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Non-polio enteroviruses (NPEVs) circulate in all populations and infection can be associated with a vast range of presentations, from asymptomatic to acute flaccid paralysis resembling polio. In general, depending on local environment and climate, 5 to 25% of stool specimens collected from healthy children can be expected to contain NPEVs. For the purposes of polio eradication it is not necessary to characterize these viruses and they can be reported simply as NPEVs. Furthermore, with the introduction and use of the L20B cell line it is no longer necessary to neutralize enterovirus cultures to exclude the possibility of a non-polio enterovirus isolate masking the presence of poliovirus in mixed cultures. However, many laboratories do wish to characterize enterovirus isolates to obtain epidemiological information. Following the recommended flowchart for isolation and typing of polioviruses and enteroviruses (Figure 7.1), enterovirus typing can be performed on the RD culture of a clinical sample.
1) Antisera
Antisera have been raised in animals against many echoviruses and coxsackieviruses. Because the large number of viruses makes it impractical to perform individual neutralization tests, these have been pooled in an overlapping scheme which allows many viruses to be identified using as few as nine tests. Interpretation of the results is done with the assistance of a list of the neutralization patterns of individual viruses. Pooled horse antisera against the most frequently isolated ECHO and Coxsackieviruses have been prepared at the National Institute of Public Health and the Environment (RIVM), Bilthoven, The Netherlands, and are supplied free of charge to WHO Polio Laboratory Network laboratories by WHO (IVB/VAM) Geneva.
Each box of RIVM enterovirus typing antisera contains anti-enterovirus pools A, B, C, D, E, F and G, an anti-Coxsackie B virus pool and a trivalent anti-poliovirus pool. These pools must be diluted before use.
The recommended dilution for all pools is 1 in 20: 0.5 ml of each pool is added to 9.5 ml of the medium specified on the insert.
Aliquot pools into clearly labelled cryovials in 1 ml volumes and store at -20°C.
For each pool A to G (10 ml), there should be enough antisera for 100 tests.
2) Neutralization test for identification of enteroviruses
Have available the following items:
· Flat-bottomed cell culture microtitre plate with cover;
· non-toxic plate sealers (if non-CO2 incubator will be used);
· 5 ml sterile tubes for dilution;
· 1 ml and 2 ml pipettes;
· sterile 50 μl droppers or pipettors with aerosol resistant tips (ARTs);
· flask of healthy RD cells;
· enterovirus serum pools at use dilution;
· maintenance medium.
Each unknown virus is tested in duplicate against a trivalent pooled polio antiserum (PP), a coxsackievirus B1-6 pool (CP), and seven pools against coxsackievirus A9 and 20 echoviruses (A–G). Non-polioviruses that fail to be identified using these antisera may be in an aggregated form which interferes with the complete neutralization by specific antisera. Isolates can be retested after emulsification with chloroform (approximately 10% by volume) and separation of the supernatant. Freezing of the chloroform-treated isolates can result in re-aggregation of the virus, so chloroform treatment should be followed by typing prior to freezing the isolate.
Do the following:
· Label the edge of the microtitre plate as indicated in Figure 7.5.
· Add 50 μl of antisera to the appropriate wells in columns 1–9.
· Add 50 μl medium to virus control wells in column 10 rows A to D.
· Add 100 μl medium to cell control wells in columns 11 and 12 rows A to D.
· Prepare 10-2 dilution of virus (it may be desirable to determine the virus titre and adjust the dilutions as needed).
· Add 50 μl virus to all wells in columns 1 to 10 of rows A to B for sample X and rows C and D for sample Y.
· Perform a back titration of virus X in rows E and F and of virus Y in rows G and H.
· Cover the plate with the lid and incubate for one hour at 36°C.
· During this incubation period, trypsinize RD cells and prepare a suspension of approximately 1.5 x 10–5 cells per ml, calculating at least 10 ml per plate.
· Distribute 100 μl of cell suspension into all wells.
· Cover the plate with a non-toxic sealer and incubate at 36°C.
· Examine daily, using an inverted microscope, and record CPE.
· Continue recording until 24 hours after CPE in the virus control wells reaches 100%.
· The virus is identified by the pattern of inhibition of CPE by antiserum pools, as shown on the table accompanying the sera.
· If CPE is seen in all wells containing virus, the isolate should be reported as a non- poliovirus.
Figure 7.5: Identification of enterovirus isolates using the microtechnique
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