Genotyping of recent measles virus strains from Russia and Vietnam by nucleotide‐specific multiplex PCR

Abstract

The nucleoprotein genes of 49 measles virus (MV) strains circulating in Russia between 2000 and 2006 and in Vietnam in 2003 were analyzed by genotype-specific PCR and the results were compared with their sequences. The sequences revealed the presence of genotypes H1 and H2 in the center (Nha Trang) and the north (Hanoi) of Vietnam, respectively. The relative diversity of the H2 strains suggested an endemic circulation of these viruses in the capital. In contrast genotype H1 strains from Nha Trang were homogenous genetically, which may indicate a recent importation. The strains obtained from 12 different regions of the Russian Federation were assigned to the genotypes H1, D4, and D6. Most strains (81.4%) were correctly genotyped by a multiplex PCR method which was sensitive to genotype-specific mutations [Kremer et al. (2004): J Clin Microbiol 42: 3017-3022]. Ambiguous or negative results for some clade H and genotype D6 strains were due to point mutations in the type-specific primer binding sites. After exchanging a single nucleotide in both the clade H- and the genotype D6-specific primers, all strains were assigned correctly to their genotype. A simplified procedure for use in Vietnam was developed to distinguish directly between genotypes H1 and H2 and any non-H genotype. These results demonstrate that our multiplex PCR method can be adapted easily to new sequence variants or specific epidemiological situations, and thus be very useful for rapid genotyping of large number of samples even in laboratories which do not have sequencing facilities.

Identification of congenital rubella syndrome in Sudan

“…The DBS samples of the 49 infants aged ≥ 6 months were also screened for rubella IgG antibodies using the Anti-rubella Virus IgG ELISA kit (Siemens, Germany; specificity 98.5% and sensitivity 100% according to the manufacturer) since the persistence of rubella IgG antibodies in infants beyond that age may be suggestive of CRS [16-18]. From five of the children who were IgM and/or IgG positive, enough OF was left for RNA extraction and reverse transcription PCR using previously published primers [19,20] and to attempt virus isolation [15]. …”

Revealing New Measles Virus Transmission Routes by Use of Sequence Analysis of Phosphoprotein and Hemagglutinin Genes

“…PCR products for NP-HVR and P were generated by nested PCRs as described before (1,4). H genes were amplified using a seminested PCR strategy, generating two overlapping fragments in a first round using primers H1 and H6, as well as primers H5 and H2.…”

Improving molecular tools for global surveillance of measles virus

“…The new nested assay demonstrated comparable sensitivity while allowing the sequencing of the entire N-450. In addition, the primers MeV210/MeV217 demonstrated fewer mismatches with the 11 genotypes than primers described in other nested assays [9,10].…”

“…Based on these sequences, 24 genotypes have been identified [6,7]. Since 1998, the standard genotyping RT-PCR assay from the CDC recommended a primer pair designated MV60/MV63.3 [8] though other primers were also used throughout LabNet [9][10][11][12][13][14]. Several new MeV genotypes have been described since the introduction of the standard genotyping method [15][16][17]; however, only limited sequence data were available for the primer binding sites of the more recent strains and the analytic sensitivity of the primers had not been evaluated.…”

“…Several new MeV genotypes have been described since the introduction of the standard genotyping method [15][16][17]; however, only limited sequence data were available for the primer binding sites of the more recent strains and the analytic sensitivity of the primers had not been evaluated. Several laboratories have introduced nested PCR assays, some of which do not amplify the complete N-450 [11,[18][19][20][21] or do not provide data for sensitivity and specificity [9,10].…”