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。     

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     

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。     

人类巨细胞病毒UL144基因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)UL144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65株HCMV临床低传代分离及7例同年龄组HCMV－DNA定量PCR方法检测阳性健康儿尿液进行HCMV-UL144 PCR扩增分析，对HCMV-UL144扩增阳性标本进行HMA－SSCP分析，并对其中32个阳性标本进行HCMV-UL144基因全序列测定及分析。结果：65株分离株中有55株UL144全序列引物PCR扩增为阳性，7份QPCR检测HCMV-DNA阳性健康儿尿液中5份UL144全序列PCR扩增为阳性，60份UL144扩增阳性标本HMA－SSCP（heteroduplex mobility assay and single-stranded conformation polymorphism)分析呈现3种典型带形，32例个测序标本序列呈现较高的多态性，差异多位于序列的前半部，种系发生图谱分析可大致分为3组，二级结构预测表现为6种类型，在重要的蛋白质功能的序列分布以Ⅰ型为主，黄患儿以Ⅲ型为主。32个HCMV-UL144序列已被GenBank收录，结论:HCMV-UL144广泛存在于临床低传代分离株中，序列呈序高度多态性，不同疾病类型的HCMV-UL144序列不同，提示UL144基因可能在HCMV致病上起一定的作用。     

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。     

人巨细胞病毒临床分离株UL131A-128mRNA转录方式的研究

目的 研究人巨细胞病毒（ human cytomegalovirns,HCMV） UL131 A-128序列在临床低传代分离株中的转录方式.方法 用3＇RACE技术扩增HCMV不同临床株,不同时期的UL131A,UL128,UL130的mRNAs并测序分析;利用PCR技术在HCMV临床株cDNA文库中筛选分别含有UL13IA,UL130,UL128序列的阳性克隆,测序并分析结果.结果 成功在临床低传代分离株中得到UL13IA,UL128,UL130基因的转录结构,UL131A,UL130的转录方式和文献报道一致,UL131A含有两个外显子,UL130为此区域内唯一不含内含子的基因.而UL128基因的转录本呈现了两种转录方式,一种结构包含三个外显子,长度为519 bp;而另一种结构包含三个外显子和第一内含子结构,长度为642 bp,在不同病毒株的不同时期均显示了包含第一内含子结构转录本的含量明显高于仅有外显子结构的UL128转录本的含量.UL131A-128三个基因在病毒的即刻早期,早期,晚期均存在.3＇RACE得到结果与HCMV cDNA文库筛选得到的结果相一致.结论 UL131A,UL130和UL128基因的mRNA结构的起始位点不同,但他们在3’末端拥有共同的终止位点.HCMV临床株UL 131A-128基因转录方式的复杂结构可能在HCMV感染的不同时期具有重要作用.     

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.     

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.     

The virulence-determining genomic BamHI fragment 4 of pseudorabies virus contains genes corresponding to the UL15 (partial), UL18, UL19, UL20, and UL21 genes of herpes simplex virus and a putative origin of replication.

The genomic BamHI-fragment 4 of pseudorabies virus (PrV) has previously been shown to encode functions necessary for expression of PrV neurovirulence (B. Lomniczi, S. Watanabe, T. Ben-Porat, and A. S. Kaplan, 1984, J. Virol. 52, 198-205). To identify proteins that might be involved in the neurotropism of PrV we sequenced the complete 9382-bp fragment BamHI-4, the longest contiguous sequence determined in the UL region of PrV so far, and analyzed its coding capacity. In an arrangement similar to that found in herpes simplex virus type 1 we identified complete open reading frames encoding proteins with strong homology to the UL18 (50% homology), UL19 (60% homology), UL20 (33% homology), and UL21 (36% homology) polypeptides and the 3'-part of a gene homologous to UL15 (67% homology) of HSV-1. In addition, a consensus sequence for an alphaherpesviral origin of replication was found at the left terminus of the fragment.     

