1769. Identification of Influenza H1N1 Quasi-Species Using Deep Targeted Next Generation Sequencing
Session: Poster Abstract Session: Viral Infections; Pathogenesis and Epidemiology
Saturday, October 5, 2013
Room: The Moscone Center: Poster Hall C

Influenza causes severe morbidity and mortality. The infidelity of RNA polymerase is responsible for antigenic drift. Influenza hemagglutinin (HA) protein attaches to sialic acid receptors on the host cell surface and is responsible for infection. Deep targeted resequencing with next generation systems can sensitively detect HA gene mutations in serial samples, revealing quasi-species within individuals.


Eight patients with 2011 H1N1 had 17 nasopharyngeal swabs collected over eight days (six patients at days 1 and 5, one at days 1 and 8, and one at days 1, 3, and 5) from the placebo arm of a phase 2B clinical trial. Samples underwent RNA extraction, HA gene amplification by RT-PCR, and sequencing using Illumina MiSeq.  We developed a next generation sequencing analysis procedure to sensitively detect minor alleles from complex mixtures. Using PROVEAN protein analysis, mutations were categorized as deleterious or neutral based on the amino acid substitution and degree of conservation of the residue in related sequences.


Greater than 20 unique coding mutations with > 1% minor allele frequency (MAF) were detected in 7 patients at a single time point, representing allelic expansions and reductions over time. A single mutation, P182Q, was found in two patients. All other mutations were present in only one patient. Six mutations in 5 patients were detected at antigenic or glycosylation sites. Seven mutations in 5 patients were detected with > 5% MAF and represent a greater allelic drift. Of these, one was found at high (56%) MAF at the second time point in an individual and was considered deleterious. The increased allelic frequency reflects an allelic expansion over time of flu quasispecies containing this mutation. Of coding mutations that appeared at <5% MAF, 12 were considered deleterious, and 2 of 12 appeared at antigenic and glycosylation sites. Two other mutations found at Cb and Sa antigenic sites were considered neutral.


Within a patient, changes in minority mutated viral populations can be detected over time during influenza infection. A small proportion of these changes located in the antigenic sites and other highly conserved regions may be deleterious to normal protein function and thus play a role in antigenic drift.

Anna M. Cushing, BS1, Mark A. Winters, MA2,3, Amanda Kamali, MD4, Erik S. Hopmans, MSc1, Ronald Moss, MD5, Mark Holodniy, MD2,6,7 and Hanlee Ji, MD1, (1)Oncology, Stanford University, Stanford, CA, (2)VA Palo Alto Health Care System, Palo Alto, CA, (3)Medicine, Stanford University, Stanford, CA, (4)Infectious Disease and Geographic Medicine, Stanford University, Stanford, CA, (5)Ansun Biopharma, San Diego, CA, (6)Department of Veterans Affairs, Stanford, CA, (7)Infectious Disease and Geographic Medicine, Stanford University, Palo Alto, CA


A. M. Cushing, None

M. A. Winters, None

A. Kamali, None

E. S. Hopmans, None

R. Moss, Ansun BioPharma: Employee, Salary

M. Holodniy, None

H. Ji, None

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