人类巨细胞病毒UL145基因在临床低传代分离株中的多态性

有研究表明人巨细胞病毒(HCMV)在体内存在不同的细胞嗜性,提示来自于不同临床症状患儿的HCMV分离株的基因结构可能存在差异.我们曾报道过HCMV UL144基因多态性[1].本文着重研究了HCMV UL145序列在临床患儿低传代分离株中的多态性.     

人巨细胞病毒UL150基因在临床低传代分离株中的多态性研究

目的 研究人巨细胞病毒(human cytomegalovirns,HCMV)UL150序列在临床低传代分离株中的多态性,探讨HCMV基因多态性与其感染引起不同临床症状之间的关系.方法 对29株经荧光定量PCR方法(Q-PCR)检测HCMV-DNA为阳性的临床低传代分离株进行HCMV UL150全序列PCR扩增,并对PCR扩增产物进行序列测定及分析.结果在29株临床低传代分离株中25株PCR扩增阳性.其中18株完成了测序.18株临床分离株HCMV UL150开放阅读框架(open reading frame,ORF)与HCMV Toledo株相应序列进行比较分析,显示18株低传代临床分离株的HCMV UL150 ORF均为1920bp,编码蛋白含有640个氨基酸,临床株的ORF及编码的氨基酸序列的长度均与Merlin株一致.结论 HCMV UL150基因在临床分离株中存在着高度的多态性,未发现其与HCMV感染不同临床症状间存在明显的关系.     

广州地区HCMV低传代临床病毒株UL143基因的多态性研究

目的研究广州地区人巨细胞病毒（HCMV）临床低传代分离株UL143基因的多态性。方法对3株经多重PCR鉴定的HCMV临床低传代分离株进行HCMV UL143基因全序列扩增，扩增产物克隆到pMD18-T载体上测序，并将其序列与GenBank中公布的其它临床分离株UL143基因一起进行分析。结果D3株UL143基因因碱基缺失形成多处终止密码无法产生有功能的蛋白；Toledo株UL143基因开放读码框由279个核苷酸组成．编码蛋白由92个氨基酸残基组成：其它临床分离株UL143基因开放读码框均由252个核苷酸组成。DNA序列比较保守，变异均为碱基替换。编码蛋白由83个氨基酸残基组成，氨基酸序列也很保守，不同临床分离株氨基酸变异率为1．2％-2．4％：HCMV UL143蛋白翻译后修饰位点在除Toledo株之外的所有分离株中均高度保守．没有缺失或新增；不同临床分离株UL143蛋白二级结构有所不同；除Toledo株外，其余分离株UL143蛋白的等电点均为8．75。结论临床低传代分离株HCMV UL143基因DNA及其编码产物的氨基酸序列极为保守。但仍存在一定多态性。     

广州地区人巨细胞病毒临床低传代株UL136基因的序列特征及多态性

目的 研究广州地区新生儿感染人巨细胞病毒(HCMV)临床低传代株UL136基因的序列特征与基因多态性.方法 从10例广州感染新生儿体内分离获得2株(D2、D3)临床HCMV分离株,经多重PCR鉴定后进行UL136基因全序列扩增.PCR产物纯化后进行基因克隆,构建HCMV UL136-pMD18-T重组质粒.经基因测序及应用生物信息学分析方法 ,分析其核酸序列稳定性、编码蛋白质的二级结构与特征.结果 成功分离2株HCMV临床分离株,测序结果 显示,D2、D3及与GenBank中公布的11株临床分离株(4J、51C、39J、33J、63J、22M、10J、32C、29C、27C、Toledo)中,UL136序列高度保守.同源性分析显示在UL136全基因序列1019个核苷酸中,存在30个位点变异,所有的变异均为碱基替换,无插入及缺失突变.编码蛋白的氨基酸序列也高度保守,240个氨基酸残基中,不同临床分离株氨基酸变异率为1.6%～3.7%.不同分离株的UL136蛋白中参与形成二级结构的氨基酸数目及等电点不同.进化树分析结果 显示D2和D3均属于1a群.结论 广州地区临床低传代分离株HCMV UL136基因核苷酸序列及其氨基酸序列极为保守,但仍存在一定多态性.其基因的稳定性提示HCMV UL136开放阅读框(ORF)可能是一个具有重要功能的基因.其编码后修饰位点提示UL136可能与膜受体介导的细胞信号转导通路有关.     

