应用聚合酶链反应检测南京地区TT病毒感染

目的：了解南京地区TT病毒感染情况。方法：采用巢式PCR方法检测血清标本中TTV－DNA。结果：163例病毒性肝炎患者血清标本中,TTV－DNA总检出率为21.5％（35／163）,其中甲型肝炎13.3％（4／30）,乙型肝炎21.3％（16／75）,丙型肝炎20.0％（3／15）,戊型肝炎5.3％（1／19）,非甲－庚型肝炎45.8％（11／24）。结论：南京地区存在TTV感染,TTV是导致非甲－庚型肝炎的重要病因,TTV可能存在非血源性传播途径。     

盐城地区孕产妇血清及乳汁中TTV感染状况

目的　了解盐城地区孕产妇血清及乳汁中 TT病毒 ( TTV)感染状况 ,探讨 TTV的传播途径。方法　采用PCR-微孔板杂交法对 1 0 5例孕产妇血清及乳汁标本进行 TTV DNA检测。结果　 1 0 5例孕产妇血清及乳汁 TTV DNA阳性率分别为 1 2 .4%和 1 0 .5 % ,1 3例孕产妇血清 TTV DNA阳性患者中乳汁同时阳性 1 0例 ,血清与乳汁感染率的差异无显著性 ( P>0 .0 5 )。结论　孕产妇存在 TTV感染 ,并可能经乳汁感染婴儿     

前瞻性追踪受血患者输血后TT病毒感染的发生率及临床表现

背景 近来发现一种新的经输血途径传播的人DNA病毒,即TT病毒(TTV)。作者试图探明受血者感染TTV的发生率及感染TTV的临床后果。研究设计和方法 应用巢式PCR方法对连续采集的血清标本进行了TTV DNA检测,作为输血后肝炎的前瞻性研究。结果:150例成人心脏病手术患者中,59例患者的输血后血样为TTV DNA阳性,其中有13例患者的输血前血清标本为TT DNA阳性。因而在137例中有46例(33.6％)以前未感染的患者在输血后发展为TTV病毒血症。在这46例输血TTV感染者中,3例伴随着HCV感染,5例伴随有HGV感染,另外的38例为单纯TTV感染。TTV感染者平均血清ALT活性为311U/1,而38例单纯TTV感染者中有34例的血清ALT活性持续在正常范围之内。在8例以后发展为非A-G的肝炎患者中,3例为TTV阳性(1/8:46/137,P=0.8)。对12例患者进行一年时间以上的随访,12例均表现为持续地TTV病毒血症。结论:在本组心脏病手术患者中,输血传播的TTV感染率大约为30％,大多数感染者为持续感染。TTV感染率高但似乎不引发肝炎。     

献血者中TT病毒检测及其致病性探讨

目的　探讨TT病毒 (TTV)在输血过程中的传播及其致病性。方法　常规酚 氯仿法抽提血清病毒DNA ,设计位于TTVORF1保守区的两对引物 ,套式PCR扩增病毒DNA。ELISA检测血清HBsAg、抗 HAV、抗 HCV和抗 HIV。结果　健康献血者血清中TTV DNA检出率为 4 3 1 % (96 / 2 2 3)。受血者输血TTV DNA阳性血后 2周 ,血清TTV DNA转阳率为 6 3 6 % (1 4 / 2 2 )。 8周血清TTC DNA阳性率仍为 4 6 4 % (1 0 / 2 2 ) ,其中两对献血者和受血者之间血清TTV DNA同源性达 1 0 0 %。上述 2 2名受血者输血 2周后血清ALT、TB和DB正常 ,血清HBsAg、抗 HAV、抗 HCV和抗 HIV均阴性。随访其中 5例至 8周 ,无肝炎症状及体征 ,血清肝功能正常。结论　TTV可通过血液传播 ,但无明显致病性     

