[Sequence analysis for Hexon genes of types 3, 7 and 11 of adenoviruses identified in Beijing].
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 electrophoresis 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 immunofluorescence assay, 37 were found as adenovirus type 3 (37/61, 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 immunofluorescence 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.


CONCLUSIONS
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.
Deng
Jie
J
Laboratory of Virology, Beijing Municipal Laboratory of Infection and Immunity, Capital Institute of Pediatrics, Beijing 100020, China.
Qian
Yuan
Y
Zhao
Lin-qing
LQ
Zhu
Ru-nan
RN
Wang
Fang
F
Sun
Yu
Y
D000256:Adenoviridae / Q000145:classification / Q000235:genetics* / Q000302:isolation & purification; D000956:Antigens, Viral / Q000032:analysis; D036022:Capsid Proteins / Q000235:genetics*; D002648:Child; D017931:DNA Primers; D004279:DNA, Viral / Q000235:genetics; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA / Q000379:methods*
D000956:Antigens, Viral; D036022:Capsid Proteins; D017931:DNA Primers; D004279:DNA, Viral; C018315:hexon capsid protein, Adenovirus