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Public Health Agency of Canada (PHAC)
Canada Communicable Disease Report

Volume 25-24
15 December 1999

[Table of Contents]

  

Appendix D - Measles Surveillance: Guidelines for Laboratory Support

MEASLES VIRUS ISOLATION

Background

Measles virus isolation is a definitive means of establishing the diagnosis of measles. Measles culture has become a more reliable tool with the use of B95-8 cell line, an Epstein-Barr virus transformed lymphoblastoid cell line known to be quite sensitive in isolating measles virus from clinical specimens. As progress is made toward elimination of measles in the Americas, it will be critical to examine virus isolates from as many outbreaks and sporadic cases as possible for strain surveillance and to identify the source of the virus. From this standpoint, viral isolation is important. Automated DNA sequencing techniques are now available for rapid genetic characterization of viral isolates. This, together with the existence of a database of nucleic acid sequence information, now makes it possible to identify the source of wild-type viruses and rapidly differentiate between wild-type and vaccine strains.

Virus isolation can take several days to weeks. Therefore, IgM serology should always be the first priority in laboratory diagnosis of measles. Specimens for virus isolation should be taken at the same time that blood is obtained, since a delay in collection will reduce the chance of isolating the virus. Nasopharyngeal, or throat swab, or urine specimens should not be substituted for blood which is required for serologic diagnosis.

Protocols for Isolation of Measles Virus*

Specimens for virus isolation should be obtained as soon as possible after the onset of rash. Always collect a urine specimen and, if possible, attempt to collect a nasopharyngeal or throat specimen.

Specimen Collection and Processing

Respiratory Specimens (nasopharyngeal or throat swabs)

  1. Obtain the sample as soon as possible after onset of rash but no later than 4 days afterwards.

  2. Use sterile swabs to obtain nasopharngeal or throat specimens. The virus is cell-associated, so attempt to swab the throat and nasal passages to collect epithelial cells. Place the swabs in a tube containing 2 mL to 3 mL of VTM (viral transport medium: PBS or suitable isotonic solution such as Hank's BBS containing antibiotics [100 units/mL penicillin, 100 mg/mL streptomycin], and either 2% fetal bovine serum [FBS] or 0.5% gelatin).

  3. Keep all specimens under refrigeration (4o C), and ship as soon as possible on wet ice to the laboratory for processing.

  4. In the laboratory, remove swabs from the VTM after allowing at least an hour for elution of the virus. Centrifuge the eluant at 2500 x g for 15 minutes at 4o C. Resuspend the pellet in 1 mL of tissue culture medium. If possible, save the supernatant in a separate tube. Either proceed directly with virus isolation (specimens may be held at 4oC for up to 24 hours), or freeze the samples at -70oC and ship on dry ice to the appropriate laboratory for virus isolation.

Urine Specimens

  1. Collect 50 mL to 100 mL of urine within 7 days after rash onset.

  2. Keep urine at 4o C and process within 48 hours at the latest.

  3. Centrifuge 50 mL urine at 2500 x g for 15 minutes at 4oC to pellet the sediment. Resuspend the sediment in 2 mL VTM or any cell culture medium (e.g. DMEM, EMEM, RPMI plus antibiotics). Either proceed directly with virus isolation or freeze the samples at -70oC and ship on dry ice to the appropriate laboratory for virus isolation.

If centrifugation cannot be done, do not freeze the urine sample. The entire urine specimen should be held under refrigeration and shipped to the appropriate laboratory on wet ice. Seal the specimen container to prevent leakage.

Virus Isolation

B95-8 (B95-a) Cell Line

B95-8 is the preferred cell line for primary isolation of measles virus(1), and this cell line is available from the American Type Culture Collection (No. CRL 1612). Note that this cell line should be handled as an infectious cell line capable of yielding Epstein-Barr virus.

The B95-8 cells grow lightly attached to the culture surface when grown in DMEM supplemented with 1 x antibiotics (100 units/mL penicillin, 100 mg/mL streptomycin), 0.25 µg/mL amphotericin (fungizone), and FBS. Cell growth is sustained by adding 5% to 10% FBS. Under these conditions, B95-8 cells grow as an adherent monolayer and are referred to as B95-a cells. FBS is used at a 2% concentration for cell maintenance during viral isolation. Grow cells in a moist CO2 incubator at 37o C. Cell stocks can be stored frozen at -70o C using standard cryoprotection medium (50% FBS, 10% DMSO, 40% DMEM).

The B95-a cells can be passaged by briefly treating the cell monolayers with 0.05% trypsin-EDTA to release cells from the tissue culture surface. Be careful not to over-trypsinize. Neutralize trypsin by adding DMEM containing 10% FBS. Usually the cells from a single monolayer culture can be split in a ratio of 1:3. The cells tend to become "floaters" growing in clumps suspended in the medium as the cell density increases. These cells are viable and can be passaged by gentle pipetting to break up the clumps then replating to a lower cell density.

The B95-a cells can be transported at room temperature in a 75 cm2 or 25 cm2 tissue culture flask with additional medium added to help keep cells attached. After shipment, look at the cell sheet. If many cells are free-floating, a light spin of the medium will recover cells, which can be added back to the flask or to another flask for passage. For maintenance, add 30mL to 50 mL of DMEM with 2% FBS to a 75 cm2 flask.

B95-a cells infected with measles virus can show syncytium formation and giant-cell cytopathic effect (CPE) as early as 48 hours after inoculation. However, isolation-attempt cultures should be followed for 7 to 8 days with two to three blind passages before isolation of measles virus is ruled out.

