Full Genome of Influenza A (H7N9) Virus Derived by Direct Sequencing without Culture - Vol. 19 No. 11 - November 2013 - Emerging Infectious Disease journal - CDC
Volume 19, Number 11—November 2013
Full Genome of Influenza A (H7N9) Virus Derived by Direct Sequencing without Culture
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.
AbstractAn 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.
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.