title: Isolation of novel sequences targeting highly variable viral protein hemagglutinin.
abstract: Rapid evolution is a hallmark of the viral kingdom and a major concern for developing universal vaccines. The isolation of substantial numbers of viral sequence variants at highly variable viral protein domains remains a major challenge. We previously developed a combinatorial method for the isolation of novel sequences to cope with rapid viral variations at the G-H loop of Foot and Mouth Disease virus VP1 protein [1]. Here we present a modification of that method in its application in the randomization of the hemagglutinin gene from a H5N2 virus, namely: •removal of potentially stressful region which harbored a stretch of basic amino acids to increase the success rates of gene cloning, and to streamline the process of future engineering of novel viral variants.•clustered randomization in a full-length gene, as the positive rate for partial gene fragment libraries was extremely low before enrichment in the previous FMDV studies.•the use of fusion partner was avoided, which was used previously for protein expression, stabilization of clones and reduction of stresses on host cells.•the use of Poisson distribution is proposed to approximate sequencing output to achieve cost effectiveness.
authors.affiliation: MOE Key Laboratory of Aquatic Product Safety, State Key Laboratory of Biocontrol, Biotechnology Research Center, The School of Life Sciences, Sun Yat-sen University, Guangzhou 510275, China.
authors.forename: Zhiwu
authors.initials: Z
authors.lastname: Xu
authors.affiliation: MOE Key Laboratory of Aquatic Product Safety, State Key Laboratory of Biocontrol, Biotechnology Research Center, The School of Life Sciences, Sun Yat-sen University, Guangzhou 510275, China.
authors.forename: Jieyu
authors.initials: J
authors.lastname: Wu
authors.affiliation: MOE Key Laboratory of Aquatic Product Safety, State Key Laboratory of Biocontrol, Biotechnology Research Center, The School of Life Sciences, Sun Yat-sen University, Guangzhou 510275, China.
authors.forename: Fan
authors.initials: F
authors.lastname: Feng
authors.affiliation: School of Life Sciences, Tsinghua University, Beijing 100084, China.
authors.forename: Xiaoxiao
authors.initials: X
authors.lastname: Zhang
authors.affiliation: MOE Key Laboratory of Aquatic Product Safety, State Key Laboratory of Biocontrol, Biotechnology Research Center, The School of Life Sciences, Sun Yat-sen University, Guangzhou 510275, China.
authors.forename: Xiaoqian
authors.initials: X
authors.lastname: Ma
authors.affiliation: MOE Key Laboratory of Aquatic Product Safety, State Key Laboratory of Biocontrol, Biotechnology Research Center, The School of Life Sciences, Sun Yat-sen University, Guangzhou 510275, China.
authors.forename: Man
authors.initials: M
authors.lastname: Tang
authors.affiliation: MOE Key Laboratory of Aquatic Product Safety, State Key Laboratory of Biocontrol, Biotechnology Research Center, The School of Life Sciences, Sun Yat-sen University, Guangzhou 510275, China.
authors.forename: Yan
authors.initials: Y
authors.lastname: Huang
authors.affiliation: Guangzhou Center for Disease Control and Prevention, Guangzhou 510440, China.
authors.forename: Ying
authors.initials: Y
authors.lastname: Zhang
authors.affiliation: MOE Key Laboratory of Aquatic Product Safety, State Key Laboratory of Biocontrol, Biotechnology Research Center, The School of Life Sciences, Sun Yat-sen University, Guangzhou 510275, China.
authors.forename: Yongchang
authors.initials: Y
authors.lastname: Cao
authors.affiliation: MOE Key Laboratory of Aquatic Product Safety, State Key Laboratory of Biocontrol, Biotechnology Research Center, The School of Life Sciences, Sun Yat-sen University, Guangzhou 510275, China ; Department of Genetics and Biochemistry, Clemson University, Clemson, SC 29634, USA.
authors.forename: Weiguo
authors.initials: W
authors.lastname: Cao
authors.affiliation: MOE Key Laboratory of Aquatic Product Safety, State Key Laboratory of Biocontrol, Biotechnology Research Center, The School of Life Sciences, Sun Yat-sen University, Guangzhou 510275, China.
authors.forename: Ran
authors.initials: R
authors.lastname: He
authors.affiliation: MOE Key Laboratory of Aquatic Product Safety, State Key Laboratory of Biocontrol, Biotechnology Research Center, The School of Life Sciences, Sun Yat-sen University, Guangzhou 510275, China ; Division of Biological Sciences, University of California, San Diego, La Jolla, CA 92093, USA.
authors.forename: Ye
authors.initials: Y
authors.lastname: Gao
authors.affiliation: MOE Key Laboratory of Aquatic Product Safety, State Key Laboratory of Biocontrol, Biotechnology Research Center, The School of Life Sciences, Sun Yat-sen University, Guangzhou 510275, China.
authors.forename: Qiuyun
authors.initials: Q
authors.lastname: Liu
keywords: Full length gene; H5N1; Hemagglutinin gene; Mutations; Novel sequences; Viral variations