人巨细胞病毒UL148A、UL148B、UL148C、UL148D基因在临床株中的多态性研究

目的研究人巨细胞病毒（human cytomegalovirus，HCMV）UL148A、UL148B、UL148C和UL148D基因序列在临床低传代分离株中的多态性。方法对22株经荧光定量PCR方法（Q-PCR）检测HCMV-DNA为阳性的临床株进行HCMV UL148A、UL148B、UL148C和UL148D基因全序列PCR扩增，并对PCR扩增产物进行序列测定及分析。结果22株临床株序列与Merlin株相比，临床分离株UL148A基因的核苷酸变异率为0．5％-8．3％，氨基酸变异率为1．3％-6．3％；UL148B基因核苷酸变异率为0．5％-4．6％，氨基酸变异率为1．3％-5．0％；UL148C基因核苷酸变异率为0．5％-3．0％，氨基酸变异率为1．3％-3．9％；UL148D基因核苷酸变异率为0．6％-8．5％，氨基酸变异率为1．7％-8．1％。22株HCMV UL148A和UL148D序列均分为3个型。与Merlin株比较，除U96株外，来自未经传代的尿标本中的UL148A、UL148B、UL148C和UL148D基因编码产物含有的翻译后修饰位点均保守。结论UL148C基因无论在核苷酸序列还是在蛋白的氨基酸结构上均较为保守。统计学分析显示HCMV UL148A和UL148D基因呈现一定的相关性。     

HMA-SSCP方法研究人类巨细胞病毒UL144基因在临床低传代分离株中的多态性

目的 探讨人类巨细胞病毒（HCMV）UL144序列在临床患儿低传代分离株中的多态性及与临床疾病的关系。方法 对65株HCMV临床低传代分离株及7例同年龄组HCMV，DNA定量PCR方法检测阳性无症状感染儿尿液进行HCMV-ULl44 PCR扩增及HMA-SSCP分析，并对其中32份阳性标本进行测序。结果 65株分离株中有55株UL144全序列引物PCR扩增阳性，7份QPCR检测HCMV—DNA阳性无症状感染儿尿液中5份UL144全序列引物PCR扩增阳性。60份UL144扩增阳性标本HMA-SSCP（异源双链泳动及单链构象多态分析）呈现3种典型带形，巨结肠患儿分离株序列、小头畸形患儿的序列分布以1型为主，巨结肠患儿分离株序列没有2型，黄疽患儿以3型为主。结论 HCMV-UL144广泛存在于临床低传代分离株中，用HMA-SSCP检测HCMV-UL144基因在临床低传代分离株中的多态性是一种可行的方法。HCMV不同疾病类型的HCMV-UL144序列不同，提示UL144基因可能对HCMV致病性起一定作用。     

先天性巨结肠患者人类巨细胞病毒UL144基因多态性的研究

目的研究人类巨细胞病毒（human cytomegalovirus,HCMV）UL144基因在先天性巨结肠（Hirschsprung＇s disease,HD）临床株中的多态性,探讨HCMV UL144基因多态性与致病性之间的关系.方法随机选取53个先天性巨结肠患儿痉挛段结肠手术标本及经荧光定量PCR方法检测HCMV DNA为阳性的4个HD患儿的尿标本,对照组为无症状或仅有皮肤轻度黄疸的6个尿标本.应用巢式聚合酶链反应的方法,扩增HCMV UL144基因开放阅读框架（ORF）,扩增阳性的临床株进行双向DNA测序,最后通过DNAclub、Bioedit、DNAstar、GeneDoc等软件进行分析.结果23份HD痉挛段肠组织（46%）及4份尿标本HCMV UL144基因扩增阳性,并且完成测序.种系进化树分析结果显示25个HD患儿的DNA序列分为3个基因型,G1A型64.0%,G2型24%,G3型12%.与对照组比较,经χ^2检验,χ^2=10.93,P为0.012;其中HD临床株G1A和G3型基因经Fisher检验,P为0.015,差异具有统计学意义.全结肠型、长段型及普通型HD分散分布于UL144各个基因型中.结论HD与HCMV感染有关,HCMV可能是HD的病因之一;在HD患儿中,HCMV感染以UL144基因G1A型为主;HD的临床分型与HCMV UL144基因分型无关.     

