北京地区近5年来儿童EB病毒感染疾病中BZLF1及其启动区Zp基因特征分析

Characterization of BZLF1 Gene and Its Promoter Zp of EBV Strains in Children with EBV-associated Diseases in Recent 5 Years in Beijing Area

为了解儿童原发性Epstein-Barr病毒(Epstein-Barr virus,EBV)感染流行株BZLF1基因及其启动区Zp的基因特征。本文对北京儿童医院2006年至2011年收治的EBV感染传染性单核细胞增多症(EBV-associated infectious mononucleosis,EBV-IM)134例和EBV相关噬血细胞性淋巴组织细胞增生症(EBV-associated hemophagocytic lymphohistiocytosis,EBV-HLH)32例患儿的外周血来源的DNA进行了EBNA3C、BZLF1和Zp基因片段的扩增分析。根据EBNA3C基因片段扩增产物的大小进行EBV-Ⅰ/Ⅱ分型,对BZLF1和Zp基因扩增产物进行直接序列测定,并用BioEdit 7.0.9进行序列分析。实验显示①儿童EBV感染流行株以EBV-I型为主,占97.2%(140/144),EBV-Ⅱ型为2.8%(4/144);②共检测出3种BZLF1基因型,12种亚型(包括6种新发现的亚型)。BZLF1-A型和BZLF1-B型两基因型在两种疾病中的分布无统计学差异(P=0.083)。BZLF1-A1是儿童EBV感染性疾病中的常见基因型。BZLF1-A型第一内含子以3个29bp重复序列为主,而B型以30bp重复序列为主(P=0.000),且重复序列的个数波动于1-13个不等;③共检测出Zp-P、Zp-V3、Zp-V4、Zp-V1四种Zp基因型,并且这四种基因型在EBV-IM和EBV-HLH两种疾病中的检出率无统计学差异(p值分别为0.272、0.252、1.0、1.0);④BZLF1和Zp基因连锁分析显示,BZLF1-A1基因型毒株的Zp基因分型以Zp-V3为主(P=0.000),而BZLF1-B4基因型以Zp-P型为主(P=0.000)。EBV-Ⅰ+BZLF1-A1与Zp分型中Zp-V3高度连锁(P=0.000);EBV-Ⅰ+BZLF1-B4与Zp-P高度连锁(P=0.000)。结果证实:①BZLF1-A1是儿童EBV感染的常见基因型。BZLF1-A型其第一内含子重复序列以29bp为主,而BZLF1-B型则以30bp的重复序列为主。②Zp-P和Zp-V3是儿童EBV感染的常见Zp基因型,二者检出率相似。③BZLF1-A1型的Zp分型以Zp-V3为主,而BZFL1-B4型则以Zp-P分型为主。EBV-Ⅰ+BZLF1-A1与Zp分型中Zp-V3高度连锁,而EBV-Ⅰ+BZLF1-B4与Zp-P高度连锁。

This study aims to investigate the genetic characteristics of BZLF1 gene and its promoter Zp of the epidemic strains in children with primary Epstein-Barr virus （EBV）-associated diseases. Total DNA was extracted from the peripheral blood of 134 children with EBV-associated infectious mononucleosis （EBV-IM） and 32 children with EBV-associated hemophagocytic lymphohistiocytosis （EBV-HLH） who were admitted to Beijing Children＇s Hospital from 2006 to 2011. The EBNA3C, BZLF1, and Zp genes were amplified by PCR assay. Typing of EBV was performed according to the size of the amplification product of EBNA3C gene; the amplification products of BZLF1 and Zp genes were subjected to direct sequencing, and sequence analysis was performed using BioEdit 7.0.9. The results were as follows： （1）EBV-1 was present in 140 samples （97.2%, 140/144） and EBV-II in 4 samples （2.8%, 4/144）. （2）Three BZLF1 genotypes and their 12 subtypes （including 6 newly found subtypes） were detected in this study; there were no significant differences in the frequencies of BZLF1-A and BZLF1-B between the children with EBV-IM and EBV- HLH （P= 0. 083）; BZLF1-A1 was the dominant genotype in children with EBV-associated diseases; t BZLF1-A mostly had three 29-bp repeats in the first intron of BZLF1 gene, and BZLF1-B mostly had 30-bp repeats （P=0. 000）, with the number of repeats varying from 1 to 13. （3）Four Zp genotypes were detected in this study, including Zp-P, Zp-V3, Zp-V4, and Zp-V1; there were no significant differences in the frequencies of these Zp genotypes between children with EBV-IM and EBV-HLH （P=0. 272, 0. 252, 1.0, and 1.0, respectively）. （4）The linkage analysis of BZLF1 gene and its promoter Zp showed that BZLF1-A1 was highly associated with Zp-V3 （P=0. 000）, while BZLF1-B4 with Zp-P （P=0. 000） ; EBV-I＋BZLF1- A1 was highly associated with Zp-V3 （P=0. 000）, while EBV-I＋BZLF1-B4 with Zp-P （P=0. 000）. The conclusions are as follows： （1）BZLF1-A1 is the dominant genotype in children with EBV-associated diseases; there are mostly 29-bp repeats in the first intron of BZLF1 gene for BZLF1-A genotype and 30-bp repeats for BZLF1-B genotype. （2）Zp-P and Zp-V3 are dominant Zp genotypes of EBV in children, which shared similar detection rates. （3）BZLF1-A1 is highly associated with Zp-V3, while BZLF1-B4 with Zp-P; EBV-I＋BZLF1-A1 is highly associated with Zp-V3, while EBV-Iq-BZLF1-B4 with Zp-P.