600484013(r-12)
xvEPA
United States
Environmental Protection
Agency
Environmental Monitoring and EPA/600/4-84/013(R-12)
Support Laboratory May 1988
Cincinnati OH 45268 Revision
Research and Development
USEPA Manual of
Methods for Virology
Chapter 12
Revised May 1988
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r*
x
o
J
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Chapter 12
Identification of Enteroviruses
May 1988
1. Introduction
A neutralization test used to identify
enteroviruses is described in this
chapter. The test utilizes reference-
typing sera directed against isolated
waterborne viruses. This renders the
viruses noninfectious when treated
with a matching serotype reagent.
Virus inactivation is ascertained
microscopically by observing the
absence of host cell destruction in the
presence of the virus-antibody mix-
ture. The procedure consists of simul-
taneously inoculating virus and anti-
serum into a microtiter plate,
incubating the virus-antibody mixture
for 2 hr, adding a suspension of host
cells to the mixture, incubating the
host cells-virus-antibody mixture for
three days, then examining the cells
daily for five more days for the
absence of cytopathic effects (CPE).
The test utilizes the Lim Benyesh-
Melnick (LB-M) antiserum pools which
consist of 61 equine antisera. They
include LB-M antiserum pools A-H for
the identification of 41 enteroviruses,
a single antiserum preparation for the
identification of coxsackievirus B3 and
LB-M pools J-P for the identification of
19 group A coxsackieviruses not iden-
tified by pools A-H. The A-H antiserum
pools, prepared by Melnick and
coworkers (1973), with instructions for
rehydration and storage and with virus
identification tables, had been pre-
viously available from the National
Institutes of Health (NIH). The stock of
these antiserum pools (including the
single coxsackievirus B3 antiserum
preparation) was depleted in 1 983.
New pools were again prepared by
Melnick and Wimberly (1985) under
the sponsorship of the World Health
Organization (WHO) using the same
bulk materials as were used to pro-
duce the earlier pools. A policy state-
ment issued by WHO, Geneva, des-
cribing conditions for their distribution
is available from Dr. C. H. Mordhorst,
Director, WHO Collaborating Centre
for Virus Reference and Research,
Statens Seruminstitut, DK-2300
Copenhagen S, Denmark. The other
set of seven antiserum pools (J-P) was
also prepared by Melnick and
coworkers (1977) under National Insti-
tutes of Health sponsorship. Informa-
tion on their distribution is available
from the National Institute of Allergy
and Infectious Diseases Repository,
Biotech Research Laboratory, Inc.,
1600 E. Gude Drive, Rockville, Mary-
land 20850. Single antiserum prepa-
rations are distributed by the Centers
for Disease Control, Biological Prod-
ucts Division, Atlanta, Georgia 30333
and the American Type Culture Collec-
tion, 12301 Parklawn Drive, Rockville,
Maryland 20852.
The microtiter method provided
herein is a modification of the method
described in the literature accompany-
ing the NIH pools. The two methods
work equally well, but the microtiter
method requires much less antiserum.
The microtiter method was also
deemed preferable to the plaque
reduction neutralization test (Lennette
and Schmidt, 1 979) based on cost,
time and supplies required for actual
analyses.
2. Procedure for Typing
Viruses
2.1 Apparatus and Materials
2.1.1 Microtiter plates, 96-well, flat
bottom.
2.1.2 Sealing tapes for microtiter
plates if plates are to be incubated in a
non-COg incubator (recommended
method), or plastic lids for microtiter
plates if plates are to be incubated in a
COa incubator.
2.1.3 Micro-pipettors or pipettes
capable of dispensing volumes of
0.025 and 0.05 mL.
2.1.4 Cornwall syringe, or equival-
ent, capable of delivering 0.2 mL
quantities.
2.1.5 Cotton-tipped applicators.
2.1.6 Magnetic stirrer and stir bars.
2.7.7 Narrow-tip felt marking pen.
2.1.8 Inverted microscope.
2.2 Media and Reagents
12-1
2.2.1 ELAH—Earle's base with 0.5%
lactalbumin hydrolysate and without
NaHC03(Hazleton Kansas City Biologi-
cal, product no. DM-303, or equiva-
lent) supplemented with antibiotics
(dihydrostreptomycin sulfate, penicillin
G, tetracycline and amphotericin B;
Sigma Chemical Co., or equivalent).
ELAH—Earle's base solution supple-
mented with antibiotics is used as a
dilution medium.
Procure 40 mL of antibiotic supple-
mented ELAH—Earle's base solution
for each virus to be identified.
