7.3 Overview and methods for determination of measles genotypes

The minimum sequence region or ‘window’ required for determination of measles genotypes is the carboxy-terminal 450 nucleotides that code for the nucleoprotein (N-450). This region of the measles genome was selected because it was determined to be the most variable coding region. The protocol used to amplify the N-450 region and sequencing primers is available in Annex 7.1.

In addition to the N-450 window, the complete protein coding sequence of the N-gene as well as the full-length hemagglutinin gene (H-gene) can be useful to provide increased discrimination between sequences within a genotype. Sequencing of the H-gene (Annex 7.2) and full-length N gene (Annex 7.3) is required when a new genotype is proposed and is recommended for strains that are associated with a large outbreak. However, the initial efforts should be directed toward completion of the N-450 region of the measles genome prior to the pursuit of additional sequence data.

Following generation of the N-450 sequence, the measles genotype is determined by comparing the virus sequence with the reference sequences for the recognized genotypes that are available from the MeaNS website. Comparisons are usually made by phylogenetic analysis although a genotype can be assigned by sequence similarity with one of the reference strains designated for the genotype or a named strain within the genotype (as implemented within MeaNS). Appropriate phylogenetic analyses can be accomplished by using bootstrapped neighbour-joining or maximum likelihood phylogenetic methods using a variety of programs (e.g. Mega version 7, http://megasoftware.net/).