Diagnostic Virology Protocols

366 Pages·2010·23.3 MB·English

by  John R. Stephenson

1
Rapid Detection of Adenovirus
from Fecal Specimens
Tanvir Tabish, Alan Warnes, and Stuart Clark
1. Introduction
In 1953, the first adenovirus was isolated from a human and, subsequently,
47 types have been shown to exist. Adenoviruses are now classified mto six
subgroups (A-F), which are based on their hemagglutmatlon properties (I).
They have a lcosahedron structure that contains double-stranded lmear
DNA of 45,000 basepan-s. Although, adenovlruses can cause a range of
infections m humans, including conJunctivltls, pharyngltls, and gastroen-
terltls, this chapter focuses on gastroenterlc adenoviruses and their rapid
detection m fecal samples.
The incidence of adenovn-al infections causing gastroenterms IS well-docu-
mented, accounting for 5-l 5% of all viral infections, which occur throughout
the year with no particular seasonal peak (2). The incubation period IS dose-
dependent, but usually takes 5-8 d to the onset of clinical symptoms. The abil-
ity of adenovu-uses to infect the intestinal tract 1sd ue to their ablllty to survive
low pH levels where high titers of virus particles can be produced and subse-
quently excreted.
Unlike respiratory adenoviruses, those causing gastroenterltls are difficult
to grow in culture, therefore, cross-reacting group proteins are commonly used
as target antigens for m vitro dlagnostlc assays.A lthough there 1sc onsiderable
genetic variation between the different types of adenoviruses, the hexon gene
contains highly conserved regions which when translated produce a cross-
reacting antigen (3). Thus, assayst hat will detect adenovuuses in fecal samples
have been deslgned using the hexon protein. Alternatively, owing to advances
m genetic engineering, it is now possible to produce recombinant proteins from
From Methods m Molecular Medune, Vol 12 Dlagnosbc Wology Protocols
Edlted by J R Stephenson and A Warnes 0 Humana Press Inc , Tolowa, NJ
1
2 Tabish, Warnes, and Clark
eukaryottc systems, and this technology 1s described m further detail m
Chapter 24.
Recent work has indicated that monoclonal antibodies (MAbs) specific for
types 40 and 4 1 can detect those adenovtrus types causmg gastroenterms, but
not those causing respiratory or ocular mfecttons (4). Subsequently, enzyme
tmmunoassays (EIAs) based on the use of these MAbs have been developed to
detect the types 40 and 41 directly from fecal samples. However, there are
reports that a number of other adenovnuses are also responsible for gastroen-
teritis, and consequently an assay that detects adenovnus group antigen may
be better suited for screening purposes (5-7).
Although there are a number of EIAs available for the detection of
adenovuuses, most require sophisticated laboratory equipment, which 1s not
always avatlable to all laboratortes We, therefore, descrtbe a latex agglutma-
tion assay for the raped detection of adenovn-uses m stool specimens that can
be as sensitive as electron microscopy (8), which is still used as the confirma-
tory test for positive fecal samples. Latex agglutination can be performed
rapidly taking only 2 mm, with 3 min for sample preparation; the result is easy
to visualize, with no requu-ement for support equipment. Upon mixmg the
extracted sample, with polyclonal antibody-coated latex particles, cross-link-
ing occurs between the anttbodies coated to the latex and the virus partrcles,
resulting in the productton of visible aggregates.
Owmg to the widespread use of adenovtral vectors as deltvery systems m
gene therapy, there is now the opportunity to utilize the rapid latex assay as an
on-line monitoring system to estimate viral load in cell culture. This has the
benefits of being able to maximize viral yield for the production of stocks,
