Whole-Genome Sequencing During Measles Outbreaks

Genetic characterization of wild-typemeasles viruses is an important compo-nent of laboratory-based surveillance formeasles virus. The genetic informationcan classify measles viruses as endemicor imported and can help map the trans-mission pathways of the virus. Since viro-logic evidence demonstrating the absenceof an endemic genotype of measles virusisoneofthekeycriteriaforveriﬁcationofmeasles elimination [1], adequate viro-logic surveillance must be conducted ona global scale. The current recommenda-tion is to obtain appropriate samples forviral genotyping from at least 80% of out-breaks[2].Inaddition,sequenceanalysisiscurrentlytheonlymethodavailabletocon-ﬁrm measles vaccine–associated rash andfever. Distinguishing between reactions tovaccine and cases caused by wild-typevirus is an important part of laboratorysupport for outbreak investigations [3].Virologic surveillance is performed bythe World Health Organization ’s(WHO’s)Global Measles and Rubella LaboratoryNetwork (LabNet). All of the LabNet lab-oratories use a standard protocol fordetermining the genotype of measlesvirus. The WHO-recommended protocolconsists of analyzing the sequence of the450 nucleotides coding for the carboxyl-terminal 150 amino acids of the nuc-leoprotein (N-450). Sequence data arecompared to a set of WHO reference se-quences representing each of the 24 rec-ognized genotypes [3–5]. Sequence dataare submitted to the WHO Global Mea-sles Sequence Database, which currentlycontains >20000 sequences. In addition,sequences may be assigned to a speciﬁcnamedlineage[4].Namedlineagesrepre-sent at least 50 identical N-450 sequencesreported within the previous 12 monthsand are named on the basis of the earliestvirus detected in the lineage. DespiteN-450 being one of the most variableregions in the measles genome, namedlineages can contain several hundredor more strains with identical N-450sequences.The measles virus genotyping proto-cols were initially developed in 1998. Al-though generating N-450 sequences wasessential for determining the overall ge-netic variability of measles viruses andfor detecting new genotypes, the use ofthis sequencing “window” has limita-tions. Because named lineages can circu-late throughout the world for severalyears, analysis of N-450 sequences isoften not sufﬁcient to distinguish be-tween continued circulation of virusesin a single chain of transmission or mul-tipleimportationsofviruses from varioussources. Since this distinction is vital forveriﬁcation of measles elimination, theLabNet began to study increasing thesize of the “sequencing window” to im-prove the resolution of the sequencedata. In addition to N-450, laboratoriescomparedthesequencesofthefull-lengthphosphoprotein and hemagglutinin genes,as well asthe sequence of an untranslatedregion that lies between the coding re-gions for the matrix and fusion proteins.The results showed that viruses withidentical N-450 sequences were oftenrepresentatives of different lineages [6,7]. Of course, increasing the size of thesequencing target increases the complex-ity of the sequencing protocols and putsgreater demand on the LabNet laborato-ries. Ultimately, the optimum sequencingwindow would be the sequence of thecomplete viral genome. Improvementsin sequencing technology now makewhole-genome sequencing (WGS) feasi-ble for studying the molecularepidemiol-ogy of measles virus.There are several different approachesto WGS for measles virus. Ampliﬁcationof the genome in several large fragmentswith virus-speciﬁc primers followed bySanger sequencing of the amplicons, asdescribed by Gardy et al, has proven suc-cessful for years and, to our knowledge,produced all of the complete genomescurrently available on GenBank. Themethods are well established, equipmentis widely available, and the procedure isrelativelyaffordable.High-throughputse-quencing of such amplicons with Illumi-na or 454 next-generation sequencingtechnologies(NGS)hasbeenusedtogen-erateWGSforotherRNAviruses,includ-ing norovirus, enterovirus A-71, hepatitisA virus, and hepatitis C virus [8–11].AnNGS approach with production of lib-raries based on random primers is also