Employ stock antibiotic and ELAH—
Earle's base solutions prepared for use
in Chapter 10 (December, 1987 revi-
sion}. Stock antibiotic solutions pre-
pared in Chapter 9 (January, 1987
revision) may also be used. If unavaila-
ble, see Chapter 10, Section 2.1.3 for
preparation of stock antibiotic solu-
tions and Section 2.1.4 for preparation
of ELAH—Earle's base solution and for
supplementation of ELAH—Earle's
base solution with antibiotics.
Remaining reagents may be stored for
subsequent use. Store antibiotic stock
solutions at -20°C and ELAH—Earle's
base solution at 4°C for periods no
greater than 4 and 2 months, respec-
tively. Reagents should be held in
tightly stoppered or capped containers.
2.2.2 Antiserum pools A-H and cox-
sackievirus B3 antiserum are prepared
as described in WHO instruction
sheets.
Store at -20° C until used.
Prepare antiserum pools J-P only
when needed to type viruses not iden-
tified by pools A -H or coxsackievirus
B3 antiserum. Antiserum pools J-P
are prepared as described in NIH
instruction sheets.
2.2.3 Growth medium supple-
mented with antibiotics (penicillin G,
dihydrostreptomycin sulfate, tetracy-
cline and amphotericin B; Sigma
Chemical Co., or equivalent) and 5%
fetal calf serum (GIBCO Laboratories,
or equivalent) prior to addition of BGM
cells.
Employ MEM/L-15 growth medium
and stock antibiotic solutions prepared
for use in Chapter 9 (January, 1987
revision). Stock antibiotic solutions
prepared in Chapter 10 (December,
-------
May 1988
1987 revision) may also be used. If
unavailable, see Chapter 9, Section
3.3.3 for preparation of growth
medium and Section 3.3.5 for prepara-
tion of stock antibiotic solutions.
Remaining reagents may be stored for
subsequent use. Store antibiotic stock
solutions at -20°C and growth
medium at 4°C for periods no greater
than 4 and 2 months, respectively.
Reagents and medium should be held
in tightly stoppered or capped
containers.
Fetal calf serum used should have
been heat inactivated at 56° C for 30
min and certified free of animal vi-
ruses, bacteriophage and mycoplasma
by the supplier.
Each liter of growth medium is sup-
plemented with 1 mL of penicillin-
dihydro-streptomycin stock, 0.5 mL of
tetracyc/ine stock and 0.2 mL of
amphotericin B stock.
Prepare 30 mL of medium for each
microtiter plate to be used.
2.3 Procedure
2.3.1 Preparation of Microtiter
Plates
Arrange each plate as indicated in Fig-
ures 12-1 and 12-2.
(a) With a narrow-tip felt marking pen,
draw lines between every two
columns along the length of the
plate.
(b) On one end of each plate, mark
identification code of samples
tested.
Four viruses can be identified
simultaneously on one plate.
Thus, number the columns 1, 2,
3, and 4 to designate duplicate
wells for each virus.
(c) Mark identity of each antiserum on
left side of plate next to each row
of wells.
(See Figure 12-1). Designate
the first eight rows as A to H to
indicate LB-M pools A -H, desig-
nate row 9 as B3 to indicate cox-
sackievirus B3 antiserum, desig-
nate row 10 as E to indicate virus
control dilution made in antibiotic
supplemented ELAH—Earle's
base solution, and designate rows
11 and 12 as El and E2, respec-
tively, to indicate serial 10-fold
dilutions of virus control in row
10.
2.3.2 Preparation of Virus for
Identification.
Figure 12-1.
Representation of micro -
titer plate preparation.
See Figure 12-2 for pho-
tographic representation
of microtiter plate prepa-
ration.
Row
E2
£1
£
B3
H
G
F
E
D
C
B
A
0 0
0 0
0 0
0 0
0 0
0 0
0 0
0 0
0 0
0 0
0 0
0 0
0 0
0 0
0 0
0 0
0 0
0 0
0 0
0 0
0 0
00
00
0 0
0 0
0 0
0 0
0 0
0 0
0 0
0 0
0 0
0 0
0 0
0 0
0 0
0 0
0 0
0 0
0 0
0 0
0 0
0 0
0 0
0 0
0 0
0 0
0 0
1234
Column
(a) Remove virus isolate from storage
in -70°C freezer, thaw, and mix
well. Designate the virus isolate
as No. 1.