Inoculation of Specimens for Measles Isolation (Flask Method - CDC)

  1. Passage cells, split into 25 cm2 flasks at 1:3 or 1:4, and incubate 24 to 48 hours. Cells should be at 75% to 85% confluency when the specimen is inoculated.

  2. For inoculation, decant medium, add 1.0 mL to 1.5 mL DMEM with 2 x antibiotics and add 0.1 mL to 1.0 mL of specimen, depending on the concentration. Save all leftover specimens as they may still be useful for PCR analysis.

  3. Incubate at 37o C for 1 hour.

  4. Decant medium into bleach water; replace medium with 5mL to 10 mL of DMEM containing 2% FBS and 2 x antibiotics.

  5. Change medium every 3 to 4 days, and passage cells by splitting at 1:3 every 7 to 9 days. Check for CPE daily.

  6. Attempt at least three blind passages before terminating isolation attempt.

  7. If CPE is visible, continue to feed the cells until the CPE becomes extensive. It may be necessary to passage the cells one more time to allow the CPE to progress. When CPE is maximal, pellet cells, resuspend the pellet in 1 mL of DMEM, and freeze at -70o C.

  8. If a culture becomes contaminated, the original specimen can be diluted in DMEM and passed through a 0.45 µm nitrocellulose filter and used to inoculate fresh B95-a cells.

  9. Confirmation of culture can be achieved by using fluorescent antibody staining or PCR.

Please remember to save some of the original clinical specimen. This material can be used for a second isolation attempt if problems occur with the first, and it can provide a specimen for direct RT-PCR analysis. (Contact the Viral Exanthemata Laboratory, LCDC, for details.)

Inoculation of Specimens for Virus Isolation (Shell Vial Method)

The shell vial method has been found to be quite convenient and highly successful by some laboratories in Canada. This method involves the following:

Maintenance of B95-8 cells

  1. B95-8 cells are routinely maintained in a 25 cm2 flask in an upright position. The cells, contained in 10 mL RPMI growth medium, settle to the bottom of the flask.

  2.  Without disturbing the cells, each week carefully remove 9 mL of growth medium and discard, leaving 1 mL of cell suspension. Add 9 mL of fresh growth medium (formula below). If additional cells are required, each flask can be split 1:3.

  3. Trypsinization is not required because these cells do not attach. Reduction in the density of the cells can be accomplished by harvesting the cells through centrifugation and resuspending them in the fresh medium to the required density.

  4. These cells can be maintained at !70o C in an appropriate cryoprotectant and easily retrieved when required.

Growth medium for B95-8 cells

  • 500 mL RPMI 1640 (Gibco Cat. No. 21870-076 without glutamine).
  • Aseptically remove and save 56 mL of the medium for other use.
  • Add 50 mL FBS (heat inactivated) to the remaining 444 mL medium.
  • Add 1 mL penicillin-gentamicin solution (final concentration: penicillin 100 units/mL, gentamicin 10 mg/mL).
  • Add 5 mL 300 mM glutamine.

Inoculation

  1. Inoculate two shell vials (containing coverslips) per specimen with 106 cells in 0.2 mL growth medium and 0.2 mL of specimen.

  2. Centrifuge 1,000 x g for 45 minutes at room temperature.

  3. Following centrifugation, remove inoculum and add 1 mL maintenance medium containing 2% FBS.

  4. Incubate at 37oC and observe for syncytium formation at 48 hours and daily for up to 6 days.

  5. When CPE is observed (at least 50% cells), or after 6 days incubation, centrifuge one vial at 900 x g for 10 minutes.

If viral isolation is needed to confirm the case, follow steps 6 and 7.

  1. Aspirate the maintenance medium from the vial, fix by adding 1 mL acetone and allow to sit for 15 minutes. Remove the acetone and wash with PBS to remove residual acetone.

  2. Aspirate PBS and stain the coverslip (within the vial) with measles immunofluorescent stain according to the manufacturer's instructions (Light Diagnostics - Chemicon; distributed in Canada by Bio/Can).

Otherwise inoculate fresh B95-8 cells in 75 cm2 flask with virus until CPE is observed, and follow steps 7 and 8 of the Flask Method protocol.

  1. If CPE is not observed after 6 days, the monolayer in the second shell vial can be scraped and passed to a fresh vial(s) for further incubation.

Remember to save some of the original clinical specimen. This material can be used for a second isolation attempt ifproblems occur with the first, as well as provide a specimen for direct RT-PCR analysis. (Contact the Viral Exanthemata Laboratory, LCDC, for details.)

Shipping of Measles Virus Isolates

Infected cells can be pelleted, resuspended in a small volume of DMEM and frozen at -70oC before shipping on dry ice.

The appropriate Transport Canada regulations for the safe shipment of infectious material and dangerous goods must always be followed.

Please contact LCDC concerning shipment of sera, clinical specimens, or virus isolates.

Viral Exanthemata Laboratory
Canadian Science Centre for Human and Animal Health
Laboratory Centre for Disease Control
1015 Arlington Street
Winnipeg, Man. R3E 3R2

Head: Dr. Graham Tipples
Telephone: 204-789-6080
Fax: 204-789-5009
E-mail: graham_tipples@hc-sc.gc.ca

* Original protocols, except for the shell vial method, are based on the method of Dr. William Bellini, Measles Section, CDC.

 

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