Volume 20, Issue 6, Pages 810-821 (December 2016) Genesis, Evolution and Prevalence of H5N6 Avian Influenza Viruses in China  Yuhai Bi, Quanjiao Chen, Qianli Wang, Jianjun Chen, Tao Jin, Gary Wong, Chuansong Quan, Jun Liu, Jun Wu, Renfu Yin, Lihua Zhao, Mingxin Li, Zhuang Ding, Rongrong Zou, Wen Xu, Hong Li, Huijun Wang, Kegong Tian, Guanghua Fu, Yu Huang, Alexander Shestopalov, Shoujun Li, Bing Xu, Hongjie Yu, Tingrong Luo, Lin Lu, Xun Xu, Yang Luo, Yingxia Liu, Weifeng Shi, Di Liu, George Fu Gao  Cell Host & Microbe  Volume 20, Issue 6, Pages 810-821 (December 2016) DOI: 10.1016/j.chom.2016.10.022 Copyright © 2016 Terms and Conditions

Cell Host & Microbe 2016 20, 810-821DOI: (10.1016/j.chom.2016.10.022) Copyright © 2016 Terms and Conditions

Figure 1 Map of Sampling Sites and Distribution of Avian Influenza Viruses in China Provinces with sampling sites were separated into six regions as indicated by color. Black dots indicate the cities (n = 39 cities in 15 provinces and 1 municipality) selected for sampling. Stars indicate locations with reported human infections of H5N6. Red regions in the pie charts on the map indicate the positive isolation rate of avian influenza viruses (AIV) in this region. “NGS (Environment)” indicates the proportion of HA and NA from environmental samples as determined by next-generation sequencing (NGS). “Virus Isolation” indicates the proportion of live AIV subtypes isolated from embryonated chicken eggs. Abbreviations for the provinces are as follows: NM: Inner Mongolia, JL: Jilin, HeN: Henan, SD: Shandong, JS: Jiangsu, SH: Shanghai, ZJ: Zhejiang, HB: Hubei, HuN: Hunan, JX: Jiangxi, AH: Anhui, FJ: Fujian, GD: Guangdong, GX: Guangxi, SC: Sichuan, YN: Yunnan. The map in the square under Guangdong province indicates islands in the South China Sea. See also Tables S1–S6. Cell Host & Microbe 2016 20, 810-821DOI: (10.1016/j.chom.2016.10.022) Copyright © 2016 Terms and Conditions

Figure 2 Virus Isolation Proportions between Chickens and Ducks The proportion of live AIV subtypes from oropharyngeal and/or cloacal samples of chickens versus ducks. The numerator and denominator in the brackets indicate the number of positive samples and the total number of the collected samples, respectively. See also Tables S1 and S2. Cell Host & Microbe 2016 20, 810-821DOI: (10.1016/j.chom.2016.10.022) Copyright © 2016 Terms and Conditions

Figure 3 Phylogenetic Analysis of H5N6 Influenza Viruses (A) Phylogenetic tree of 505 HA genes of H5N6 viruses sequenced in this study, in addition to all publicly available H5N6 and closely related H5 sequences available from GenBank and GISAID (a total of 1,063). The H5 clade classification reference data set recommended by the WHO, Food and Agriculture Organization of the United Nations, and OIE was also included. Red lines represent the 505 HA genes of H5N6 viruses sequenced in this study. (B) Phylogenetic analysis of the 505 NA genes of the H5N6 viruses sequenced in this study, in addition to all publicly available NA gene sequences of the N6 Eurasian lineage from GISAID (a total of 1,273). Red lines represent the 505 NA genes of the H5N6 viruses sequenced in this study. (C) The part of the tree in (A) representing Clade 2.3.4. For mammalian strains, they are marked by circles with different colors (Human H5N6, red; Feline H5N6, blue; Swine H5N6, green). For the 505 H5N6 viruses sequenced in this study, the clade origins of each gene segment are indicated by different colored bars. The white bars respect the gene segments of the reference strains downloaded from GISAID and GenBank. Detailed phylogenetic trees are available as supplementary files. See also Figure S1 and Table S7. Cell Host & Microbe 2016 20, 810-821DOI: (10.1016/j.chom.2016.10.022) Copyright © 2016 Terms and Conditions

Figure 4 Schematic of the H5N6 AIV Evolutionary Pathway The eight gene segments are (horizontal bars starting from top to bottom of the virion) PB2, PB1, PA, HA, NP, NA, M, and NS. Different colors represent different virus lineages. Arrow thickness is an approximation of the number of H5N6 isolates in this pathway. See also Figure S1 and Table S7. Cell Host & Microbe 2016 20, 810-821DOI: (10.1016/j.chom.2016.10.022) Copyright © 2016 Terms and Conditions

Figure 5 Genotype of H5N6 Viruses in Different Hosts (A) Schematic for the genetic source of human H5N6 isolates. (B) The genotypes of H5N6 viruses isolated in humans, pigs, cats, domestic ducks, domestic chickens, domestic geese, domestic pigeons, and wild birds. The eight gene segments are (horizontal bars starting from top to bottom of the virion) PB2, PB1, PA, HA, NP, NA, M, and NS. Different colors represent different virus lineages. See also Table S7. Cell Host & Microbe 2016 20, 810-821DOI: (10.1016/j.chom.2016.10.022) Copyright © 2016 Terms and Conditions

Figure 6 The Geographical Distribution of Genotypes of H5N6 AIVs in China by Province The eight gene segments are (horizontal bars starting from top to bottom of the virion) PB2, PB1, PA, HA, NP, NA, M, and NS. Different colors represent different virus lineages. See also Table S7. Cell Host & Microbe 2016 20, 810-821DOI: (10.1016/j.chom.2016.10.022) Copyright © 2016 Terms and Conditions