Variability of UL18, UL40, UL111a and US3 immunomodulatory genes among human cytomegalovirus clinical isolates from renal transplant recipients.

BACKGROUND: Variability of human cytomegalovirus (HCMV) genes counteracting immune responses is poorly investigated in non-cultured clinical strains. OBJECTIVES: In HCMV-infected renal graft recipients, we aimed to (i) investigate the variability of four HCMV immunomodulatory genes, without any culture-related viral selection, (ii) provide evolutionary sequence data, and (iii) study co-existing HCMV variants and their evolution. STUDY DESIGN: UL18, UL40, UL111a and US3 were sequenced in 31 blood samples from 17 patients (8 with sequential samples). Cloning of UL40 PCR products was performed in one donor-positive/recipient-positive (D+/R+) patient's samples. RESULTS: Each patient harboured a unique strain (combination of four genes), however single identical genes were demonstrated among various patients, suggesting recombination events. Sequencing showed in D+/R- recipients, either complete gene stability (four patients) or significant variability (one patient); in three D+/R+ patients, multiple gene variations, possibly linked to super- or co-infections. Cloning evidenced different variants at each time point with an increasing variability over time, illustrating possibly viral reactivations and the subsequent evolution of the variants mixture. CONCLUSION: A noticeable HCMV natural polymorphism was shown, with different evolutive patterns. Moreover, we described the co-evolution of variants mixtures in one patient. Consequences on HCMV infection and graft function deserve further studying.     

Distribution of UL144, US28 and UL55 genotypes in Polish newborns with congenital cytomegalovirus infections

Human cytomegalovirus (HCMV) is the most common congenital infection. HCMV strains display genetic variability in different regions. Distribution of HCMV genotypes in the population of congenitally infected newborns from Central Poland and viral load in newborns' blood is described and discussed. HCMV isolates were analysed by sequencing at three sites on the genome: the UL144 tumour necrosis factor-alpha (TNFα)-like receptor gene, the US28 beta-chemokine receptor gene and the UL55 envelope glycoprotein B (gB) gene. The newborns' blood was examined for HCMV DNA with a nested (UL144, UL55) or heminested (US28) polymerase chain reaction, and the genotypes were determined by sequence analysis. HCMV DNA was detectable in 25 out of 55 examined newborns born by HCMV-infected mothers (45.5%). The blood viral load in mother-infant pairs was determined. Most of the newborns had identical virus genotype, gB2 (96%), UL144 B1 (88%) and US28 A2 (84%). These genotypes were detected in all newborns with asymptomatic congenital infection. The occurrence of UL144 B1 or US28 A2 genotypes in the babies examined was significant in comparison to other genotypes (p=0.0002 and p=0.040 respectively). There was no association between specific gB subtypes in all patients groups (p=0.463). There was no correlation between HCMV genotypes and the outcome.     

Association of Two Membrane Proteins Encoded by Herpes Simplex Virus Type 2, UL11 and UL56

Herpes simplex virus (HSV) acquires envelope by budding into trans-Golgi network (TGN)-derived vesicles. Previous studies showed that the UL11 gene product enables efficient virion envelopment and export from infected cells and is incorporated into virions as tegument protein. At its N-terminus, UL11 is dually acylated by myristoic and palmitoic acids. Fatty acylation of UL11 provides both membrane binding strength and Golgi-targeting specificity. We show here that UL11 interacts with UL56 protein, a tail-anchored type II membrane protein encoded by HSV, which associated with the Golgi apparatus and cytoplasmic vesicles. We previously showed that UL56 is involved in vesicular transport in infected cells. The UL11–UL56 complex localized to the perinuclear region of the cytoplasm in infected cells. Fatty acylation of UL11 was important for the formation of the UL11–UL56 protein complex. Taken together, our results identify a novel interaction between two HSV proteins facilitated by mutual interactions with Golgi-derived vesicles.