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rename figures to match manuscript, add SRA accessions to README

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lmoncla committed Jun 11, 2019
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# H5N1 within-host diversity in humans and poultry in Cambodia
# Quantifying within-host evolution of H5N1 influenza in humans and poultry in Cambodia

#### Louise H. Moncla<sup>1</sup>, Trevor Bedford<sup>1,2</sup>, Philippe Dussart<sup>3</sup>, Philippe Buchy<sup>4</sup>, Huachen Zhu<sup>5,6</sup>, Thomas C. Friedrich<sup>7,8</sup>, Paul F. Horwood<sup>9,10</sup>
#### Louise H. Moncla<sup>1</sup>, Trevor Bedford<sup>1,2</sup>, Philippe Dussart<sup>3</sup>, Srey Viseth Horm<sup>3</sup>, Sareth Rith<sup>3</sup>, Philippe Buchy<sup>4</sup>, Erik A Karlsson<sup>3</sup>, Lifeng Li<sup>5,6</sup>, Yongmei Liu<sup>5,6</sup>, Huachen Zhu<sup>5,6</sup>, Yi Guan<sup>5,6</sup>, Thomas C. Friedrich<sup>7,8</sup>, Paul F. Horwood<sup>9,10</sup>

<sup>1</sup>Fred Hutchinson Cancer Research Center, Seattle, Washington, United States, <sup>2</sup>University of Washington, Seattle, Washington, United States, <sup>3</sup>Virology Unit, Institut Pasteur du Cambodge, Phnom Penh, Cambodia, <sup>4</sup>GlaxoSmithKline, Vaccines R&D, Singapore, Singapore,<sup>5</sup>Joint Influenza Research Centre (SUMC/HKU), Shantou University Medical College, Shantou, People's Republic of China,<sup>6</sup>State Key Laboratory of Emerging Infectious Diseases/Centre of Influenza Research, School of Public Health, The University of Hong Kong, Hong Kong, SAR, People's Republic of China,<sup>7</sup>Department of Pathobiological Sciences, University of Wisconsin School of Veterinary Medicine, Madison, WI, United States,<sup>8</sup>Wisconsin National Primate Research Center, Madison, WI, United States,<sup>9</sup>Papua New Guinea Institute of Medical Research, Goroka, Paula New Guinea,<sup>10</sup>Australian Institute of Tropical Health and Medicine, James Cook University, Cairns, Australia.

## Abstract
H5N1 viruses periodically cross species barriers and cause disease in humans. The likelihood that an avian influenza virus will acquire mammalian-adapting mutations and evolve enhanced mammalian transmissibility depends on its ability to acquire and select mutations within hosts during spillover. We use deep sequence data from infected humans and poultry in Cambodia to examine how H5N1 viruses evolve during natural spillover infection. We find that viral populations in both species are characterized by predominantly low-frequency (<10%) variation shaped by a combination of purifying selection and genetic drift. Human samples had a greater number of within-host polymorphisms on average, although the distribution of single nucleotide polymorphism (SNP) frequencies and overall mean SNP frequency was similar in both host species. We detect a handful of mutations in humans at sites explicitly linked to H5N1 mammalian adaptation (PB2 627 Lys, HA 150 Val, and HA 238 Leu), although these mutations were present at low frequencies, despite ≥8 days of infection. Finally, we show that mutations detected within-host are not enriched among viruses that have caused spillover infections in the past. By comparing signatures of diversity among humans and poultry, we show that H5N1 viruses are capable of generating known markers of human adaptation during natural spillover infection. However, a short duration of infection, randomness and purifying selection together severely limit the evolutionary capacity of H5N1 viruses to evolve extensively within human hosts.
Avian influenza viruses (AIVs) periodically cross species barriers and infect humans. The likelihood that an AIV will evolve mammalian transmissibility depends on acquiring and selecting mutations during spillover. We analyze deep sequencing data from infected humans and ducks in Cambodia to examine H5N1 evolution during spillover. Viral populations in both species are predominated by low-frequency (<10%) variation shaped by purifying selection and genetic drift. Viruses from humans contain some human-adapting mutations (PB2 E627K, HA A150V, and HA Q238L), but these mutations remain low-frequency. Within-host variants are not enriched along phylogenetic branches leading to human infections. Our data show that H5N1 viruses generate putative human-adapting mutations during natural spillover infection. However, short infections, randomness, and purifying selection limit the evolutionary capacity of H5N1 viruses within-host. Applying evolutionary methods to sequence data, we reveal a detailed view of H5N1 adaptive potential, and develop a foundation for studying host-adaptation in other zoonotic viruses.
@@ -8,7 +8,7 @@ All consensus sequences are available [here](https://github.com/blab/h5n1-cambod


## Within-host data
All within-host variants reported in the manuscript and analyzed are available [here](https://github.com/blab/h5n1-cambodia/blob/master/data/within-host-variants-1%25.txt). This data file includes all variants present at a frequency of at least 1% in all human and duck samples. Fastq files were processed and variants called using [this pipeline](https://github.com/lmoncla/illumina_pipeline), briefly outlined below:
Human reads were removed from all raw fastq files by mapping to the human reference genome GRCh38 with bowtie2. Only unmapped reads were further processed and used for data analysis. The raw fastq files with human reads filtered out are all publicly available in the Sequence Read Archive under the accession number [PRJNA547644](https://www.ncbi.nlm.nih.gov/sra/?term=PRJNA547644), accession numbers SRX5984186-SRX5984198. All within-host variants reported in the manuscript and analyzed are available [here](https://github.com/blab/h5n1-cambodia/blob/master/data/within-host-variants-1%25.txt). This data file includes all variants present at a frequency of at least 1% in all human and duck samples. Fastq files were processed and variants called using [this pipeline](https://github.com/lmoncla/illumina_pipeline), briefly outlined below:

1. Adapter and quality trimming with [Trimmomatic](http://www.usadellab.org/cms/?page=trimmomatic )
2. Mapping with [bowtie2](http://bowtie-bio.sourceforge.net/bowtie2/index.shtml)

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