1例人感染腺病毒55型病原体的分离鉴定及进化分析

Isolation, identification and evolutionary analysis of a human infected by adenovirus type 55

目的对1例人感染腺病毒55型病原体进行分离鉴定及进化分析。方法选取2015年5月安庆市某流感监测哨点医院采集的一例被诊断为急性上呼吸道感染患者的咽拭子标本作为研究样本，提取标本总核酸，采用人腺病毒通用引物进行real—time荧光定量PCR检测。将检出的阳性标本感染Hep－2细胞进行病毒分离。对成功分离的腺病毒毒株进行六邻体基因的扩增、纯化并测序。测序结果提交GeneBank上Blast，同源分析确定型别、制作系统进化树以及分析六邻体基因和氨基酸序列。结果对咽拭子标本中的病毒核酸进行荧光定量PCR检测，结果显示，腺病毒核酸DNA检测呈阳性（Ct〈24）。同源比对结果显示，该株的六邻体（Hexon）基因与辽宁株、江苏株、河北株、山西株、安庆株（GenBank号分别为KP896483、KX289874、KP896478、FJ643676、KP279748）的同源性可达100％，与安徽株（KC551973）可达99．96％，存在一个核苷酸C→T的无义突变。进一步的分析显示，该株与参考株在系统发生树上处于同一进化分支，Hexon蛋白氨基酸的同源性为100％。结论此ADR标本由人腺病毒55型感染引起，其Hexon蛋白氨基酸没有出现变异。

Objective To isolate, identify and to perform evolution analysis of an adenovirus type 55 strain from one human case of infection. Methods The swab specimen from a patient diagnosed as acute upper respiratory tract infection was collected by an influenza surveillance sentinel hospital in Anqing city in May 2015 and was selected as the research sample. Total nucleic acids were extracted from the specimen. Human adenovirus universal primer was used to detect the virus by the real- time fluorescence quantitative PCR. The sample was then innoculated to Hep-2 cells for virus isolation. The hexon gene of the isolated strain were amplified and the DNA product was purified and sequenced. The result was submitted to GeneBank to determine the genotype, and to made phylogenetic trees for the analysis of hexon gene and the amino acid sequence. Results The resultof viral nucleic acid in throat swab specimens by fluorescent quantitative PCR was positive （Ct 〈 24）. Homologous comparison results showed that the hexon gene of the strain was up to 100% of similarity to that of Liaoning strain, Jiangsu strain, Hebei strain, Shanxi strain and Anqing strain （GenBank accession number： KP896483, KX2898744, KP896478, FJ643676 and KP279748）, and was 99.96% similar to Anhui strain （KC551973）. There was a a nonsense mutation of C→T. Further analysis showed that the strain was in the same branches of the reference strains in the phylogenetic tree. The amino acid homology of hexon protein was 100%. Conclusions This ADR case was caused by human adenovirus 55 infection. No mutation of amino acids sequence of the hexon protein were found.