(b) Dilute thawed virus to 10~5 in anti-
biotic supplemented ELAH—
Earle's base solution.
Prepare 2 mL of 10~5 dilution of
virus. The 10~5 dilution is the
working dilution of virus that will
be mixed with the antiserum pools
in the microtiter plate wells.
(c) From the 10~5 dilution, prepare a
1:2 dilution in Earle's Balanced
Salt Solution.
This dilution will be transferred
to row E of the microtiter plate
later.
(d) From the 1:2 dilution of virus pre-
pare two serial 10-fold dilutions
(1:20 and 1:200).
These dilutions will be trans-
ferred to rows E1 and E2 of the
microtiter plate later.
(e) Repeat Steps (a) to (d) with each
virus isolate to be identified.
designate these isolates 2 through
4, and proceed to Section 2.3.3.
2.3.3 Addition of Antiserum Pools to
Microtiter Plate
(a) Thaw the antiserum pools, and mix
each antiserum pool well.
(b) With a micro-dilutor tip or pipette,
dispense 0.025 mL of antiserum
from pool A into each well in row
A.
It is important to place tip of di-
lutor or pipette into the bottom of
the well and to expel all of the
antiserum in the pipette into the
well.
(c) Repeat Section 2.3.3, Steps (a) and
(b) with antiserum pools B to H
and with the antiserum for cox-
sackievirus B3, placing anti-
serums into designated wells, and
proceed to Section 2.3.4.
2.3.4 Addition of Virus to Microtiter
Plates
(a) Add 0.025 mL of the 10"5dilution of
virus No. 1 from Section 2.3.2,
Step (b) to each well in rows A to
B3 of column 1.
Take care to introduce the virus
at the top of the wells. Do not
allow tip of dilutor or pipette to
touch the pooled antiserum within
a well and thereby possibly cross-
contaminate other antiserum
pools.
(b) Into the two wells marked E in
column 1, add 0.05 mLof the 1:2
dilution of virus No. 1 from Sec-
tion 2.3.2, Step (c).
(c) Into the two wells marked E1 in
column 1, add 0.05 mLof the 1:20
dilution of virus No. 1 from Sec-
tion 2.3.2, Step (d).
(d) Into the two wells marked E2 in
column 1, add 0.05 mL of the
1:200 dilution of virus No. 1 from
Section 2.3.2, Step (d).
(e) Repeat Steps (a) to (d) with viruses
No. 2 through 4, adding the
appropriate dilutions of the vi-
ruses to the appropriate wells
(See Figure 12-1).
(f) Gently tap the sides of the microt-
iter plate with index finger to mix
the contents of the wells.
12-2
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May 1988
Figure 12-2, Photographic representation of microtiter plate preparation. See Figure
12-1 for schematic representation of microtiter plate preparation.
(g) Cover microtiter plates with lids or
with a loose sterile cover, and
incubate plates at 36.5°C + 1 °C
for two hours.
2.3.5 Preparation of Cell Suspension
and Completion of Microtiter Test.
Many host cell types, primary and
continuous, are available for propaga-
ting viruses. Usually, the host cell type
in which a virus is recovered from the
environment is suitable for identifying
that virus by the microtiter neutraliza-
tion test. See Chapter 9 for procedures
and media for culturing Buffalo green
monkey (BGM) kidney cells. See
Lenette, E. H. and Schmidt, N. J.,
Diagnostic Procedures for Viral, Rick-
ettsial and Chlamydial Infections,
American Public Health Association,
Washington, DC, 1979, for methods
for preparation of primary and other
continuous cell types, for suckling
mouse procedures necessary for iden-
tifying most Group A coxsackieviruses,
and for methods for identifying viruses
other than enteroviruses.
(a) Trypsinize sufficient cells to yield a
final cell count appropriate for the
cells used in the test.
For BGM cells a count of
30,000 to 50.000 cells per 0.2 mL
of cell culture medium is appro-
priate. The number of cells
required for this test differs with
different cell types.
(b) Add cells to appropriate volume of
cell culture medium.
(c) Mix cells on a magnetic stirrer and
stir for at least 1 5 minutes at
speed sufficient to develop vortex.
A longer period of mixing will
generally not injure cells.
(d) Dispense 0.2 mL of cell suspension
into each well with a Cornwall
syringe after completing the two
hour incubation of virus-
antiserum mixtures in Section
2.3.4, Step (g).
Do not allow tip of syringe to
touch contents of a well and
thereby possibly cross-
contaminate the contents of other
wells. With cotton-tipped applica-
tors, wipe up spilled cells on the
top of plates between and around
wells.
