腺病毒55型感染肺炎患者的病原学鉴定及全基因序列分析

Identification and complete genome sequencing of human adenovirus type 55 isolated from a patient with pneumonia

目的对1例不明原因肺炎患者进行病原学诊断,分析其病原学特征及重组特点,为其防治提供参考。方法采集患者的咽拭子标本,提取DNA,采用Real-time荧光定量PCR法检测病原体特异性片段;采用Hep-2细胞培养分离病毒;通过Sanger测序hexon基因进行型别鉴定,通过高通量测序技术对病毒进行全基因组测序,采用MEGA6.0软件进行序列比对,通过邻近归并法(Neighbor-Joining)构建种系发生进化树分析基因特征,运用Simplot软件进行序列重组分析。结果从患者咽拭子标本中检测并分离到腺病毒,hexon基因测序后进行比对分析,其与腺病毒B组55型及11型同源性较高;全基因测序显示该病毒与HAdV-55QS-DLL及Shanxi/QZ01/2011株同源性较高,在E1B、DNA-Polymerase、100Ka Hexon和Fiber编码区存在氨基酸变异;种系发生进化树显示该毒株与腺病毒14型同属一支,与腺病毒11型进化分支较近;相似性曲线及BootScan分析显示该病毒株于hexon编码区存在HAdV-11与HAdV-14型间重组。结论本例患者不明原因肺炎由腺病毒B组55型感染引起,该病毒由HAdV-11与HAdV-14重组产生,其生物学特性有待进一步研究。

Objectives To identify a possible pathogen from a patient with pneumonia and to analyze its etiological characteristics and recombination features. Methods DNA was extracted from throat swabs of patients, and real-time qPCR was used to examine the unknown pathogen. The sample was inoculated into Hep-2 ceils and cultured to isolate the unknown virus. The genotype of a human adenovirus （HAdV） isolate was determined based on hexon genes identified using Sanger sequencing. The genomic sequence of the isolate was obtained using the next-generation sequencing （NGS） Illumina MiSeq Platform. MEGA6.0 software was used to align nucleotides. The phylogenetie and molecular characteristics of the isolate were analyzed using the neighbor-joining method, and recombination was analyzed using Simplot software. Results A specific DNA fragment of the HAdV was detected with real-time qPCR. The hexon gene of the HAdV isolate had a high level of sequence similarity to HAdV-B55 and HAdV-11. The complete genome sequence had 99.99G nucleotide sequence similarity to the QS-DLL strain of HAdV-55 and 99. 98% nucleotide similarity to the Shanxi/QZ01/2011 strain of HAdV-55. The amino acid sequences had several mutations in regions coding for E1B, DNA polymerase, 100-ka hexon, and fiber. Phylogenetic analysis indicated that this strain belonged to the same clade as strains of HAdV-14 and that it was closely related to strains of HAdV-11. Similarity plots and bootsean analysis revealed a recombinant hexon gene from HAdV-11 and HAdV-14. Conclusion Pneumonia was caused by HAdV-55, and HAdV- B55 evolved from an intertypic recombination event between HAdV-11 and HAdV-14. Its biological characteristics need to be studied further.