特异引物PCR和基因型特异探针杂交法在HBV基因分型中的临床应用

分别采用特异引物PCR和基因型特异探针杂交（试剂盒）对99例慢性乙肝病毒感染（慢乙肝）患者血清HBV进行基因分型。结果特异引物PCR法鉴定为B型34株,C型59株,D型1株,AB混合型1株,BC混合型3株,未被分型1株;特异探针杂交法鉴定为B型33株,C型61株,D型1株,BC混合型3株,未被分型1株。两种分型方法一致率为95．96％（95／99）。认为两种分型方法各有优缺点,特异引物PCR法较为适用且经济。     

新疆汉族人群HBV基因型与临床病情的关系

目的了解新疆汉族人群乙型肝炎患者乙型肝炎病毒(HBV)基因型的分布及与临床病情的关系.方法新疆HBV感染者的血清226份,包括慢性乙型肝炎轻度、中度、重度、重型肝炎、肝硬化、肝癌患者.采用型特异性引物巢式PCR方法对血清中的HBV进行基因分型.结果新疆汉族人群HBV基因型以B、C混合型为主,占43.8%(99/226),B型31.46%(71/226),C型24.34%(55/226),未分型1例(0.44%),未见有A、D、E、F型分布.随机选取的4株HBV株经S基因序列的测定证实与本检测结果一致.C型HBV感染者的平均ALT、AST、γ-GT、AKP水平及HBV DNA浓度均高于B型、B+C型者(P＞0.05),C、B+C型TBIL平均水平高于B型,差异有统计学意义(P＜0.05).C型HBV感染者TP平均水平低于B、B+C型患者(P＞0.05).随着病情的加重,C型所占百分比逐渐高于B、B+C型,在LC、HCC中分别为36.4%,27.3%,C型感染者抗HBe阳性率逐渐增高,在HCC中B、C、B+C混合型者中抗HBe阳性率分别为26.7%、42.9%和32.6%(P＞0.05).结论新疆汉族人群HBV患者存在B、C与B+C基因型,以B、C混合型为主,基因型的分布与国内其它地方汉族人群相比有自身特点,且与临床病情有关,除TBIL外,各型在临床生化指标方面差异虽无统计学意义,但认为C型可能与较严重肝病有关,B+C型与HBV感染后病情的关系有待进一步明确.     

B和C基因型乙型肝炎病毒对阿德福韦酯治疗的病毒学应答比较

目的 阐明不同基因型HBV对阿德福韦酯治疗反应是否存在差异.方法 首先利用型特异引物PCR法结合型特异核苷酸分析法检测HBV基因型,然后根据基因型对阿德福韦酯Ⅲ期临床资料进行分析及统计学处理(计量资料用t检验,计数资料用卡方检验).结果 177例临床标本检出B基因型HBV感染者102例,C基因型感染者65例,B+C混合型感染者6例,B+D混合型感染者4例.治疗第12、24周时,B基因型组和C基因型组血清HBV DNA下降均值分别为2.2log10>拷贝/ml、2.1log10拷贝/ml和2.7log10拷贝/ml、2.4log10拷贝/ml,两组差异均无统计学意义(P>0.05),第48周时两组HBV DNA分别下降3.6log10拷贝/ml和3.1log10拷贝/ml,差异有统计学意义(P<0.05).治疗结束时(48周)B基因型组和c基因型组分别有43例(42.2%)和22例(33.8%)出现血清HBV DNA转阴,差异有统计学意义(P<0.05);两组患者HBeAg阴转率抗-Hbe血清转换率分别为30.4%、29.2%和21.6%、20.0%,差异无统计学意义(P>0.05).两组患者血清ALT复常率在治疗第12、24、36周和48周时分别为35.3%、33.9%,51.0%、53.9%,63.4%、61.5%和83.3%、81.5%,各时间段两组ALT复常率差异均无统计学意义(P>0.05).结论 阿德福韦酯治疗慢性乙型肝炎48周时,部分病毒学指标(如血清HBV DNA下降均值和HBV DNA阴转率)B基因型优于C基因型HBV感染者.但由于阿德福韦酯起效较慢,抑制病毒作用相对较弱,有必要延长治疗时间进一步证实这一现象.     

