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。     

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。     

人巨细胞病毒UL136基因在临床低传代分离株中多态性分析

目的 研究人巨细胞病毒(human cytomegalovirus，HCMV)UL136基因在临床低传代分离株中的多态性，探讨其多态性与HCMV先天性感染不同致病性之间的关系。方法 对48株经荧光定量PCR方法检测HCMV DNA为阳性的临床低传代分离株进行HCMV ULl36全序列PCR扩增，对于扩增阳性的12株PCR产物进行ULl36基因全序列测定及结果分析。结果 48株临床低传代分离株ULl36 PCR扩增，12株阳性，阳性率25％，以HCMV Toledo株作为参考株，进行序列比较分析表明，12株临床分离株ULl36开放阅读框架(open reading frame，ORF)长度均与Toledo株相同，为723bp，编码241个氨基酸的蛋白。DNA序列变异均为碱基替换，不同临床分离株ULl36基因与Toledo株进行同源性比较，结果在核苷酸水平为97．7％～99．3％，氨基酸水平为96．6％～99．1％。ULl36编码蛋白的氨基酸变异率为0．83％～3．3％。二级结构预测分为两种构象。大多数HCMV ULl36蛋白翻译后修饰位点在所有分离株中均高度保守，仅几个位点在一些分离株中存在缺失或新增。Toledo株及12株临床分离株核苷酸及氨基酸序列系统进化树分析表明：45J最接近Toledo株。结论 12株临床低传代分离株HCMV ULl36基因DNA及其编码产物的氨基酸序列比较保守，但仍存在一定多态性。未发现不同临床分离株ULl36基因多态性与HCMV先天性感染的表现关系。     

人巨细胞病毒UL138～142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)UL138～UL14 2基因在临床低传代分离株中的多态性及其与HCMV先天感染致病性之间的关系。方法　对经荧光定量PCR方法检测HCMV DNA为阳性的临床分离株进行UL138～UL14 2基因全序列PCR扩增 ,对扩增阳性的标本进行全序列的测序及结果分析。结果　HCMV临床分离株的UL138、UL14 2ORF(openreadingframe)高度保守 ;UL139ORF呈现高度多态性 ,可被明确划分为 3个基因型 ,且核苷酸及氨基酸的变异主要集中在序列的 5′端 ;所有临床分离株的UL14 0ORF在Toledo株第 174位核苷酸处插入 1个胞嘧啶核苷酸 ,其ORF较Toledo株增加了 2 31个核苷酸 ;所有临床分离株的UL14 1ORF在Toledo株第 2 2 7位核苷酸处缺失了 1个胸腺嘧啶核苷酸 ,故形成UL14 1a及UL14 1b 2个新的ORF。HCMV临床分离株的UL14 0蛋白较Toledo株新增了ScAMP磷酸化和酪蛋白激酶Ⅱ磷酸化位点 ,其它基因编码蛋白的重要功能区域相对保守。结论　HCMV临床分离株UL139ORF的 5′端呈现高度多态性 ,而且被明确地分成 3个基因型 ,故其可能在HCMV先天感染的致病性差异方面起一定作用 ;尚未发现UL138～UL14 2中某个特定基因与HCMV先天感染致病性有本质联系     

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.     

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。     

人类巨细胞病毒编码趋化因子HCMV UL 146序列在临床低传代分离株中的多态性研究

目的探讨人类巨细胞病毒(human cytomegalovirus,HCMV)UL146序列在临床患儿低传代分离株中的多态性及其与临床疾病的关系。方法对23株HCMV临床低传代分离株及2例同年龄组HCMV-DNA定量PCR方法检测阳性健康儿尿液进行HCMV-UL146 PCR扩增及测序分析。结果UL146序列呈现较高的多态性,UL146的序列可以分为3组,所有巨结肠患儿标本均分布在G2组,无症状感染儿均在G2B组,而α化学因子功能域在各序列中保守。结论序列的变异可能会影响UL146吸附中性粒细胞,影响病毒扩散。     