与人巨细胞病毒UL128两种不同结构蛋白相互作用蛋白的筛选与分析

目的 利用酵母双杂交系统从人胎脑cDNA文库中筛选与两种不同转录结构的人巨细胞病毒(HCMV)UL128编码蛋白相互作用的蛋白,比较两者相互作用蛋白之间的异同点.方法 通过3'RACE和5'RACE技术扩增出两种HCMV UL128片段,其大小分别为519 bp和642 bp,并将其成功构建到酵母诱饵表达载体pGBKT7中.将以上两种酵母表达载体分别转化到酵母菌AH109中,再将文库DNA转化到已含有酵母表达载体的AH109中,筛选与两种片段大小不同的UL128编码蛋白相互作用的人胎脑蛋白,并对筛选得到的阳性克隆进行测序和生物信息学分析.结果 筛出EFEMP2与UL128-519 bp编码蛋白相互作用,THY-1与UL128-642 bp编码蛋白相互作用.结论 成功构建pGBKT7 UL128-519 bp和pGBKT7 UL128-642 bp,并应用酵母双杂交系统分别筛选出EFEMP2和THY-1与UL128-519 bp和UL128-642 bp编码蛋白相互作用,在所筛选得到的相互作用蛋白之间未发现有相同的蛋白,UL128-519 bp和UL128-642 bp所编码的蛋白在HCMV感染致病过程中可能发挥不同的作用.     

人巨细胞病毒UL148基因在临床低传代分离株中的多态性研究

目的 研究人巨细胞病毒(HCMV)UL148序列在临床低传代分离株中的多态性。方法 对38株经荧光定量PCR方法(Q-PCR)检测HCMV-DNA为阳性的临床低传代分离株的细胞培养上清液进行HCMV UL148全序列PCR扩增，并对PCR扩增产物进行序列测定及分析。结果 38株临床低传代分离株有17株PCR扩增阳性，与HCMV Toledo株进行序列比较分析，17株临床分离株ULl48开放阅读框架(ORF)长度均与Toledo株相同。其编码蛋白的氨基酸变异率为0．3％～2．3％。所有分离株均有蛋白翻译后修饰位点的新增或缺失。与Toledo株相比17株临床分离株UL148蛋白质二级结构预测结果均为第15～18位之间由α-螺旋变为β-折叠。结论 17株HCMV临床低传代分离株UL148基因及其编码产物的氨基酸序列比较保守，但仍存在一定程度的多态性。     

人巨细胞病毒UL149基因在临床低传代分离株中的多态性研究

目的研究人巨细胞病毒(humancytomegalovirus,HCMV)UL149序列在临床低传代分离株中的多态性,探讨HCMV基因多态性与其感染引起不同临床症状之间的关系。方法对29株经荧光定量PCR方法(QPCR)检测HCMVDNA为阳性的临床低传代分离株的细胞培养上清液进行HCMVUL149全序列PCR扩增,并对PCR扩增产物进行序列测定及分析。结果29株临床低传代分离株有26株PCR扩增阳性,与HCMVToledo株进行序列比较分析,26株临床分离株UL149开放阅读框架(openreadingframe,ORF)之间存在着高度的多态性,种系进化树分析结果显示26个序列可分为3个型,黄疸患儿分布以G1型为主;小头畸形以G3型为主;巨结肠仅见于G1、G2型,G3型未见。部分临床分离株存在CKP位点的缺失及TKP位点增加。结论HCMVUL149基因在临床分离株中存在着高度的多态性。来自不同临床症状分离株的UL149基因及其编码蛋白具有一定的结构特点,并与基因型呈一定的相关性。     

Human cytomegalovirus tropism for endothelial/epithelial cells: scientific background and clinical implications

