Genetic Characterization of the H Gene of MeV Strains (H1, B3, and D4) Recently Circulated in Iran for Improving the Molecular Measles Surveillance in the National Measles Lab

Abstract

Background: Despite decreasing the global burden of measles disease after the introduction of vaccination, measles remains one of the most devastating childhood diseases. Since genotype B3 is reported as a predominant Measles Virus (MeV) genotype recently, the current study aimed to better understand MeV genetic variation by analyzing the complete sequence of Hemagglutinin (H) gene associated with outbreaks of circulated genotypes in Iran.

Methods: Nine positive measles specimens were selected from three circulated different genotypes H1, B3, and D4. Two different regions of MeV RNA were detected by RT-PCR assay. Sequence data and phylogenetic trees were analyzed and constructed by MEGA X software program. Moreover, missense and silent mutations in critical positions of the MeV-H protein were investigated.

Results: The result of phylogenetic analysis from the C-terminus of the Nucleoprotein gene (NP-450) and the complete H gene revealed that the mean sequence diversity was 0.06%-0.08% and 0.04%, respectively. Genotype H1 had the highest mutation in this study; however, the substitutions in genotype B3 fundamentally occurred in critical epitopes. Moreover, genotype D4 was more stable than genotypes B3 and H1.

Conclusion: Mutations were investigated in the whole sequence of H protein. Moreover, the mutations that occur in the critical sites of the protein have an important effect on the pathogenicity of the virus. In this way, we were able to illustrate why genotype B3 is more transmissible than other measles genotypes and is the most important circulating genotype around the world.

Keywords: Amino acid substitution; B3 genotype; Hemagglutinin protein; Molecular analysis; Vaccine efficacy.

Fig. 1:

Agarose gel electrophoresis of complete MeV-H gene (1854 bp) and 450 nucleotides from C-terminus of the MeV-N gene (NP-450): RT-PCR amplification of measles species that circulated in Iran (genotypes H1, B3 and D4) and the ALK-C, vaccine strain (A genotype). Three overlapping amplicons for each sample (630,818 and 762bp fragments respectively) to the complete sequence of MeV-H protein (a) a couple of amplicons for the 450 nucleotides C-terminus of N gene (b) C -: negative control C ₊: positive control MW: Molecular Weight (1kb)

Fig. 2:

Phylogenetic trees: The complete MeV-H gene (A) the 450 nucleotides of C-terminus region of MeV-NP gene (B). Nine representative wild-type measles isolates from different outbreaks in Iran (genotypes D4, B3, and H1) and ALK-C vaccine strain in compared with all WHO reference measles strains

Fig. 3:

Ribbon diagram of MeV-H protein structure and substitutions in its critical sites: PDB ID: 2RKC. Genotype D4 (A), Genotype B3 (B) and Genotype H1 (C). Total overlapping area that involved in critical epitopes of RBE, NE, HNE, and SSE are shown purple. The illustrated substitutions obtained from nine representative measles isolates from different outbreaks in Iran