肝细胞癌组织中HBV基因型的检测及分析

目的 了解肝细胞癌组织中HBV基因型分布状况及特点.方法 应用型特异性引物分型法和基因测序与生物信息学相结合的分型方法分析肝细胞癌组织中HBV基因型,并与HBV携带者血清中HBV基因型进行比较.结果 在63份肝细胞癌组织标本中,B、C、混合型B C和混合型B D基因型分别占20份(31.8%)、37份(58.7%)、4份(6.3%)和2份(3.2%).与HBV携带者血清相比,两者基因型构成比无显著性差异(P＞0.05).结论 中国南部地区的肝细胞癌组织中存在HBV基因型B、C、混合型B C和混合型B D,以C基因型为主,其次为B基因型.肝细胞癌组织中HBV基因型分布与当地HBV携带者基因型密切相关.     

乙型肝炎病毒基因型与病毒复制的关系

检测慢性乙型肝炎患者血清乙型肝炎病毒(HBV)基因型和病毒载量(HBV-DNA定量),探讨它们之间的关系。用微板核酸杂交-ELISA方法对HBV进行基因分型,用PCR荧光定量检测血清HBV-DNA水平,共318例。检测到HBV B型111例(35％);C型128(40％);混合型(B+C,C+D,B+C+D)45例(14％);D型2例;F型2例;未分型30例(9.4％);没有发现A、E型。结果发现,C型和混合型HBV的血清DNA水平高于B型(1.14×107vs2.2×107vsl.60×106拷贝/ml,P<0.05);而混合型HBV的血清DNA水平与C型无差别(P=0.127)。研究提示,我国HBV以B,C两基因型为主,HBV基因型可能是影响HBV复制的重要因素之一。     

基因芯片技术检测西藏拉萨地区的乙型肝炎病毒基因型

目的:研究西藏拉萨地区乙型肝炎病毒基因型的分布与特点.方法:采集92份西藏拉萨地区乙型肝炎患者的血清,参照GenBank中HBV DNA序列设计寡核苷酸探针并制备HBV基因分型芯片,利用套式PCR扩增HBV S基因部分片段,结合基因芯片、DNA测序和BioEdit软件进行基因分型检测,并对其与乙肝标志物、DNA含量、性别和民族之间的关系进行分析.结果:在92例血清标本中,套式PCR检测73例HBV DNA阳性可进行基因分型检测.其中B型13例(17.8%),C型18例(24.7%),D型39例(53.4%)和B/D混合基因型3例(4.1%).统计学分析3种基因型分布在不同乙肝标志物阳性、不同DNA含量和不同性别之间无差异,但与民族存在统计学差异(x~2=7.179,P<0.05).B型以汉族为主(9/13),而C、D型以藏族为主(12/18、28/39).将基因芯片分型的B、C、D型和B/D混合型进行DNA序列分析,表明两种分型方法的结果完全一致.结论:PCR结合基因芯片技术可用于HBV基因分型.西藏拉萨地区HBV基因型包括B、C、D和B/D混合型,其中以D型为主.     

HBV基因型与阿德福韦酯抗病毒疗效的关系

目的 探讨HBV不同基因型对阿德福韦酯抗病毒疗效的影响.方法 选取42例应用阿德福韦酯治疗的慢性乙肝患者作为研究对象,观察治疗24周及48周时抗病毒疗效.采用型特异性引物进行巢式PCR,对患者血清中的HBV进行基因分型,采用荧光定量PCR检测患者血清HBV DNA复制水平,HBV血清病毒标志物采用双抗体夹心ELISA法检测.结果 42例慢性乙肝患者的HBV基因型分布为:B型23例,C型10例,B C混合型9例,未发现A、D、E、F基因型.不同基因型患者治疗前HBVDNA水平无统计学差异(P＞0.05).HBV基因型B型、C型和B C混合型患者治疗24周及48周时HBV DNA载量的变化、丙氨酸氨基转移酶(ALT)复常及HBeAg血清转换均无统计学差异(P＞0.05).结论 HBV基因型B型或C型对阿德福韦酯治疗的病毒学应答和生化应答相当,提示HBV基因型可能对阿德福韦酯的疗效无影响.     