HBsAg阴性、HBV DNA阳性献血者病毒感染特征研究

目的了解上海地区献血人群中血清HBsAg阴性、HBV DNA阳性感染的流行率、病毒血清学和分子生物学特点。方法选取HBsAg阴性、核酸筛查确认为HBV DNA阳性的献血者血清标本,采用酶联免疫法（ELISA）检测病毒血清标志物抗-HBc和抗-HBs;PCR扩增HBV S区基因片段,对扩增后的产物进行测序分析后,将产物序列与Genbank中HBV序列进行Blast比对,获得标本HBV毒株的基因型;采用DNAMAN软件进行蛋白表达分析,确定标本毒株的血清型,并与Genbank中野生型HBV序列进行比较,获得S区基因编码蛋白突变情况。结果上海地区HBsAg阴性献血人群HBV DNA阳性感染率约为0.045%。18例HBsAg阴性、HBV DNA阳性标本血清病毒载量均低于200IU/ml,且大部分低于20IU/ml;其中14例（77.8%）病毒血清标志物为抗-HBc和/或抗-HBs阳性。18例样本全部为B和C基因型,主要为adw和adr血清型。14例血清抗-HBc和/或抗-HBs阳性样本中,13例样本发生了S蛋白氨基酸突变,其中8例B基因型较多发生Q101K/H、M103T/I、F134L、D144A突变,5例C基因型较多发生S114T/A、T118K/R、K141T、S143T突变;4例血清阴性样本中,均未发生S区蛋白位点突变。结论上海地区献血者存在HBsAg阴性、HBV DNA阳性感染者,其血清病毒载量低,感染病毒全部为B和C基因型,主要为adw和adr血清型;其中大部分为隐匿性HBV感染,少数可能为窗口期感染;隐匿性HBV感染病毒多发生S蛋白氨基酸位点突变。     

人类肝炎相关病毒——TT病毒研究现状

TT病毒(TTV)是1997年底新发现的无包膜、单链DNA肝炎相关病毒,可能属于微小病毒科。在暴发型肝炎病人、慢性不明原因肝病病人、经血感染高危人群、甚至健康献血者中均可检出TTV DNA。研究发现,TTV既可通过非肠道途径传播,也可通过粪-口途径传播,其核酸序列变异较大,存在不同的基因型。我国是病毒性肝炎高发区,有必要对TTV进行深入的研究。本文介绍TTV的发现、特性及分类地位、检测方法、基因分型及其传播途径和致病性。     

慢性乙型肝病患者外周血单个核细胞以及血清中HBVcccDNA的对照检测

目的评价慢性乙型肝病患者外周血中HBVcccDNA存在状态及临床意义。方法采用微粒子酶免分析法（MEIA）、荧光聚合酶链反应（PCR）法、PCR-微流芯片法检测17例慢性乙型肝炎患者、12例终末期肝病肝移植术后患者血清HBVM、HBVDNA、HBVcccDNA、外周血单个核细胞（PBMC）中HBVcccDNA。结果17例慢性乙型肝病患者、12例肝移植患者外周血血清、白细胞中HBVcccDNA检出率分别为10／17、7／17和1／12、8／12,血清HBVDNA检出率分别为12／17、0／12。结论血清HBVcccDNA的来源可能不只是肝组织;慢性乙型肝病患者外周血PBMC中HBVcccDNA低于血清HBVcccDNA及HBV DNA,HBVcccDNA、HBV DNA二者联合检测更能准确判断病毒复制;肝移植术后血清病毒复制不活跃,外周血低滴度HBVcccDNA的存在可能为乙型肝炎的复发提供了基础。     

不同地区人群TT病毒感染的分布

用 PCR方法测定十个不同国家人群的 TT病毒 (TTV) ,感染率极高。结果提示 TTV是一种普通单链无包膜的 DNA病毒 ,与人类疾病关系尚不清楚。血清标本采自日本 (2 33例无肝病者 ) ,Myanmar(5 1例健康人 ,92例肝病患者 ) ,尼泊尔 (177例献血员 ) ,越南 (6 2例高危人群即医务工作者 ) ,朝鲜 (73例血透析病人 ) ,柬浦寨 (18例 HIV患者 ) ,加纳 (95例HIV患者 ) ,美国 (6 8例 HIV患者 )及埃及 (95例献血者 )。血清标本置-2 0℃或更低温下贮存。取 10 0μl血清标本用核酸提取剂提取 DNA,用 Takahashi等介绍的方法 :正义引物为 5′-GCTA…     

