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.

Keywords

• Hemagglutinin gene
• H5N1
• Novel sequences
• Viral variations
• Mutations
• Full length gene