厦门市乙型肝炎患者病毒基因型的分析

目的:采用多对型特异性引物,通过巢式PCR法检测厦门市乙型肝炎患者血清中乙型肝炎病毒(HBV)基因型的分布情况.方法:收集250例HBV感染患者血清,提取血清中HBV DNA作为模板,设计HBV前S1基因和S基因中区域内设计出10条内外引物,并将其中8条型特异性内引物分成A,B两组,分别扩增A,B,C和D,E,F型HBV,然后将第2轮PCR产物以用30g/L琼脂糖进行电泳,根据PCR产物电泳显示的产物长度判定HBV基因型,以了解厦门HBV基因型分布情况.结果:共120例确定了HBV基因型.患者群中慢性乙型肝炎90例,占75.0%,急性乙型肝炎、肝炎肝硬化、原发性肝癌分别占5.8%(7/120)、6.7%(8/120)和12.5%(15/120).分型结果:B型58例(48.3%)、C型30例(25.0%),B/C混合型32例(26.7%).HBeAg阳性患者中B基因型占63.8%,B/C型混合感染21.9%;抗-HBe阳性患者中以B/C型混合感染68.8%,B型25.9%,HBeAg阳性组与抗-HBe组之间比较发现B型和B/C混合型之间(P<0.05).结论:厦门乙型肝炎患者HBV基因型以B型为主,B/C混合感染是一个值得重视的问题.     

青海地区乙型肝炎感染者C/D基因型重组分布

目的区分D基因型重组类型,了解青海地区乙型肝炎病毒(hepatitis B virus,HBV)感染者C/D基因型重组情况.方法收集217份青海地区慢性乙型肝炎感染者样品,PCR扩增535-1460 nt和1779-2400 nt2个片段的乙型肝炎病毒基因序列,分2区(593-799 nt、799-1450 nt和1799-2400 nt)构建进化树,确定青海地区HBV基因重组类型.利用INNO-Li PA HBV分型试剂检测青海地区的C/D基因型重组样品,计算符合率.结果青海地区利用HBV不同区域进化树分型,结果显示各基因型比例为CD1(61.9%)、CD2(8.4%)、C(27.0%)和B(2.8%).基因型分布在藏族和汉族乙型肝炎感染者中的差异具有显著的统计学意义(?2=17.9,P0.01),藏族乙型肝炎感染者中C/D基因型重组和C基因型比例为9 1.5%和8.5%;在汉族中的比例为68.5%和32.3%.C/D基因型重组和C基因型HBV e抗原阴阳性的差异(?2=0.28,P0.05)以及病毒载量的差异(t=1.125,P0.05)均没有统计学意义.C/D重组样品INNO-Li PA分型试剂检测结果为D基因型的理论符合率为87.8%.结论青海地区乙型肝炎感染者以CD1(10-799 nt)基因型重组以主,对其临床意义应进一步的研究分析.该地区的HBV分型检测应注意基因重组对分型试剂的影响.     