免疫抑制心脏移植受者及免疫活性个体的TT病毒血症和排泄

背景 TT病毒(TTV)发现于有输血后肝炎症状的患者中,但许多的病毒血症个体并未表现有症状。因此,有必要研究输血传播感染的情况,作者在献血员及心移植受者中筛选TTV感染。方法 对600例献血员、100例健康个体和495例心移植受者的血浆、血清、尿及粪便样品中,采用巢式聚合酶链反应检测TTV DNA。结果 3.2％的献血员,25％的心移植受者有病毒血症。2组中均观察到G1a/b和G2a/b TTV亚型,亚型的分布有     

我国部分地区人群中TT病毒感染的分子流行病学

目的　了解我国不同地区和人群中 TTV DNA的感染情况。方法　采用巢式 PCR方法检测血清标本 TTVDNA。结果　在 2 1 4例各型病毒性肝炎患者中 ,检出 TTV DNA阳性 5 7例 ,阳性率为 2 6.64%。在甲～戊型肝炎、非甲～戊型肝炎、有偿献血者和正常人群中 ,TTV DNA流行率分别为 2 5 .97% ( 4 0 /1 5 4)、2 8.3 3 % ( 1 7/60 )、3 9.2 9% ( 2 2 /5 6)和 1 7.86%( 1 0 /5 6) ,四组差异亦无显著性 ( P>0 .0 5 ) ;我国北京、沈阳、南京、合肥和深圳的非甲～戊型肝炎患者中 TTV DNA流行率为 2 9.5 7% ( 68/2 3 0 ) ,与上述甲～戊型肝炎组比较差异亦无显著性 ( P>0 .0 5 )。结论　我国不同地区人群中存在 TTV DNA感染 ,各型病毒性肝炎患者和正常人群中 TTV DNA流行率较高。     

Prevalence of TT virus in serum and peripheral mononuclear cells from a CAPD population.

BACKGROUND: A novel virus named TT virus (TTV) has been isolated recently from patients with posttransfusional hepatitis of unknown etiology. The prevalence of TTV in several groups at risk has been reported, however, there is no information about the prevalence of TTV in patients on continuous ambulatory peritoneal dialysis (CAPD) without blood transfusions or hemodialysis antecedents. OBJECTIVE: To study the incidence of TTV in serum and peripheral blood mononuclear cells (PBMC) of CAPD patients. DESIGN: TTV DNA was detected by polymerase chain reaction, using primers from the open reading frames (ORF) 1 and 2, in serum and PBMC from 22 CAPD patients who had not received blood transfusions or hemodialysis therapy prior to CAPD. As controls, sera from 20 patients with chronic viral hepatitis (10 with HBV and 10 with HCV) and 20 healthy donors were included in the study. RESULTS: TTV DNA was detected in the serum of 5 of 22 (22.7%) CAPD patients with both sets of primers. Four of the 5 (80%) patients with TTV DNA in their serum were TTV positive in their PBMC with primers from ORF1 and ORF2. Five of 20 (25%) patients with chronic viral hepatitis (2 patients with HBV and 3 with HCV) and 4 of 20 (20%) healthy donors were TTV DNA positive in serum. No relation was found between TTV infection and the underlying kidney disease, previous surgery, and abnormal alanine aminotransferase levels. CONCLUSION: We have found a relatively high prevalence of TTV that is similar to that found in healthy donors and in patients with chronic viral hepatitis.     

Clinical manifestations and histopathologic features of chronic liver disease with serum TT virus (TTV) DNA positive alone as measured by first generation primer sets]

To determine the clinical manifestations and histopathologic features of serum TTV DNA-positive chronic liver disease, we investigated eleven patients who showed serum TTV DNA alone, as detected by the polymerase chain reaction using first generation primer sets (RD primer series). Clinical manifestations were as follows: (1) biochemical abnormalities of the ALT-dominant and gamma-GTP-dominant types; (2) persistently elevated gamma-GTP despite normalization of ALT in gamma-GTP-dominant type patients; (3) association of TTV with pathogenesis of fatty liver or alcoholic liver dysfunction; and (4) response to liver-protective medicines. Histopathologic features were as follows: (1) inflammation-related cell infiltration scattered within the portal area, with distinguishable necroinflammation in the lobular region; (2) pathologic findings on biliary epithelium; (3) high incidence of steato-metamorphosis involvement; and (4) histologic characteristics undistinguishable from "viral" chronic hepatitis in some liver specimens.     

