Improving molecular tools for global surveillance of measles virus

Abstract

Background: The genetic characterization of wild-type measles viruses plays an important role in the description of viral transmission pathways and the verification of measles elimination. The 450 nucleotides that encode the carboxyl-terminus of the nucleoprotein (N-450) are routinely sequenced for genotype analysis.

Objectives: The objectives of this study were to develop improved primers and controls for RT-PCR reactions used for genotyping of measles samples and to develop a method to provide a convenient, safe, and inexpensive means to distribute measles RNA for RT-PCR assays and practice panels.

Study design: A newly designed, genetically defined synthetic RNA and RNA isolated from cells infected with currently circulating genotypes were used to compare the sensitivity of primer pairs in RT-PCR and nested PCR. FTA® cards loaded with lysates of measles infected cells were tested for their ability to preserve viral RNA and destroy virus infectivity.

Results: A new primer pair, MeV216/MeV214, was able to amplify N-450 from viruses representing 10 currently circulating genotypes and a genotype A vaccine strain and demonstrated 100-fold increased sensitivity compared to the previously used primer set. A nested PCR assay further increased the sensitivity of detection from patient samples. A synthetic positive control RNA was developed that produced PCR products that are distinguishable by size from PCR products amplified from clinical samples. FTA® cards completely inactivated measles virus and stabilized RNA for at least six months.

Conclusions: These improved molecular tools will advance molecular characterization of circulating measles viruses globally and provide enhanced quality control measures.

Keywords: CDC; Centers for Disease Control and Prevention; Ct; FTA(®) cards; Genotyping; LabNet; MeV; Measles; Molecular diagnostics; RT; WHO; WHO Measles and Rubella Laboratory Network; World Health Organization; cpe; cytopathic effect; measles virus; nt; nucleotides; room temperature; threshold cycle.

Fig. 1

Genetic variability of primer binding sites among 11 genotypes. (A) Schematic representation of measles genome and location of N-450. N, P/C/V, M, F, H, L are measles genes. (B) Nucleotide sequence of primer binding sites of 11 genotypes. Left side of figure depicts forward primer binding sites, right side of figure depicts reverse primer binding sites. Dots below the consensus sequence indicate variable positions. Numbers above the consensus sequence refer to nucleotide positions in the measles genome.

Fig. 2

Schematic representation of synthetic RNA MeV-N3in. RNA consists of nucleotides 533–1765 of the measles genome including a 220 nucleotide insert derived from the bacterial chloramphenicol-acetyl-transferase gene. The location of the qRT-PCR product and the product of genotyping RT-PCR are also indicated.

Fig. 3

RT-PCR with primers MV60/MV63.3. (A) Viral RNA. Lanes 1–11: genotypes as listed in Table 1, 10⁶ copies/reaction. (B) MeV-N3in. Lanes 1–6: 10-fold serial dilution of MeV-N3in, 10⁶–10 copies/reaction. un, RNA from uninfected cells; H₂ O, water control.

Fig. 4

RT-PCR with primers MeV216/MeV214. (A) Viral RNA. Lanes 1–11: genotypes as listed in Table 1, 10⁴ copies/reaction. (B) MeV-N3in. Lanes 1–6: 10-fold serial dilution of MeV-N3in, 10⁶–10 copies/reaction. un, RNA from uninfected cells; H₂ O, water control.

Fig. 5

Nested PCR with primers MeV210/MeV217. (A) Lanes 1–11: genotypes as listed in Table 1, 10³ copies/reaction. (B) MeV-N3in. Lanes 1–6: 10-fold serial dilution of synthetic RNA, 10⁶–10 copies/reaction. un, RNA from uninfected cells; H₂ O, water control.

Fig. 6

Stability of RNA on FTA^® disks. RNA extracted from one FTA^® disk loaded with the indicated genotype (Table 1) was analyzed by RT-qPCR after incubation at 4 °C for one day or six months. Ct values are averages of three replicate wells. Error bars indicate one standard deviation.