Full Genome of Influenza A (H7N9) Virus Derived by Direct Sequencing without Culture

Xianwen Ren¹, Fan Yang¹, Yongfeng Hu¹, Ting Zhang¹, Liguo Liu¹, Jie Dong, Lilian Sun, Yafang Zhu, Yan Xiao, Li Li, Jian Yang, Jianwei Wang, and Qi Jin

Author affiliations: MOH Key Laboratory of Systems Biology of Pathogens, Beijing, China

Abstract

An epidemic caused by influenza A (H7N9) virus was recently reported in China. Deep sequencing revealed the full genome of the virus obtained directly from a patient’s sputum without virus culture. The full genome showed substantial sequence heterogeneity and large differences compared with that from embryonated chicken eggs.

Recently, a novel influenza A (H7N9) virus infected humans in China (1,2), leading to great concerns about its threat to public health (3). However, almost all the current genomes of the novel subtype H7N9 virus have been sequenced after culture in embryonated chicken eggs or mammalian cells. Switching the evolutionary selection pressure from in vivo human respiratory tract to embryonated chicken eggs might introduce mutations into the final genome sequences during culture (4). We report determination of the full genome of the influenza A (H7N9) virus derived directly by deep sequencing, without virus culture, from a sputum specimen of an infected human. Deep sequencing provides a direct way to evaluate the genome characteristics and potential virulence and transmissibility of the novel influenza A (H7N9) virus.

The Study

We collected a sputum specimen from a 54-year-old woman with fever, cough, sputum production, and pneumonia. Influenza A (H7N9) virus was detected in the specimen by specific real-time reverse transcription PCR (RT-PCR). The specimen was then processed with a viral particle–protected nucleic acid purification method (5). Total RNA was extracted and amplified by sequence-independent PCR (5) and then sequenced with an Illumina/Solexa GAII sequencer (Illumina, San Diego, CA, USA). Reads generated by the Illumina/Solexa GAII with lengths of 80 bases were directly aligned to those nucleotide sequences of influenza A viruses in the National Center for Biotechnology Information nonredundant nucleotide database by the blastn program in the BLAST (6) software package, version 2.2.22 (www.ncbi.nlm.nih.gov/blast) with parameters −e 1e−5 −F T (−e 1e−5 for selection of highly similar reads and −F T for masking the low-complexity reads) after filtering of the sequence adapters and RT-PCR primers. No assembly was performed before alignment. We obtained 19,177 reads aligned to influenza A viruses.