Human cytomegalovirus (HCMV) has been routinely isolated from and propagated in vitro in human embryonic lung fibroblast (HELF) cell cultures, while in vivo it is known to infect predominantly endothelial and epithelial cells. In recent years, genetic determinants of the HCMV tropism for endothelial/epithelial cells were identified in the UL131A/UL130/UL128 locus of HCMV genome of wild‐type strains. UL131A‐UL128 gene products form a complex with glycoprotein H (gH) and L (gL) resulting in a gH/gL/UL131A‐UL128 complex that is required for HCMV entry into endothelial/epithelial cells. In contrast, virus entry into fibroblasts has its genetic determinants in the complex gH/gL/gO (or gH/gL). During primary HCMV infection, the neutralising antibody response measured in endothelial cells (EC) is potent, occurs very early and is directed mostly against combinations of two or three gene products of the UL131A‐128 locus. On the contrary, neutralising antibodies measured in fibroblasts appear late, are relatively weak in potency and are directed against gH and gB. The T‐cell immune response to UL131A‐UL128 gene products remains to be investigated. Recently, a role has been proposed for neutralising antibody in conferring prevention/protection against HCMV infection/disease in pregnant women with primary HCMV infection. However, the level of cooperation between humoral immunity and the well‐established T‐cell protection remains to be defined. Copyright © 2010 John Wiley & Sons, Ltd.     

Role of human cytomegalovirus genotype polymorphisms in AIDS patients with cytomegalovirus retinitis

Although several host factors have been identified to influence the course of HCMV infection, it still remains unclear why in AIDS patients without highly active antiretroviral therapy human cytomegalovirus (HCMV) retinitis is one of the most common opportunistic infections, whereas in other immunosuppressed individuals it has a low incidence. It was suggested that HCMV glycoprotein B strains may be suitable as marker for virulence and HCMV retinitis. Moreover, UL144 ORF, a member of the TNF-α receptor superfamily, may play a crucial role in innate defences and adaptive immune response of HCMV infection. Furthermore, sequence analyses of HCMV genes UL128, UL130, and UL131A as major determinants of virus entry and replication in epithelial and other cell types were performed. To evaluate the association of sequence variability of depicted viral genes with HCMV retinitis and in vitro growth properties in retinal pigment epithelial cells (RPE) and human foreskin fibroblasts (HFF), we compared 14 HCMV isolates obtained from vitreous fluid and urine of AIDS patients with clinically proven HCMV retinitis. Isolates were analyzed by PCR cycle sequencing and phylogenetic analysis. In addition, sequences of HCMV strains AF1, U8, U11, VR1814, and its cell culture adapted derivates were included. Sequence analysis of gB yielded three genetic subtypes (gB type 1 (5 isolates), gB type 2 (12 isolates), and gB type 3 (5 Isolates)), whereas sequence analysis of UL144 showed a greater diversity (7 isolates type 1A, 2 isolates type 1C, 7 isolates type 2, and 3 isolates type 3). In contrast, the UL128, UL130, and UL131A genes of all low-passage isolates were highly conserved and showed no preferential clustering. Moreover, in HFF and RPE cells, all of our HCMV isolates replicated efficiently independently of their genetic subtype. In conclusion, beside a possible link between the gB subtype 2 and HCMV retinitis, our study found no direct evidence for a connection between UL144/UL128/UL130/UL131A genotypes and the incidence of HCMV retinitis in AIDS patients.     

Human cytomegalovirus UL131A, UL130 and UL128 genes are highly conserved among field isolates

Summary. Coding sequences of the UL131A, UL130, and UL128 genes of human cytomegalovirus (HCMV) were found to be highly conserved among 34 field isolates from pregnant women with primary HCMV infection and their fetuses or newborns, as well as from solid organ transplant recipients and patients with AIDS. No strain clustering was observed. In contrast, sequencing of UL55 (gB coding gene) allowed the 34 isolates to be clustered into 4 genotypes. The conservation of the UL131A-UL128 locus is consistent with the conclusion that the three encoded proteins are all essential for growth of HCMV in endothelial cells and virus transfer to leukocytes.