晚期黄疸新生儿人巨细胞病毒感染与US3基因多态性的关系

目的了解晚期黄疸新生儿人巨细胞病毒（HCMV）感染状况,探讨HCMV US3基因多态性与致病性之间的关系。方法应用巢式PCR法检测2010年1～6月在本院新生儿科就诊的79例晚期黄疸新生儿样本HCMV US3基因,阳性标本结果进行双向DNA测序,通过BioEdit、DNAstar、GeneDoc等软件进行序列分析。结果 79例晚期黄疸新生儿中20例HCMV US3基因PCR扩增阳性,阳性率25.3%。以Towne作为参考株,序列比对分析显示,20株临床分离株US3的ORF长度均与参考株相同,为561bp,编码186个氨基酸蛋白。US3核酸变异比较普遍,变异主要集中在序列的N端,大部分是同义突变,US3氨基酸序列高度保守,仅几个位点在少数分离株中存在变异。未发现US3基因多态性与临床致病性的联系。结论 HCMV感染是导致新生儿晚期黄疸的重要原因之一。20株临床分离株HCMV US3基因编码的氨基酸序列比较保守,但仍存在一定的多态性。未发现不同临床分离株US3基因多态性与HCMV引起的新生儿晚期黄疸的联系。     

人巨细胞病毒UL132基因在先天感染患儿临床株中的变异研究

目的研究人巨细胞病毒(human cytomegalovirus,HCMV)UL132基因序列在先天性感染患儿中的多态性,探讨HCMV基因多态性与其感染引起不同临床症状的关系。方法对30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 UL132全序列PCR扩增,并对PCR扩增产物进行序列测定及分析。结果30株HCMV临床株的测序结果与HCMV Toledo株进行序列比较分析显示,临床株UL132开放阅读框架(open reading frame,ORF)间存在着高度的多态性,基因变异主要集中在ULl32 ORF的5’端。30株HCMV临床株种系进化树分析结果显示,UL132序列可分为3个基因型,黄疸患儿分布以G1型为主;神经损伤患儿以G2型为主;巨结肠患儿分别见于G1、G2、G3 3个基因型。所有临床株的ULl32编码蛋白是疏水性蛋白,含有信号肽及跨膜蛋白区域,并在信号肽和跨膜蛋白区域之间存在2个保守的N-糖基化位点。结论HCMV UL132基因在临床株中存在着高度的多态性。来自不同临床症状的HCMV UL132基因及其编码蛋白具有一定的结构特点,提示观132基因及其所编码的蛋白在HCMV的致病性上可能起重要作用。     

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。     

Sequence variability of the human cytomegalovirus UL141 Open Reading Frame in clinical strains

Summary. Human cytomegalovirus (HCMV) displays genetic polymorphisms. HCMV disease and tissue tropism may be related to specific genomic variability among strains. This work analyzed the genetic polymorphism of UL141 open reading frame (ORF), one of the genes in HCMV UL/b′ region, from 21 clinical strains. 8 previously published UL141 sequences in the GenBank were used for sequence comparison. Detailed sequence analysis showed that the UL141 gene was highly conserved at both the nucleotide and amino acid level. The coding regions were identical in size. The nucleotide and amino acid sequence identities among all strains were 96.9–100% and 97.6–100%, respectively.     

