also suggested that HCV infection can potentially trigger auto-immune hepatitis.     

Clinical characteristics and distribution of genotypes of TT virus infection in a hepatitis C virus-hyperendemic township of a hepatitis B virus-endemic country (Taiwan)

BACKGROUND: The prevalence of TT virus (TTV) viremia, without definite clinical significance, has been reported to be higher among chronic hepatitis C patients. The status and clinical characteristics of TT virus (TTV) infection and distribution of TTV genotypes in a hepatitis C virus (HCV) hyperendemic township (Masago community) in a hepatitis B virus (HBV) endemic country (Taiwan) were investigated. METHODS: Sera from 100 Masago residents were tested for alanine aminotransferase (ALT) and markers of HBV, HCV and GB virus C/hepatitis G virus (GBV-C/HGV) and TTV-DNA. Sera of 250 blood donors as a control group were tested for TTV-DNA. Sera of Masago residents and blood donors with positive TTV-DNA were directly sequenced, and phylogenetic analyses were performed subsequently. RESULTS: The prevalences of TTV viremia in different age groups among individuals from Masago were significantly higher than that among blood donors. In regard to the subtypes of TTV, 23, seven, two, eight, one, six and one isolate were related to the genotypes 1a, 1b, 2a, 2b, 3, 4 and 5, respectively, from Masago and 21, 14, one, nine and three isolates were related to the genotypes 1a, 1b, 2a, 2b, and 4, respectively, from donors. No clinical or virological factor was associated with TTV viremia or TTV genotypes. CONCLUSIONS: TT Virus prevalence was higher among HCV hyperendemic township residents than blood donors with similar genotype distributions (genotype 1 was the most prevalent) in Taiwan. Neither TTV viremia nor a particular genotype was associated with HBV, HCV or GBV-C/HGV infection and abnormal ALT levels.     

Replication status and histological features of patients with triple (B, C, D) and dual (B, C) hepatic infections

In patients with multiple hepatotropic viral infections (B and C, or B, C and D), the reciprocal influence of each virus remains controversial. The aims of this study were twofold: first, to determine the impact of multiple infection on the replication status of B, C and D viruses and on histological features; and second, to compare patients with multiple infection to patients infected only with the hepatitis C virus (HCV). We retrospectively included 50 patients with multiple infection and 50 control HCV patients, who were matched on independent factors associated with fibrosis, such as age, gender, alcohol consumption and duration of infection. The replication status of hepatitis B virus (HBV), HCV and hepatitis D virus (HDV), and histological lesions, were determined. In patients with multiple infection, HCV RNA was present less frequently (44% vs 98%, P  < 0.001) and the prevalence of cirrhosis was higher (35% vs 8%, P  < 0.001). Among patients with triple infection ( n  = 16), HBV replication was observed in 25%, HCV RNA was detectable in only two ( P  < 0.0001) and HCV viremia was significantly lower than in the matched HCV patients (0 vs 54.7, P  < 0.0001). Among patients with dual infection ( n  = 34), HCV RNA was present less frequently in those with serological markers of active HBV infection than in those without (30% vs 79%, P  = 0.01). Hence, multiple infection is associated with a decrease of HCV replication. Cirrhosis seems to be more frequently observed in patients with multiple infection. In patients with triple infection, serum HCV RNA and markers of HBV replication were absent in 80%, suggesting that HDV acts as a dominant virus. In patients with dual infection, HBV and HCV exert an alternative, dominant replication.     

血液透析患者HCV感染的危险因素

目的 研究血液透析患者HCV感染的相关因素。 方法 血清采自血透前、血透后2-60月的10例肾衰患者,用ELISA检测抗HCV,用PCR法检测HCV,RNA和HCV基因型。 结果 10例患者透析前无HCV感染。透析后2-60月,3例抗HCV阳性(30％),2例HCV RNA阳性(20％),其基因型均为HCVⅡ型。10例患者中7例有输血史,其中4例(57％)HCV感染,所有抗HCV阳性和HCV RNA阳性患者均有输血史。而无输血史的3例患者未见HCV感染。两组有非常显著性差异(P<0.001)。HBV感染与HCV感染无相关性。 结论 反复输血是血透患者感染HCV的高危因素,长期血液透析是参考因素。