(e) Seal each plate with sealing tape
and incubate plates at 36.5°C
±1°C.
If plates are to be incubated in a
COz incubator, do not seal plates.
(f) After three days of incubation,
examine cells in wells daily for
five or more days for the appear-
ance of CPE.
Use an inverted microscope to
examine cells.
(g) When CPE develops, use identifica-
tion tables provided with anti-
serum pools to identify viruses.
Virus identification is based
upon the absence of CPE in those
wells containing ant/serum and
virus.
If a/1 wells evidence CPE, titrate
virus and repeat entire test with a
virus dilution calculated to add
100 infective doses to each well
in Row E. Follow this same proce-
dure if all virus control wells in
Rows El and E2 are negative and
the pattern of results does not
allow identification with identifi-
cation tables.
Where an appropriate quantity
of viral infective doses has been
used in test and cells in only one
of the duplicate wells containing
antiserum show no CPE. repeat
tests with antiserum pools A -H
and B3. If CPE still appears in all
wells containing virus and anti-
serum, repeat test but with anti-
serum pools J-P instead of A-H
and B3. If CPE continues to be
observed in all wells, one must
take into consideration the possi-
bility that more than one virus
was present in the original test
sample. Jo eliminate the possibil-
ity of a mixture of viruses being
present, dilute the original test
sample with ELAH—Earle's base
solution to a viral concentration of
between 5 and 10 plaque forming
12-3
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May 1 988
units per mL Plaque purify the
virus using the cell monolayer
plaque assay technique described
in Chapter 10 (December 1987
revision). Pick at least three well-
separated plaques and prepare
sufficient virus quantities from
each plaque in accordance with
the procedures given in Chapter
11 (March 1988 revision). Retest
each of the new viral stocks utiliz-
ing the Lim Benyesh-Melnick
antiserum pools. If the viral iso-
lates are still not neutralized by
known antisera, record the virus
in question as unidentified.
If it is essential that all isolates
be identified in a monitoring pro-
gram, store the virus at -70°C for
later studies. The virus in question
may either be further tested
against single antiserum prepara-
tions of serotypes not included in
the pools or used to prepare
antiserum to characterize the iso-
late against known enteric
viruses.
Combination Pools of Enterovirus
Equine Antisera: Preparation and
Test Procedures for the Identifica-
tion of Field Strains of 19 Group A
Coxsackievirus Serotypes. Intervirol.
8:172-181.
Melnick, J. L. and I. L. Wimberly.
1985. Lyophilized Combination
Pools of Enterovirus Equine Antis-
era: New IBM Pools Prepared from
Reserves of Antisera Stored Frozen
for Two Decades.fiu//. Wld. Hlth.
Org. 63:543-550.
3. Bibliography
Casals, J. 1967. Immunological Tech-
niques for Animal Viruses, p. 113-
198. In K. Maramorosch and H.
Koprowski (eds.). Methods in Virol-
ogy, Vol. 3. Academic Press, New
York, NY.
Habel, K. 1969. Virus Neutralization
Test, p. 288-296. In K. Habel and N.
P. Salzman (eds.), Fundamental
Techniques in Virology. Academic
Press, New York, NY.
Laboratory Manual in Virology. 1974.
Edition Two. Ontario Ministry of
Health, Toronto, Ontario, Canada,
375 pp.
Lamb, G. A., K. Plexico, W. P. Glezen,
and T. D. Y. Chin. 1 965. Use of
Micro Technique for Serum Neutral-
ization and Virus Identification. Pub-
lic Health Rep. 60:463-466.
Lennette, E. H. and N. J. Schmidt.
1979. Diagnostic Procedures for
Viral, Rickettsial and Chlamydial
Infections, American Public Health
Association, Washington, DC,
1138pp.
Melnick, J. L., V. Rennick, B. Hampil,
N. J. Schmidt, and H. H. Ho. 1973.
Lyophilized Combination Pools of
Enterovirus Equine Antisera: Prepa-
ration and Test Procedures for the
Identification of Field Strains of 42
Enteroviruses. Bull. Wld. Hlth. Org.
48:263-268.
Melnick, J. L, N. J. Schmidt, B. Ham-
pil, and H. H. Ho. 1977. Lyophilized
12-4
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xvEPA
United States Environmental Monitoring and EPA/600/4-84/013(APP)
Environmental Protection Support Laboratory June 1988
Agency Cincinnati OH 45268 Revision
Research and Development-
USEPA Manual of
Methods for Virology
Appendix
Revised June 1988
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June 1988
Appendix
Vendors
List of vendors only indicates one possible source of products used in this Manual.
