Genetic Analysis of Measles Viruses
Detection of measles RNA in a clinical specimen can provide laboratory confirmation of infection. Real–time RT–PCR to detect measles RNA and endpoint RT–PCR to determine the genotype are performed at CDC and the four Vaccine-Preventable Diseases (VPD) Reference Centers (CA, MN, NY, and WI) of the American Public Health Laboratory Association (APHL). The real-time RT-PCR is more sensitive than endpoint RT–PCR assay for detection of measles RNA in clinical specimens, while the endpoint assay is routinely used to amplify the region of the measles genome required to determine the genotype.
Molecular epidemiology of measles viruses is an important component of outbreak investigations and is used for global surveillance of circulating wild–type measles strains.
Wild-type measles viruses have been divided into eight clades containing 24 genotypes based on the nucleotide sequences of their hemagglutinin (H) and nucleoprotein (N) genes, which are the most variable genes in the viral genome. The eight clades are designated A to H, with numerals to identify the individual genotypes. For each genotype, a reference strain is designated for use in genetic analysis (phylogenetic analysis), usually the earliest known virus isolate of that group. Within a genotype there may be multiple distinct genetic lineages.
The 450 nucleotides encoding the carboxyl-terminal 150 amino acids of the nucleoprotein (N-450) have up to 12% nucleotide variation between genotypes. The entire N-450 sequence is required for determination of the genotype (1).
The following 19 genotypes have been detected since 1990:
A*, B2, B3, C1, C2, D2, D3, D4, D5, D6, D7, D8, D9, D10, D11, G2, G3, H1, H2
*All vaccine strains (e.g. Moraten, Edmonston-Zagreb) are genotype A.
Since 2018, four genotypes were identified by global surveillance:
B3, D4, D8, H1
Laboratorians are requested to consult with the CDC Measles Laboratory at email@example.com before reporting a new measles genotype.
Measles virus genotyping can play an important role in tracking transmission pathways during outbreak investigations. Genotyping results can help confirm, disprove, or detect connections among cases. If two cases have matching N-450 sequences, they may be connected even if the connection is not obvious.
Genotyping can also distinguish whether a person has wild-type measles virus infection, or a rash caused by a recent measles vaccination. A small percentage of measles vaccine recipients experience rash and fever 10 to 14 days following vaccination. During outbreaks, measles vaccine is administered to help control the outbreak, and in these situations, vaccine reactions may be mistakenly classified as measles cases. The vaccine strain of measles virus can be distinguished from wild-type viruses by determination of the genotype from clinical specimens or virus isolates. (See Specimen Collection, Storage, and Shipment). Vaccine strains can also be distinguished from wild-type viruses by real-time RT-PCR (see Molecular Diagnostics).
Lastly, measles virus genotyping can help establish which foreign country may be the source of an imported U.S. case. However, genotyping alone is not sufficient, since each genotype can circulate in multiple countries and even in different regions of the world. Genotype data should be reviewed in conjunction with epidemiological information, such as travel and exposure histories, to determine which country may be the source of an imported US case.
It is important to note that genotyping information does not change the basic steps of public health investigations of measles, which include laboratory confirmation of measles infection, obtaining immunization histories for confirmed cases, identifying sources of infection, assessing potential for transmission and identifying contacts without presumptive evidence of immunity, classifying importation status, and obtaining specimens for viral isolation.
The strain names will provide information that is essential for interpretation of the molecular data. Since sequence data may be derived from viruses isolated in cell culture or from RNA extracted directly from clinical material, strains or sequences will be designated as either:
- MVi: sequence derived from RNA extracted from measles virus isolate in cell culture or
- MVs: sequence derived from RNA extracted from clinical material
Other information to be included in the strain/sequence name:
- City or state/province where the case of measles occurred. The full name should be given. (required)
- Country, use 3-letter ISO designation (required)
- Date of rash onset if known, otherwise date of specimen collection by epidemiological week (1-53) and year. Detailed instructions can be found in the Measles Virus Nomenclature Update (2). (required)
- Specimen number if more than 1 specimen was sequenced from the same epidemiological week and location (where required)
- Genotype in square brackets [optional]
- Special designation for sequences derived from measles inclusion-body encephalitis (MIBE), subacute sclerosing panencephalitis (SSPE) cases or vaccine reactions (VAC) (optional)
The following examples illustrate the WHO-approved nomenclature:
- MVi/NewYork.USA/03.98/2 [D2]
- MVs/London.UNK/17.97 [G3] SSPE
In order to maintain reference stocks of viruses, two global Measles Strain Banks have been established. The Viral Vaccine-Preventable Diseases Branch of the Centers for Disease Control and Prevention (CDC) in Atlanta, Georgia, USA, and the Virus Reference Department, Public Health England (PHE) in London, UK, were selected to serve this purpose.
Upon request, viral sequencing and analysis can be provided for measles virus characterization as well as storage of viral strains.
