北京地区2006年流行的3、7和11型腺病毒部分六邻体基因序列分析

Sequence analysis for Hexon genes of types 3, 7 and 11 of adenoviruses identified in Beijing

目的分析2006年北京地区流行的3、7和11型腺病毒（ADV）的部分六邻体基因序列，了解该地区ADV流行型别及六邻体基因变异情况。方法采集急性呼吸道感染患儿咽拭子或鼻咽分泌物标本，分离病毒及免疫荧光快速抗原检测。对抗原检测和／或病毒分离为ADV阳性的61份标本进行PCR分型检测。随机选取分离到的3株ADV3，3株ADV7和3株ADV11毒株，扩增其六邻体近5＇端1278bp基因片段进行序列分析并与GenBank发表的部分序列进行比较和进化树分析。结果61份ADV检测为阳性的标本中，ADV3占60．73％（37／61）；ADV7占27．9％（17／61）；ADV3和ADV7混合感染占1．6％（1／61）；ADV11占4．9％（3／61）；非3、7、11和21型4．9％（3／61）；未检测到21型ADV。3株ADV3分离株与2005年广州分离株（AY878716）亲缘关系最近，与其他株比较，3株北京分离株氨基酸的变异位点主要有3个。3株ADV7分离株与1998年日本分离株（AF053086）的亲缘关系最近。与其他株比较，3株北京分离株氨基酸的变异位点主要有1个。3株ADV11分离株与2004年日本分离株（AB162772）的亲缘关系最近（100％），但是这4株（包括日本的毒株）与其他毒株比较其氨基酸的变异位点有12个。相对于3型和7型，11型ADV的变异最大，表现为变异的氨基酸位点的分布较为分散，其次是3型ADV，而7型ADV的变异较小。结论2006年北京地区引起婴幼儿急性呼吸道感染的ADV以3型为主，7型为辅，11型较为少见，未发现21型；3、7和11型ADV北京分离株与GenBank中其他序列比较虽然有着较高的同源性，但是还都有一定的核苷酸和氨基酸的变异；变异多发生在抗原决定簇密集的HVR1区和HVR7区。

Objective The objective of this study was to develop a rapid, sensitive and specific method for identifying and typing for adenovirus from clinical specimens and to learn about the viruses identified in Beijing on the molecular bases. Methods Primers were designed using hexon gene of adenovirus as target. One primer pair was designed as universal primers for amplifying a 1278 bp gene fragment located at the hexon gene of adenovirus types 3,7,11 and 21. Four primer pairs located within the region of this 1278 bp fragment were designed specifically for amplifying adenoviruses types 3, 7, 11 and 21, which were used for a multiplex nest-PCR in a single tube. The products from this multiplex nest-PCR were 502 bp （for type 3）, 311 bp （for type 7）, 880 bp （for type 11） and 237 bp （for type 21）, respectively. The type of the adenovirus being tested could be determined after agarose eleetrophoresis analysis of the PCR products. Sequencing was performed for part of the Hexon genes at the 5＇ end from types 3, 7 and 11 strains isolated from clinical specimens and the sequences were compared with corresponding genes published in GenBank. Results PCR products with predicted sizes were visualized in the agarose gel for prototype strains of adenovirus types 3, 7, 11 and 21, but not for other respiratory viruses, indicating that the technique is specific for typing without cross reaction with other viruses. Out of 61 clinical specimens which had been proved to be adenovirus positive by tissue culture and/or immunofluerescence assay, 37 were found as adenovirus type 3 （37161, 60.73 % ）, 17 as adenovirus type 7 （17/61, 27.9% ）, 3 was adenovirus type 11 （3/61, 4.9% ）. 1 was positive for both type 3 and 7 （1/61, 1.6 % ）, suggesting that the patient was co-infected with type 3 and 7 adenoviruses. No adenovirus type 21 was detected. Out of the 61 positive specimens, three showed positive on both tissue culture and immunofluerescence but could not be identified under the methods we used, suggesting that these 3 strains （4.9%） were with the types other than types 3, 7, 11 and 21. Data from sequence analysis indicated that adenoviruses types of 3, 7 and 11 in this study shared high homology with corresponding types of the strains published in GenBank. Three of the type 3 adenovirus in this study shared highest homology with the adenovirus type 3 identified in Guangzhou, China in 2005. Three of the type 7 adenovirus shared highest homology with the adenovirus type 7 identified in Japan in 1998 and 3 of type 11 adenovirus shared highest homology with the adenovirus type 11 identified in Japan in 2004. Comparing with types 3 and 7, the type 11 in this study showed highest diversity with the corresponding type in GenBank, indicated by the dispersing of the varied amino acids within the region of HVR1 and HVR7 of the Hexon genes. Conclusion This multiplex nest-PCR method had the advantages of rapid, sensitive and specific and could be used for identifying types of adenoviruses in clinical specimens. Although adenovirus types 3, 7 and 11 from Beijing strains shared high homology with the corresponding genes in GenBank, some variances were noticed,especially in type 11 strains.