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

A study on variability of human cytomegalovirus UL144 gene in low-passage clinical isolates

目的 探讨人类巨细胞病毒 (humancytomegalovirus ,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 6 5株HCMV临床低传代分离株及 7例同年龄组HCMV DNA定量PCR方法检测阳性健康儿尿液进行HCMV UL144PCR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 6 5株分离株中有 5 5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全序列引物PCR扩增为阳性。 6 0份UL144扩增阳性标本HMA SSCP (heteroduplexmobilityassayandsingle strandedconformationpolymorphism)分析呈现 3种典型带形 ,32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区氨基酸序列高度保守。各病种间结果比较显示 ,巨结肠患儿分离株序列没有Ⅱ型 ,小头畸形患儿的序列分布以Ⅰ型为主 ,黄疸患儿以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 ,序列呈现高度多态性 ,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。

Objective Human cytomegalovirus (HCMV) is an important infectious factor that results in neonatal disease and congenital deformity. HCMV may invade many organs. The different symptoms and tissue tropism of HCMV infection perhaps result from the genetic polymorphism of HCMV. Recent study showed that Toledo genome contained 19 open reading frames (denoted UL131 to 151) which were not present in the AD169 genome, leading us to focus on the relationship between HCMV disease and the products of these 19 open reading frames. UL144 open reading frames encode a homologue of the tumor necrosis factor receptor. It seems important to study the strain-specific variability of UL144 sequence in low-passage clinical isolates and to discuss if the variability related to the clinical HCMV infection. Methods HCMV-UL144 gene was amplified by PCR assay in 65 low-passage clinical isolates and urine from 7 healthy children, which were HCMV-DNA positive as shown by QPCR. All the positive PCR products were analyzed by HMA-SSCP (Heteroduplex mobility assay, single-stranded conformation polymorphism) and 32 of them were sequenced. The UL144 sequences of 32 clinical isolates have been assigned GenBank accession No. AF382014 to AF382031 and AF447377 to AF447390. Results 55 isolates and 5 urine samples were HCMV-UL144 positive by PCR. Sequencing and HMA-SSCP analysis showed that significant strain-specific variability was present in UL144 ORFs. Phylogenetic analysis indicated that the nuclear sequences could be classified into 3 major genotypes. Comparing between UL144 sequences and the corresponding symptoms showed that genotype 2 did not exist in megacolon isolates. Genotype 1 and 3 were the major types among microcephaly and jaundice isolates respectively. Conclusion HCMV-UL144 exist in almost all the low passage isolates and the sequence is hypervariability. The characters of sequences in different kinds of isolates showed that UL144 gene might play an important role in HCMV infection. [