Dr. Paul Rota
Centers for Disease Control and Prevention
Viral Vaccine-Preventable Diseases Branch
Atlanta, Georgia, 30329
Dr. Bettina Bankamp
Centers for Disease Control and Prevention
Viral Vaccine-Preventable Diseases Branch
Atlanta, Georgia, 30329
Dr. Kevin Brown
Virus Reference Department
Public Health England
61 Colindale Avenue
Genotyping is routinely performed on RNA isolated from clinical specimens. However, virus isolates provide more genetic material for sequencing, which is especially important for extended sequencing windows or whole genome sequencing. Virus isolates also contribute to the strain bank at CDC and provide valuable research material. The Vero/hSLAM cell line [hSLAM=human signaling lymphocytic activation molecule] is recommended for use in the laboratories that provide measles surveillance as part of the World Health Organization’s Global Measles and Rubella Laboratory Network (WHO GMRLN) (3). Vero/hSLAM cells are Vero cells that have been stably transfected with a plasmid encoding the gene for the human SLAM molecule (4). hSLAM is a receptor for both wild-type and laboratory-adapted strains of measles. The sensitivity of Vero/hSLAM cells for isolation of measles virus is equivalent to that of B95a cells, which were recommended in the past. The advantage to the Vero/hSLAM cells is that, unlike the B95a cells, they are not persistently infected with Epstein-Barr virus. This provides a significant safety advantage for laboratorians and greatly facilitates international shipments of virus isolates. Vero/hSLAM cells must be cultured in medium containing the antibiotic geneticin to retain SLAM expression. This increases the cost of the cell culture medium. Protocols for culture of Vero/hSLAM cells are available from CDC.
WHO GMRLN members should only accept Vero/hSLAM cells from a WHO-approved source (Regional Reference Laboratory or Global Specialized Laboratory). Laboratories in the US can request Vero/hSLAM cells from CDC. Due to restrictions imposed by the CITES convention, CDC is not able to ship Vero/hSLAM cells internationally.
Left: Microscopic image of uninfected Vero/hSLAM cells. Right: Microscopic image of measles-infected Vero/hSLAM cells.
Vero/hSLAM cells were developed by Dr. Yusuke Yanagi, Kyushu University, Kukuyoka, Japan. He has kindly agreed to allow the WHO GMRLN to use these cells under the following conditions:
- The cell line Vero/hSLAM is used only for laboratory diagnosis of measles and rubella viral infection by virus isolation and/or investigation of measles or rubella strains for molecular epidemiological purposes.
- The cell line is not used for commercial purposes.
- The cell line is not distributed to laboratories outside the WHO GMRLN without Dr. Yanagi’s and WHO’s permission.
- Any publication of work using the Vero/hSLAM cell line acknowledges the original publication (Ono et al. J. Virol. 2001;75:4399-4401).
In countries that have eliminated measles or are close to elimination, it is important to identify every case of measles. Detection of anti-measles IgM in serum of suspected cases is the routine test to confirm a case. However, on days 0-2 after rash onset, 23% of measles patients do not have detectable levels of IgM in their serum (5), leading to a possible false-negative case classification. Detection of viral RNA in clinical specimens by real-time RT-PCR can support case confirmation and is especially sensitive if specimens are collected as early as possible after rash onset.
Because measles is rare in the US, it is not cost-effective for many laboratories to maintain testing capability. Instead, laboratories may refer specimens from suspected measles cases to the APHL-VPD Reference Centers. The APHL-VPD Reference Centers perform molecular testing for measles and other vaccine-preventable diseases on behalf of public health laboratories that have enrolled as submitting sites. They also offer proficiency testing for molecular testing for VPDs. Find further information about the VPD Reference Centers and enrollment as a submitter.
Identification of measles vaccine reactions
About 5% of recently vaccinated persons develop symptoms of rash and fever. Because persons with vaccine reactions are not contagious, it is not necessary to conduct outbreak control measures such as contact investigations. In outbreak situations, some people may be recently vaccinated and exposed to a measles case. Since it is important to determine very rapidly if suspected cases are true measles cases or vaccine reactions, the CDC and APHL-VPD Reference Centers offer the Measles Vaccine (MeVA) Assay. The MeVA assay is a real-time RT-PCR assay that detects only measles vaccine strains. It must be performed together with a standard real-time RT-PCR assay that detects all measles strains, such as the CDC real-time RT-PCR assay. The MeVA assay allows the identification of vaccine reactions in about two hours. Additional guidance for use of the MeVA assay can be found on the APHL web site.
- Bellini, WJ, Rota PA. Genetic Diversity of Wild-Type Measles Viruses: Implication for Global Measles Elimination Programs. Inf. Dis. 1998; 4 (1) 29-35
- Measles Virus Nomenclature Update. 2012, Weekly Epidemiological Record (WER), 2 March 2012; 87(9):73-80.
- Featherstone DA, Rota PA, Icenogle J, Mulders MN, Jee Y, Ahmed H et al. Expansion of the global measles and rubella laboratory network 2005-09. J Infect Dis. 2011:204(Suppl 1); S491-S498
- Ono N, Tatsuo H, Hidaka Y, Aoki T, Minagawa H, Yanagi Y et al. Measles viruses on throat swabs from measles patients use signaling lymphocytic activation molecule (CDw150) but not CD46 as a cellular receptor. Virol. 2001; 75;4399-4401.
- Helfand RF, Heath JL, Anderson LJ, Maes EF, Guris D, Bellini WJ. 1997. Diagnosis of measles with an IgM capture EIA: the optimal timing of specimen collection after rash onset. Infect. Dis. 175;195-199.
- Rota PA, Brown K, Mankertz A, Santibanez S, Shulga S, Muller CP et al. Global distribution of measles genotypes and measles molecular epidemiology. Infect. Dis. 2011; 204 Suppl 1:S514-23