Detection of TT virus DNA in patients with non-A to G liver diseases]

A novel single-stranded DNA virus, TT virus(TTV), has been reported recently. We detected TTV viral sequences by polymerase chain reaction using primers derived from nucleotide sequences of ORF1 and the 5' noncoding region of ORF2. Using primers of the 5' noncoding region, TTV DNA was detected in 21 of 25(84%) healthy individuals, suggesting that most TTV strains detected by these primers are almost harmless. In contrast, using primers of ORF1, which detect genotype 1a TTV that was reported to be a causative agent of posttransfusion hepatitis, TTV DNA was detected in only 3 of 25 healthy subjects and 3 of 27 acute and 9 of 72(12%) chronic non-A to G hepatitis patients. Whether these TTV strains actually cause hepatitis remains to be determined.     

Pathogenic significance and organic virus levels in patients infected with TT virus.

Previous reports documented the recovery of a DNA virus from a patient with posttransfusion non-A-G hepatitis and named TT virus (TTV). Although the virus was initially detected as a causative agent of hepatitis, there is doubt about its pathogenicity. The aim of this study was to clarify the relationship between TTV and liver diseases. Histopathological examination of liver biopsies from 14 patients with TTV genotype 1 positive non-B, non-C and non-G chronic hepatitis showed mild fibrosis and periportal/piecemeal necrosis. Using the real-time detection (RTD)-PCR method, we found that TTV DNA levels of genotype 1 in liver samples from 3 such patients were 100- to 1,000-fold higher than those in the paired serum samples. Further investigation using various tissues from 2 autopsies of patients with hepatitis C with hepatocellular carcinoma revealed that the concentrations of TTV DNA in the liver were also higher than in serum samples. However, the highest TTV DNA concentrations in these 2 autopsies were found in the lung and bone marrow, respectively. Our results suggest that TTV may replicate in various tissues including the liver and may cause only mild liver damage.     

72例肝炎患者TT病毒DNA检测及部分基因序列分析

目的 了解肝炎患者中的TTV的感染情况并对部分分离株进行基因分型。方法 设计通用引物，采用半套式聚酶链反应检测肝炎患者血清标本中TTVDNA，并对部分PCR产物进行直接测序和序列分析。结果 在72例不同肝炎患者血清中，共检出TTVDNA阳性血清39份，总检出率为54.16%.其中,在非甲-非庚型肝炎患者中,TTVDNA阳性率为87.5%;在甲-庚型肝炎患者TTVDNA的阳性率(50.0%).对4株TTV序列分析结果显示,它们与日本分离株TA278、NA004、TKB555、PT3最高的同源性分别为97.7%、99.1%、96.8%、91%。经系统发育分析。其中有1株属G1型，有3株属G2型。结论 本次研究的肝炎人群中，TTV感染率较高，。感染的TTV颁发于G1及G2两个不同的基因型，TTV可能是非甲-非庚肝炎致病因素之一。     

Detection and typing of TT virus DNA genotype by the PCR-RFLP method

TT virus (TTV) is a novel single-stranded DNA virus that was identified in patients with post-transfusion-hepatitis of non-A-G type in Japan in 1997. We developed a new detection and typing method for TTV DNA strains, by combining polymerase chain reaction (PCR) and restriction fragment length polymorphism (PCR-RFLP) techniques. TTV DNA was amplified by nested-PCR with TTV-specific mixed primers derived from the conserved open reading frame 1 (ORF1) region of the published TTV DNA sequences. Using the enzymes Hae III, Dra I, Eco RI and Pst I, we were able to distinguish between the four TTV genotypes. In 200 serum samples obtained from healthy pregnant women at an outpatient clinic, the positive rate of TTV detection was 27.0%, almost identical to the positive rate of the detection of ORF1-PCR in Japanese healthy individuals. The strains detected in the positive serum samples were genotype 1a (9.3%), genotype 1b (11.1%), genotype 2 (51.9%), genotype 3 (7.4%) and other genotypes (1.9%). This results indicate that genotype 1 and genotype 2 were the two major strains in the Japanese population. Moreover, 10 serum samples (18.5%) presented RFLP patterns in which some genotypes were mixed.     

