人巨细胞病毒无症状性感染者UL144基因多态性研究

Analysis of Polymorphisms in the HCMV UL144 Gene in Clinical Isolates from Children with Asymptomatic Infection

为探究人巨细胞病毒（HCMV）无症状性感染者UL144基因多态性,本研究采用聚合酶链反应扩增HCMVDNA阳性无症状性感染者尿沉渣标本UL144基因开放读码框（ORF）,阳性结果进行双向DNA测序,通过生物信息学工具BioEdit、DNAstar、Mega5.0、GeneDoc等软件进行序列特征分析。结果显示,在50例HCMV-DNA阳性无症状性感染者尿沉渣标本中,21例标本UL144基因扩增阳性,阳性率为42%,并完成测序。同源性比较显示核苷酸同源性为80.2%～100%,氨基酸同源性为77.8%～100%。种系进化树分析显示21例无症状性感染者DNA序列主要分为2个基因型,A型（10例,47.61%）和B型（11例,52.38%）,未见其他基因型。Expasy数据库预测分析UL144基因编码产物重要功能基团,结果显示ASN、PKC、TNFR、NCD3G等翻译后修饰位点高度保守。与UL144A型相比,UL144B型在1-16和30-96氨基酸残基之间各增加了一个PROKAR_LIPOPROTEIN位点和ZF_CTCHY位点。与Toledo标准病毒株比较,UL144A型的CRD1和CRD2区域高度保守,UL144B型的CRD1和CRD2功能区变异较大,但跨膜区（transmembrane domain）和胞浆区（cytoplasmic domain）在UL144A型和B型中均高度保守。UL144A型和B型碱基变异未对UL144蛋白的预测等电点和二级结构造成较大改变。总之与HCMV显性感染者不同,HCMV无症状性感染者UL144基因型为A型和B型。

The purpose of this study was to examine polymorphisms in the human cytomegalovirus（HCMV）UL144gene in children with asymptomatic HCMV infection.PCR was performed to amplify the UL144 open reading frame（ORF）from urine specimens of asymptomatic HCMV-DNA positive children and both strands of the amplicon were sequenced.Sequence analysis was performed with software including BioEdit,DNAstar,Mega5.0and GeneDoc.Twenty-one of 50 clinical strains were successfully amplified and sequenced,giving apositive rate of detection of 42%.Nucleotide sequence homology ranged from 80.2%to100% and amino acid sequence homology ranged from 77.8%to 100%.The UL144 sequences were distributed among two genotypes,type A（47.61%）and type B（52.38%）.The Expasy database was used to analyze the important functional motifs of the UL144 protein.These results revealed that there was a high level of conservation of post-translational modification sites including ASN, PKC, TNFR,and NCD3 G.UL144type B added a PROKAR-LIPOPROTEIN site and ZF-CTCHY site between amino acid residues 1 and 16 and between amino acid residues 30 and 96,respectively,as compared with type A.Compared to the UL144 gene from the Toledo strain,there was a high level of conservation in the CRD1 and CRD2of UL144 type A,while significantly more variability was observed in CRD1 and CRD2of UL144 type B.The transmembrane and cytoplasmic domains were highly conserved in both UL144 type A and type B.Variation in nucleotide sequences of UL144 type A and type B did not cause major changes to the predicted isoelectric point or secondary structure of the UL144 protein.The UL144 genotype of children with asymptomatic HCMV infection was divided into type A and type B,which was different from children with symptomatic HCMV infection.