An alternative method to determine the 5′ extremities of non-segmented, negative sense RNA viral genomes using positive replication intermediate 3′ tailing: Application to two members of the Paramyxoviridae family

Determining the sequence of non-segmented, negative sense RNA viral genomes is far from routine and often requires the application of several techniques. In this study, an existing method used currently just for determination of the genomic 3′ extremity was used to determine both the 3′ and 5′ sequence extremities of a Newcastle disease virus and an avian metapneumovirus. This was achieved with a single 3′ nucleotide tailing reaction of both the genomic RNA and the full length, positive sense, antigenomic RNA, followed by a single reverse transcription reaction targeted to the common polynucleotide tails, and then individual PCRs specific for each extremity using PCR primers derived from the sequence of the RT primer or from neighbouring virus sequences known previously. For each virus the method was employed separately. Sequences from both viruses were in agreement with those reported previously for other paramyxoviruses, yet one extra base at the 3′ and one extra base at the 5′ were identified for the avian metapneumovirus. In this study, importantly, the newly determined extremities maintained the complementarity known to exist between the extremities of these viruses. The method was equally successful with both viruses and can be tailored easily to function with other non-segmented, negative sense viruses through minor modification of only the primer sequences.