Quantitative and genotypic analysis of TT virus infection in Chinese blood donors

BACKGROUND: The TT virus (TTV) is a member of a newly described family of human viruses related to the C ircoviridae viruses. Its association with specific diseases has not been established, and screening of blood donors has not been implemented. To date, 16 genotypes have been identified. STUDY DESIGN AND METHODS: Sera from 471 healthy blood donors (aged 11-58 years) were randomly selected and tested for TTV by the use of two sets of primers: NG59d/NG61d/NG63d primers and T801/T935 primers. Quantitative competitive PCR (QC-PCR) was developed to measure the TTV DNA concentration among the blood donors. Sequencing of a part of the genome was performed to identify the various genotypes. Several samples showed a mixed genotype infection. RESULTS: TTV was detected in 251 (53.3%) of 471 healthy Hong Kong blood donors by the use of NG59d/NG61d/NG63d primers. The prevalence of the virus increased steadily with age (p = 0.03). TTV DNA was detected in 90 percent (90 of a randomly selected 100) of samples by the use of T801/T935 primers. TTV DNA concentration was also measured by QC-PCR in the blood donors who were positive for TTV DNA in the first round of the heminested PCR. TTV titers ranged from 4.8 x 10(2) copies per mL to 6 x 10(4) copies per mL, with a median value of 1.2 x 10(4) copies per mL. Sequencing and phylogenetic analysis of a 223-bp fragment from open reading frame 1 showed three main genotypes (G1 [60.7%], G2 [24.3%], and G3 [14%]) and a new genotype 17 (G17), with the latter bearing 60-percent nucleotide homology with other genotypes deposited at GenBank. In addition, a new TTV subtype, G2f, was found. CONCLUSION: The prevalence of TTV is high in healthy Chinese blood donors. Three main genotypes (G1, G2, and G3) were detected. In addition, a new TTV genotype, tentatively designated as G17, and a new subtype, G2f, were identified.     

血液透析患者单个核细胞中TTV-DNA的研究

目的 :探讨血液透析患者血清和外周血单个核细胞 (PBMC)中输血传播病毒 (TTV)检测状况及意义。方法 :采用聚合酶链反应 (PCR)对 6 6例血液透析患者血清及PBMC进行TTV DNA检测 ,同时采用酶免疫分析 (EIA)检测血清中HBsAg和抗 HCV。结果 :血液透析患者TTV、HCV、HBV检出率分别为 18.2 %、2 4 .2 %、7.6 %。血清TTV DNA阳性与阴性患者 ,抗 HCV阳性率分别为 9.1%与 2 7.3% ,HBsAg阳性率分别为 0 %与 9.1% ,经统计学分析均无显著差异。PBMC中TTV DNA检出率 2 2 .7% ,PBMC中TTV DNA检出率在血清TTV DNA阳性者显著高于血清TTV DNA阴性者 (5 8.3%vs 14 .8% ,P<0 .0 5 )。结论 :血液透析患者TTV感染不依赖于HCV或HBV而存在。PBMC中TTV DNA主要在血清TTV DNA阳性患者中检出 ,PBMC可能是TTV的一个贮存场所。     

聚合酶链反应斑点杂交法检测TT病毒感染

目的研究ＴＴ病毒在肝炎发病中的作用机制及其临床病理特征。方法采用聚合酶链反应（ＰＣＲ）技术将地高辛素标记到ＴＴ病毒ＤＮＡ上,制备成地高辛素探针,建立斑点杂交方法,并与套式ＰＣＲ方法进行比较。结果此探针具有一定的特异性和较高的敏感性,采用斑点杂交方法对２９１例各种肝炎血清标本进行检测,ＴＴ病毒阳性率为１３．７％（４０／２９１）,其中甲～戊型肝炎为５．３％（９／１７０）,庚型肝炎为１５．０％（３／２０）,非甲～戊型肝炎阳性为２７．７％（２８／１０１）。斑点杂交与套式ＰＣＲ方法相符率为９８．３％（２８６／２９１）。结论ＴＴ病毒在各型肝炎中均有感染,且斑点杂交方法作为ＴＴ病毒的检测方法是可行的。