Rapid sequencing of the non-coding regions of influenza A virus

The non-coding regions (NCRs) of influenza A virus gene segments play a crucial role in the viral replication cycle. Although the NCRs are considered to be conserved, some variation does exist, that affects viral replication. Therefore, a rapid method to sequence the 5′ and 3′ NCRs was designed. This method is based on ligation of viral RNA, RT reactions and subsequent PCR with primersets consisting of a gene segment specific primer and a primer designed across the junction of the 5′ and 3′ ends. These PCR fragments can be sequenced directly without the need for cloning PCR fragments first. This method was used to sequence the NCRs of A/Bilthoven/16190/68 (H3N2) and A/Turkey/Turkey/1/05 (H5N1).