Wild-type measles viruses with non-standard genome lengths

Abstract

The length of the single stranded, negative sense RNA genome of measles virus (MeV) is highly conserved at 15,894 nucleotides (nt). MeVs can be grouped into 24 genotypes based on the highly variable 450 nucleotides coding for the carboxyl-terminus of the nucleocapsid protein (N-450). Here, we report the genomic sequences of 2 wild-type viral isolates of genotype D4 with genome lengths of 15,900 nt. Both genomes had a 7 nt insertion in the 3' untranslated region (UTR) of the matrix (M) gene and a 1 nt deletion in the 5' UTR of the fusion (F) gene. The net gain of 6 nt complies with the rule-of-six required for replication competency of the genomes of morbilliviruses. The insertions and deletion (indels) were confirmed in a patient sample that was the source of one of the viral isolates. The positions of the indels were identical in both viral isolates, even though epidemiological data and the 3 nt differences in N-450 between the two genomes suggested that the viruses represented separate chains of transmission. Identical indels were found in the M-F intergenic regions of 14 additional genotype D4 viral isolates that were imported into the US during 2007-2010. Viral isolates with and without indels produced plaques of similar size and replicated efficiently in A549/hSLAM and Vero/hSLAM cells. This is the first report of wild-type MeVs with genome lengths other than 15,894 nt and demonstrates that the length of the M-F UTR of wild-type MeVs is flexible.

Figure 1. Location of insertions and deletions in the M-F UTR of MVi/New York.USA/26.09/3 and MVi/Florida.USA/19.09.

A. Schematic representation of the MeV genome. The genome is depicted in the positive sense antigenomic orientation. Coding regions of each gene are in shades of grey, non-coding regions are white. Asterisk marks position of M-F intergenic trinucleotide at nucleotides 4872–4874. B. Insertion of 7 nucleotides in cytidine-rich region between nucleotides 4751–4775. C. Deletion of 1 nucleotide between nucleotides 5059–5072. The alignment was generated with MEGA5.10. Numbers under the alignment indicate nucleotide positions in aligned full-length genomes.

Figure 2. Neighbor joining phylogenetic tree based on the N-450 sequences from selected genotype D4 viruses and reference strains.

Bootstrap values of at least 70% are shown. The scale bar shows the number of expected substitutions per site. Red dots indicate viruses sequenced in this study that share the indel in the M-F UTR; blue dots indicate viruses sequenced in this study that do not have the indel in the M-F UTR. Black triangles indicated named lineages in MeaNS.

Figure 3. Multi-step growth curves of MeVs with and without the indels.

MVi/New York.USA/26.09/3, MVi/Florida.USA/19.09, MVi/Washington.USA/07.10 and MVi/Virginia.USA/21.11/1 were used to infect Vero/hSLAM cells (A) and A549/hSLAM cells (B) at an MOI of 0.001. At the indicated time points, cells were scraped into the medium and viral titer was measured by endpoint dilution on Vero-hSLAM cells. Titers represent averages of duplicate wells. Error bars depict one standard deviation. Blue symbols and lines indicate viral isolates without indels and red symbols and lines indicate viral isolates with indels.

Figure 4. Cytopathic effect and lateral spread of genotype D4 viruses with or without indels.

(A) Vero/hSLAM cells and (B) A549/hSLAM cells in chamber slides were infected with MVi/New York.USA/26.09/3, MVi/Florida.USA/19.09, MVi/Washington.USA/7.10 and MVi/Virginia.USA/21.11/1. Cells were overlaid with medium containing CMC agarose. Cells were fixed and immunostained 24 hours after infection. Representative fields are shown at 200× magnification. Green: MeV nucleoprotein, blue: nuclei.