新疆维吾尔族慢性乙型肝炎病毒感染者病毒基因型及亚型分布特点

目的 了解新疆维吾尔自治区(简称新疆)维吾尔族慢性HBV感染者基因型、基因亚型分布情况及其与预后的关系.方法 采用聚合酶链反应限制性片段长度多态性分析方法,分析109例新疆维吾尔族慢性HBV感染者感染病毒的基因型及基因亚型.多标本均数比较采用单向方差分析.结果 109例新疆维吾尔族慢性HBV感染者,其中慢性乙型肝炎88例,乙型肝炎肝硬化17例,肝癌4例.13基因型HBV感染者9例,占8.3%,C基因型感染者50例,占45.9%,C/D基因型重组体32例,占29.4%,D基因型感染者18例,占16.5%,C基因型、C/D基因型重组体为主要基因型.经过聚合酶链反应限制性片段长度多态性分析及测序检测,鉴定9份B基因型HBV,均为Ba亚型.50例C基因型HBV感染者病毒基因亚型分布情况:Cl亚型27例,占54%,C2亚型23例,Cl亚型比C2亚型感染者多.在慢性乙型肝炎、乙型肝炎肝硬化、肝癌患者中,HBV Ba基因亚型感染者分别为8例(9.1%)、1例(5.8%)和0例;C2基因亚型感染者分别为17例(19.3%)、8例(47.5%)和2例(50%);C/D基因型感染者分别为29例(33.0%)、2例(11.9%)和1例(25%),随着病情加重,Ba基因亚型和C/D基因型感染者所占比例呈下降趋势,C2基因亚型感染者所占比例增加.结论 新疆地区维吾尔族慢性HBV感染者以Cl基因亚型为主,新疆维吾尔族慢性HBV感染者存在C/D基因型重组体.C2基因亚型HBV感染预示病情严重,预后差.     

Genotyping 238 HBV strains using type-specific primer PCR combined with type-specific nucleotide analysis]

OBJECTIVE: To establish a set of suitable and reliable methods for HBV genotyping and to study the distribution of HBV genotypes. METHODS: Type-specific nucleotides were searched through alignment of S genes (more than 1000 sequences) listed in GenBank. Then, type-specific primers were designed and type-specific primer PCR was used to genotype the 238 HBV strains. S genes of the untyped strains were further amplified and sequenced to find out their genotypes with type-specific nucleotide analysis. RESULTS: All the 238 HBV strains were genotyped. 159 (66.8%) cases were genotype B, 69 (28.9%) were genotype C, 6 (2.5%) were mixtures of genotypes B and C and 4 (1.6%) were mixtures of genotypes B and D. No genotypes of A, E, F, G, and H were found. CONCLUSION: Genotypes B and C are the most common types for HBV strains. Mixtures of genotypes B and C or genotypes B and D coinfection rarely existed. There is no relationship between the gender of the patients and HBV genotypes (X2 = 0.794, P more than 0.05).     

Human parainfluenza virus induces a type-specific protective immune response

Induction of type-specific and cross-protective immune responses against human parainfluenza viruses have been investigated. The envelope glycoproteins HN (76 kDa) and F0 (62 kDa) from parainfluenza type 2 virus were selectively solubilized with octylglucoside. Detergent-soluble envelope glycoproteins were used as vaccine antigens for intranasal immunization of hamsters. The immunized animals showed complete protection from challenge infection with prototype live virus but failed to demonstrate a significant level of protection against either human parainfluenza type 1 or type 3 virus. The sera and bronchial lavages of immunized animals also showed type-specific neutralizing antibodies. A similar type-specific protective immune response was also noted after primary infection with live virus. The results indicate that a multivalent parainfluenza virus vaccine is probably required for protection against natural infection.     

Horizontal cells reveal cone type-specific adaptation in primate retina

The human cone visual system maintains contrast sensitivity over a wide range of ambient illumination, a property known as light adaptation. The first stage in light adaptation is believed to take place at the first neural step in vision, within the long, middle, and short wavelength sensitive cone photoreceptors. To determine the properties of adaptation in primate outer retina, we measured cone signals in second-order interneurons, the horizontal cells, of the macaque monkey. Horizontal cells provide a unique site for studying early adaptational mechanisms; they are but one synapse away from the photoreceptors, and each horizontal cell receives excitatory inputs from many cones. Light adaptation occurred over the entire range of light levels evaluated, a luminance range of 15-1,850 trolands. Adaptation was demonstrated to be independent in each cone type and to be spatially restricted. Thus, in primates, a major source of sensitivity regulation occurs before summation of cone signals in the horizontal cell.     

