Measles Genotyping: How Labs Trace Outbreaks and Spot Vaccine Reactions

Finding measles virus genetic material (RNA) in a patient's specimen can confirm an infection. Two lab methods are used: real-time RT-PCR, which is more sensitive for detecting measles RNA, and endpoint RT-PCR, which routinely amplifies the part of the genome needed to work out the virus's genotype. Studying the genetics of measles viruses — molecular epidemiology — is an important part of outbreak investigations and of global surveillance of the wild strains in circulation.

Uninfected (left) and measles-infected (right) Vero/hSLAM cells under the microscope (CDC).

Clades and genotypes

Wild-type measles viruses are grouped into eight clades (A to H) containing 24 genotypes, based on the sequences of two genes that vary most in the viral genome: the hemagglutinin (H) and nucleoprotein (N) genes. Genotypes are named with a letter and a numeral, and each has a reference strain, usually the earliest known isolate. A genotype may contain several distinct lineages.

The key stretch is N-450: the 450 nucleotides encoding the last 150 amino acids of the nucleoprotein, which differ by up to 12% between genotypes. The entire N-450 sequence is needed to assign a genotype.

• 19 genotypes have been detected since 1990: A, B2, B3, C1, C2, D2 through D11, G2, G3, H1 and H2.
• 4 have been identified by global surveillance since 2018: B3, D4, D8 and H1.
• All vaccine strains, such as Moraten and Edmonston-Zagreb, are genotype A.

Laboratories are asked to consult the CDC Measles Laboratory before reporting a new genotype.

What genotyping can do in an investigation

• Connect cases. Matching N-450 sequences can reveal links between cases even when the connection isn't obvious — or rule links out.
• Separate measles from a vaccine rash. A small share of vaccine recipients — about 5% — develop rash and fever, typically 10 to 14 days after vaccination. During outbreaks, when vaccine is given to help control spread, such reactions can be mistaken for measles. Genotyping, or real-time RT-PCR, can tell the vaccine strain from wild virus.
• Point to the source of an imported case. Genotypes circulate in many countries and regions, so the genotype alone isn't enough; it must be combined with travel and exposure histories.

Genotyping doesn't change the basic steps of a measles investigation: confirming infection in the lab, getting vaccination histories, finding sources, judging the potential for spread, identifying contacts without evidence of immunity, classifying importation status and collecting specimens for virus isolation.

Naming strains and sequences

Names begin with MVi (sequence from a virus isolated in cell culture) or MVs (sequence from RNA taken directly from clinical material), followed by:

• the city or state/province where the case occurred, in full (required);
• the country's three-letter ISO code (required);
• the date of rash onset — or, if unknown, of specimen collection — as epidemiological week (1–53) and year (required);
• a specimen number, if more than one specimen from the same week and place was sequenced (where needed);
• optionally, the genotype in square brackets, and a label for sequences from measles inclusion-body encephalitis (MIBE), subacute sclerosing panencephalitis (SSPE) or vaccine reactions (VAC).

WHO-approved examples: MVi/NewYork.USA/03.98/2 [D2] and MVs/London.UNK/17.97 [G3] SSPE.

Strain banks

Two global Measles Strain Banks keep reference stocks of virus: CDC's Viral Vaccine-Preventable Diseases Branch in Atlanta and the Virus Reference Department of Public Health England in London. On request they can sequence and analyze measles viruses and store strains.

Growing the virus: Vero/hSLAM cells

Genotyping is usually done on RNA straight from clinical specimens, but isolating the virus yields more genetic material — especially useful for longer sequences or whole-genome sequencing — and adds to CDC's strain bank.

The recommended cell line for laboratories in the WHO Global Measles and Rubella Laboratory Network is Vero/hSLAM: Vero cells engineered to carry the gene for the human signaling lymphocytic activation molecule (SLAM), a receptor used by both wild and lab-adapted measles strains.

• They isolate measles as well as the formerly recommended B95a cells, but unlike those cells they are not persistently infected with Epstein-Barr virus — safer for lab staff and much easier to ship internationally.
• They must be grown with the antibiotic geneticin to keep expressing SLAM, which raises costs.
• Network members should get them only from WHO-approved sources. U.S. labs can request them from CDC, but international restrictions under the CITES convention prevent CDC from shipping them abroad.

The cells were developed by Dr. Yusuke Yanagi of Kyushu University in Japan, who allows the network to use them only for diagnosing measles and rubella and for molecular epidemiology — not commercially, not shared outside the network without his and WHO's permission, and with his original publication acknowledged.

Molecular diagnosis

Where measles has been eliminated or nearly so, every case matters. The routine confirmation test detects anti-measles IgM antibodies in blood, but in the first two days after the rash appears, 23% of patients have no detectable IgM — a possible false negative. Real-time RT-PCR for viral RNA can support confirmation and is especially sensitive when specimens are taken soon after rash onset.

Because measles is rare in the United States, many labs can't justify keeping the test in-house and instead send specimens to the APHL-VPD Reference Centers, which do molecular testing for measles and other vaccine-preventable diseases for enrolled public health labs and offer proficiency testing.

Vaccine reaction or measles? The MeVA assay

People with vaccine reactions are not contagious, so no contact investigation is needed — which makes it important to decide quickly, especially in outbreaks where some people may be both recently vaccinated and exposed. CDC and the APHL-VPD Reference Centers offer the Measles Vaccine (MeVA) assay, a real-time RT-PCR test that detects only vaccine strains. Run alongside a standard test that detects all measles strains, it can identify a vaccine reaction in about two hours.

Questions about measles lab testing, protocols or positive controls can go to the CDC Measles Laboratory at CDCMeaslesLab@cdc.gov.

Sources

• Centers for Disease Control and Prevention: "Genetic Analysis of Measles Viruses"; a work of the United States government in the public domain.