Appendix 7-1

Measles Virus Isolation

(Updated 2009)

I. Background

Specimens for virus isolation should be taken at the same time that serum is obtained, since 

a delay in collection will reduce the chance of isolating the virus. While direct RT-PCR 

methodologies exist to detect and characterize measles viruses from clinical samples, successful 

virus isolation provides unlimited material for characterization of the virus and is not generally 

subject to the scrutiny given to RT-PCR results if contamination issues arise or results cannot 

be replicated. 

The establishment of automated DNA sequencing techniques has allowed for rapid genetic 

characterization of a large number of wild-type strains of measles virus. Genetic analysis of the 

nucleotide sequence obtained from the virus can often identify the source of wild-type viruses 

and also may be important in distinguishing between wild-type strains and vaccine virus. 

II. Samples for Virus Isolation (or RT-PCR Detection)

Throat (oropharyngeal) swabs (and/or nasopharyngeal [NP] swabs) are the preferred clinical 

samples for measles virus. Urine samples can be collected in addition to the throat swab. While 

throat swabs are generally more easily collected, processed and transported, urine samples 

provide an additional opportunity for successful isolation of virus and may prove superior to 

throat swabs if collection is delayed beyond about 5 days after onset of rash. 

Respiratory specimens

Materials:

●

Viral transport medium (VTM): Commercial sources of VTM are available. As an alternative, 

sterile phosphate buffered saline (PBS) or suitable isotonic solutions such as Hanks balanced 

salt solution or cell culture medium can be utilized. Addition of antibiotics (100 units/ml 

penicillin, 100 μg/ml streptomycin) and a source of protein (either 2 % fetal bovine serum or 

0.5% gelatin) is recommended to support viability of the virus. 

●

Sterile swabs (Dacron or synthetic)

Attempt to obtain the sample as soon as possible after onset of rash. Samples collected 5 

days after rash onset have much lower chances for successful isolation of virus. Sterile 

swabs (Dacron or synthetic fiber) are used to absorb secretions and collect infected cells in 

the posterior nasal passages. The virus is extremely cell-associated, so the aim is to collect 

epithelial cells in the throat and nasal passages. Place both swabs in a tube containing 2–3 ml of 

VTM. If the specimen is to be shipped without freezing, the swab can be broken off and left in 

the tube of VTM.

Keep all specimens on wet ice or at 4° C and ship as soon as possible on cold packs. If 

immediate, cold shipment (within 48 hrs) cannot be arranged or is not convenient, nose and 

throat swabs should be removed from the VTM. Gently vortex or swirl the swab in the fluid and 

ream the swab against the side of the tube. These samples should be frozen and shipped at 

-70° C (dry ice).

Urine specimens

Although a respiratory sample (throat swab) is adequate as a viral sample, urine samples 

may serve as an additional source of virus. First morning voided specimens are ideal as these 

may contain greater numbers of sloughed cells, but urine collection at any time is adequate, 

more important is to collect as soon as measles is suspected. Collect 10–50 ml of urine in a 

urine specimen container. It is best to centrifuge the urine specimen as soon after collection 

as possible. After collection, keep the specimen cold (refrigerator or wet ice). If facilities are 

available, centrifuge the urine at 400 x g for 10 minutes at 4° C to pellet the sediment. Add 2 

ml of VTM (above) or any cell culture medium (DMEM, EMEM, RPMI plus antibiotics) to 

resuspend the sediment and ship.

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Appendix 7-2

Preferably, specimens that have been centrifuged and resuspended should be frozen at 

-70° C and shipped on dry ice. If dry ice is not available, however, they can be stored at 4° C and 

shipped on cold packs. Avoid repeated freeze–thaw cycles. If centrifugation is not available, do 

not freeze the urine sample. The entire urine specimen should be stored at 4° C, and shipped to 

the lab on cold packs. Most urine collection cups are not leak-proof. Transfer the urine to sterile 

plastic centrifuge tubes.

III. Vero/SLAM Cell Line

The Vero/SLAM cell line is now recommended for routine isolation of measles. These cells are 

Vero cells which have been transfected with a plasmid encoding the gene for the human SLAM 

(signaling lymphocyte-activation molecule) protein (Ono et al., 2001). SLAM has been shown 

to be a receptor for both wild-type and laboratory-adapted strains of measles. The sensitivity of 

Vero/SLAM cells for isolation of measles virus is equivalent to that of B95a cells, and measles 

infection of Vero/SLAM cells produces the typical cytopathic effect that is readily observed, 

usually within 1–2 passages of the infected cells. 

The advantage of the Vero/SLAM cells compared to B95a cells is that they are not persistently 

infected with Epstein-Barr virus, and therefore, are not considered as hazardous material. This 

provides a significant safety advantage for laboratory workers and greatly facilitates shipments. 

The disadvantage of the Vero/SLAM cells is that they must be cultured in medium containing 

the selective antibiotic G418 sulfate (Geneticin®) to retain SLAM expression. This does result 

in an increase in the cost of the tissue culture medium over that required for the B95a cell line. 

Contact the Measles Virus Laboratory for protocols and information on obtaining the 

Vero/SLAM cell line.

III. Shipping

Please attempt to notify CDC before virus isolates or clinical specimens are shipped. The CDC 

specimen submission form (Form No. 50.34; see Appendix 23) should include the mailing 

address of the submitting laboratory. If possible, supply a contact person and telephone number 

and e-mail address in case additional information is required. 

Ship to:

Centers for Disease Control and Prevention

DASH Unit # 81—Att: Dr. William Bellini

1600 Clifton Rd.

Atlanta, GA 30333 

Phone: 404-639-1156 or 404-639-3512 

Fax: 404-639-4187 or 404-639-1516

IV. Reporting

Depending on the workload and the quality of the specimens, completion of the virus isolation 

and subsequent genetic analysis can take 1–3 weeks. The Measles Virus laboratory at CDC will 

send a report describing virus isolation and PCR results for all specimens received. Because 

PCR is more sensitive than virus isolation, all specimens from the U.S. will be screened using 

real-time RT-PCR to detect measles RNA. These results are generally available within 3 days 

to 1 week of receipt of sample, depending on current workload. Standard RT-PCR and genetic 

analysis may take an additional 1–2 weeks. Failure to detect virus by RT-PCR or by virus 

isolation should NOT be interpreted as evidence to rule out a clinically diagnosed measles case.