Human cytomegalovirus a sequence and UL144 variability in strains from infected children

Human cytomegalovirus (HCMV) displays genetic polymorphisms. This variability may contribute to strain-specific tissue tropism and disease expression in HCMV-infected humans. To determine strain variability in a sequence and UL144 gene regions, 51 low-passage isolates from 44 HCMV-infected children were studied. Isolates were obtained from 28 healthy children attending child care centers in Iowa and from 16 congenitally infected infants born in Texas. Isolates demonstrated substantial nucleotide variation in each gene region. Phylogenetic analysis of a sequence variability allowed 39 isolates to be grouped into six clades. The largest clade contained 16 isolates with > or = 95% nucleotide homology. Forty-eight of the 49 HCMV isolates yielding UL144 amplicons was grouped according to the clades described a few years ago [Lurain et al. (1999) Journal of Virology 73:10040-10050]. No linkage was observed among a sequence, UL144, and glycoprotein B (gB; UL55) polymorphisms. Four Texas and 11 Iowa isolates displayed > or = 95% sequence homology for a sequence and UL144 regions and possessed identical gB genotypes. No relationship between UL144 polymorphisms and outcome of congenital HCMV infection was observed. These data indicate that HCMV strains circulating among young children have UL144 polymorphisms similar to those of HCMV strains excreted by immunocompromised adults. Identification of conserved nucleotide sequences among Iowa and Texas children suggests genetic stability and biologic importance of these gene regions.     

Polymorphisms of human cytomegalovirus UL148A, UL148B, UL148C, UL148D genes in clinical strains.

BACKGROUND: Clinical isolates of human cytomegalovirus (HCMV) display polymorphisms in multiple genes. Some authors have suggested that polymorphisms are implicated in HCMV-induced immunopathogenesis, as well as in strain-specific behaviors, such as tissue-tropism and the ability to establish persistent or latent infections. OBJECTIVE: To describe the features of HCMV UL148A, UL148B, UL148C and UL148D open reading frames (ORFs) and the variable sites within the frames in clinical strains. STUDY DESIGN: PCR was performed to amplify these ORFs in 22 clinical strains. PCR amplification products were sequenced directly and analyzed. RESULTS: The nucleotide diversity of UL148A, UL148B, UL148C and UL148D ORFs in studied strains is 0.5-8.3%, 0.5-4.6%, 0.5-3% and 1.7-8.1%, respectively; the amino acid diversity of their putative proteins is 1.3-6.3%, 1.3-5.0%, 1.3-3.9% and 1.7-8.1%, respectively, related to the Merlin strain. The modification sites of UL148A, UL148B, UL148C and UL148D predicted proteins from strains in unpassaged urine samples were conserved, except for strain U96, compared with that of the Merlin strain. By phylogenetic and statistical analysis, the UL148A and UL148D sequences of clinical strains were classified into three groups. CONCLUSION: Compared to the UL148A, UL148B and UL148D ORFs, the UL148C ORF was relatively conserved, as was the amino acid sequence of the UL148C putative protein. Isolates that have been passaged several times in human embryonic lung fibroblasts (HELF) showed some changes of modification sites, however. A discrete linkage was found between the groups of UL148A gene and those of UL148D gene.     

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 genetic variability in strains isolated from Japanese children during 1983-2003

The genetic variability of 74 human cytomegalovirus (HCMV) clinical isolates from 60 Japanese infants and children during 1983-2003 was investigated, and the relevance to their clinical course was studied. The patients consisted of 10 asymptomatic congenitally infected babies, 45 infected perinatally or postnatally resulting in HCMV mononucleosis/hepatitis and 5 immunocompromised hosts. The hypervariable region of the HCMV genome, that is the a sequence and UL144 region was analyzed using the polymerase chain reaction (PCR) and unrooted phylogenetic trees. HCMV glycoprotein B (gB) polymorphism was also studied. Unrooted phylogenetic trees of a sequence and UL144 allowed the isolates to be grouped to 5 and 3 clades, respectively. Three gB genotypes were also determined. However, there was no correlation between specific genotypes of these three genes and clinical forms, except for congenital infection which fell into one of three clades of the UL144 gene. In addition, the variability of the three genes had no correlation with each other. This implies that study of a single gene is insufficient for investigating the molecular epidemiology of HCMV. This study provides basic data on the genetic variability of HCMV in an Asian population and should help to determine the strains for vaccine candidates.