In most instances, many other vendors can supply the same materials listed or
acceptable alternatives.
American Type Culture Collection
12301 Parklawn Drive
Rockville, Maryland 20852
800-638-6597
Curtin Matheson Scientific, Inc.
999 Stuebner Airline
Houston, Texas 77038
713-820-9898
AMF Cuno Division
400 Research Parkway
Meriden, Connecticut 06450
203-237-5541
Difco Laboratories
P.O. Box 1058
Detroit, Michigan 48232
313-961-0800
Badger Meter Utility Prod.
Flow Products Division
4545 West Brown Deer Road
Milwaukee, Wisconsin 53223
414-355-0400
Beckton Dickinson Consumer Products
Franklin Lakes, New Jersey 0741 7
201-848-6800
Bellco Glass, Inc.
P.O. Box B
340 Edrudo Road
Vineland, New Jersey 08360
800-257-7043
Brockway Inc.
Route 10, Box 13
Parkersburg, West Virginia 26101
304-295-9311
Carborundum Company
Commercial Filters Division
Lebanon, Indiana 46052
317-482-3900
Centers for Disease Control
Biological Products Division
Atlanta, Georgia 30333
404-639-3355
The Clorox Company
1221 Broadway
Oakland, California 94623
415-271-7000
Corning Glass Works
(see Fisher Scientifc,
Thomas Scientific, or
VWR Scientific)
Du Bois Chemicals
Division of Chemed Corporation
1300 Du Bois Tower
Cincinnati, Ohio 45202
513-762-6000
Eli Lilly and Company
307 E. McCarty Street
Indianapolis, Indiana 46285
317-261-2000
Falcon Labware
Becton Dickinson Labware
1950 Williams Drive
Oxnard, California 93030
800-235-5953
Filterite Corporation
2033 Greenspring Drive
Timonium, Maryland 21093
301-252-0800
Fisher Scientific
711 Forbes Avenue
Pittsburgh, Pennsylvania 15219
412-562-8300
Gelman Sciences, Inc
600 South Wagner Road
Ann Arbor, Michigan 48106
313-665-0651
GIBCO Laboratories
Life Technologies, Inc.
3175 Staley Road
Grand Island, New York 14072
800-828-6686
Hana Biologies, Inc.
850 Marina Village Parkway
Alameda, California 94501
800-772-4262
A-1
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June 1988
Hazleton Research Products, Inc.
(formerly K C Biological, Inc.)
P.O. Box 14848
13804W. 107th Street
Lenexa, Kansas 66215
800-255-6032
ICN Nutritional Biochemicals
P.O. Box 28050
Cleveland, Ohio 44128
800-321-6842
Johanson and Son Machine Corporation
259 Allwood Road
Clifton, New Jersey 0701 2
201-773-6160
Millipore Corporation
80 Ashby Road
Bedford, Massachusetts 01 730
617-275-9200
The Vollrath Company
1236 North 18th Street
Sheboygan, Wisconsin 53081
414-457-4851
VWR Scientific
P.O. Box 7900
San Francisco, California 94120
415-468-7150
WHO Collaborating Centre
for Virus Reference and Research
ATTN: Dr. C. H. Mordhorst, Director
Statens Seruminstitut
DK-2300
Copenhagen S, Denmark
(45)01-952817
National Institute of Allergy
and Infectious Diseases Repository
Biotech Research Laboratory, Inc.
1600 E. Gude Drive
Rockville, Maryland 20850
301-251-0800
New Brunswick Scientific Co., Inc.
Box 4005
44 Talmadge Road
Edison, New Jersey 0881 8
800-631-5417
Pfizer Laboratories Division
235 East 42nd Street
New York, New York 10017
212-573-2323
Polychem Corporation
12 Lyman Street
New Haven, Connecticut 06511
800-243-3093
Sigma Chemical Company
P.O. Box 14508
St. Louis, Missouri 63178
800-325-3010
E. R. Squibb and Sons, Inc.
P.O. Box 4000
Princeton, New Jersey 08540
609-921-4000
Thomas Scientific
99 High Hill Road at I-295
P.O. Box 99
Swedesboro, New Jersey 08085
609-467-2000
Van London Company
6103 Glenmont
Houston, Texas 77081
713-772-6641
A-U.S. GOVERNMENT PRINTING OFFICE: !»«« - Stt-IM/87030
A-2
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