Clinical uses of herpes simplex virus type-specific serology.

Type-specific serological tests to distinguish between infection with herpes simplex virus type 1 (HSV-1) and herpes simplex virus type 2 (HSV-2) have largely been used for studies which have contributed to our knowledge of the epidemiology and natural history of these infections. Such tests could be used as diagnostic tools in clinical situations provided that clinician and patient are aware of the test characteristics, which may result in false negative and false positive results. The use of serological tests in the screening of populations or sub-populations, such as pregnant women, has been advocated to attempt to halt the worldwide spread of the infection. However, there is little evidence currently to support this strategy.     

Cell type-specific metabolism of peptidylglycine alpha-amidating monooxygenase in anterior pituitary

Peptidylglycine alpha-amidating monooxygenase (PAM) is a bifunctional enzyme expressed in each major anterior pituitary cell type. We used primary cultures of adult male rat anterior pituitary to examine PAM expression, processing, and secretion in the different pituitary cell types and to compare these patterns to those observed in transfected AtT-20 corticotrope tumor cells. Immunostaining and subcellular fractionation identified PAM in pituitary secretory granules and additional vesicular compartments; in contrast, in AtT-20 cells, transfected PAM was primarily localized to the trans-Golgi network. PAM expression was highest in gonadotropes, with moderate levels in somatotropes and thyrotropes and lower levels in corticotropes and lactotropes. Under basal conditions, less than 1% of the cell content of monooxygenase activity was secreted per h, a rate comparable to the basal rate of release of individual pituitary hormones. General secretagogues stimulated PAM secretion 3- to 5-fold. Stimulation with specific hypothalamic releasing hormones demonstrated that different pituitary cell types secrete characteristic sets of PAM proteins. Gonadotropes and thyrotropes release primarily monofunctional monooxygenase. Somatotropes secrete primarily bifunctional PAM, whereas corticotropes secrete a mixture of mono- and bifunctional proteins. As observed in transfected AtT-20 cells, pituitary cells rapidly internalize the PAM/PAM-antibody complex from the cell surface. The distinctly different steady-state localizations of endogenous PAM in primary pituitary cells and transfected PAM in AtT-20 cell lines may simply reflect the increased storage capacity of primary pituitary cells.     

Systemic and cell type-specific gene expression patterns in scleroderma skin

We used DNA microarrays representing >12,000 human genes to characterize gene expression patterns in skin biopsies from individuals with a diagnosis of systemic sclerosis with diffuse scleroderma. We found consistent differences in the patterns of gene expression between skin biopsies from individuals with scleroderma and those from normal, unaffected individuals. The biopsies from affected individuals showed nearly indistinguishable patterns of gene expression in clinically affected and clinically unaffected tissue, even though these were clearly distinguishable from the patterns found in similar tissue from unaffected individuals. Genes characteristically expressed in endothelial cells, B lymphocytes, and fibroblasts showed differential expression between scleroderma and normal biopsies. Analysis of lymphocyte populations in scleroderma skin biopsies by immunohistochemistry suggest the B lymphocyte signature observed on our arrays is from CD20+ B cells. These results provide evidence that scleroderma has systemic manifestations that affect multiple cell types and suggests genes that could be used as potential markers for the disease.     

Population-based type-specific prevalence of high-risk human papillomavirus infection in Estonia

Background  

Effective prophylactic vaccines are available against human papillomavirus (HPV) types 6, 11, 16, and 18 which are licensed for routine use among young women. Monitoring is needed to demonstrate protection against cervical cancer, to verify duration of protection, and assess replacement frequency of non-vaccine types among vaccinated cohorts.