using an assay that takes only 2 mm to perform.
2. Materials
1. Beckman5 2-21 centrifuge (Fullerton, CA).
2. Dialysis tubing BDH Stze 5 Dram 24/32-19.0 mm (BDH, Poole Dorset, UK)
3. Nalgene glass fiber pre-filters: Sybron International (Rochester,N Y).
4 Amicon 76mm Ultracentrifugation cell (Amicon Ltd, Stonehouse, Gloucester-
shire, UK)
5. Ultrafiltration membrane (PM30 43mm) (Amicon).
6. Roller mix.
7. Sonicator MSE Soniprep 150 (MSE Scientific Instruments, Crawley, UK)
8. Sephacryl S-400-HR (Sigma S400-HR) (Sigma Chemical Company Ltd, Poole,
Dorset, UK).
9 Glycme-buffered salme (GBS). 100 mA4 glycine, 171 mM sodium chloride,
15 mA4 sodium azide, pH to 8.2 with 10 A4 sodmm hydroxide solution (BDH).
10. Latex; Prolabo K080: 0.8 pm (Estapor, Manchester, UK).
11 Glacial acetic acid (BDH).
Detection of Adenovirus 3
12. n-Octanoic acid (BDH)
13. Phosphate-buffered saline (PBS): 150 rnA4 sodium chlortde, 4 rnM potassium
dihydrogen phosphate, 11 mM disodium hydrogen phosphate dehydrate (BDH).
14. Ethylenedtamine tetra-acetic acid (EDTA): 10 mM stock solution
15 Acetate buffer, pH 4.0. 100 mM sodium acetate, 5 75 mM glacial acetic acid
16 Acetate buffer, pH 4.8: 100 mA4 sodium acetate, 5 75 n&Y glacial acetic acid
17. 0.1% Bovine serum albumin (BSA) solution (Bayer Diagnostics, Nankakee, IL)
m GBS
18 Normal rabbit serum (NRS).
19. Cell growth media (EMEM): 10% fetal bovine serum, 1X nonessential ammo
acids, 10 nM HEPES,2 rnM L-glutamme.
20. Beta-propiolactone.
2 1. Extraction buffer: available from Microgen Bioproducts Ltd (Camberley, Surrey).
22 Mixing cards (Microgen Bioproducts).
23. Filtration units (Microgen Bioproducts).
3. Methods
The following list of protocols describes the procedures involved in purify-
ing hexon proteins required as immunogen to raise polyclonal anti-adenovirus
hexon antisera.
3.1. Production of Adenovirus
1 Grow a Hep2 cell monolayer to 80% confluency in 175 cm2 tissue-culture flasks
m cell-growth media sparged with 95% sir/5% CO2 at 37°C.
2. Infect cells with a multtpltcity of infection of 0.01 of adenovuus type 5 and mcu-
bate for 1 h at 37°C.
3. Remove the vnus and wash the cells with prewarmed PBS.
4. Add 60 mL of fresh prewarmed cell-growth media, gas wtth 95% sir/5% CO2
and incubate at 37°C
5. Examme the cell monolayer daily and harvest, when the cell monolayer shows
100% cytopathic effect.
6. Freeze thaw the culture four times and then inactivate the vnus with 0.4% beta-
propiolactone.
7. Store crude vnus stockp reparationsa t -20°C.
3.2. Purification of Adenovirus Hexon Protein
and Production of Polyvalent Sera
A key stage is the purification of hexon protein and the subsequent prodwc-
tion of polyvalent sera; although the precise method cannot be mentioned here,
purification processes are now well-advanced and detailed elsewhere (9). The
use of genetic engineering should also not be forgotten because these methods
now offer the productton and purification of protems as diagnostic antigens
with relative ease (see Chapter 24). The purified adenovirus hexon protem can
4 Tabwh, Warnes, and Clark
be evaluated for purity using polyacrylamlde gel electrophoresis (PAGE) or
immunoblotting if polyclonal antibodies are available (see Note 1).
Polyclonal anti-hexon sera can then be obtained by immumzation of am-
mals, and the serum stored at -4OOC.
3.3. Fractionation of IgG
from Rabbit Antiserum Using n-Octanoic Acid
1 Pour the rabbit antiserum mto appropriate plastic containers; add twice the vol-
ume of acetateb uffer, pH 4.0.
2. Stir each pot vigorously at room temperature with the aid of a magnetic stirrer
3. For every 10 mL of rabbit serum, add 0 75 mL n-octanolc acid in a dropwlse manner.
4 Leave containers to stir at room temperature for 30 mm
5. Centrifuge m Beckman J2-21, for 20 mm, at 15,300g and 10°C
6. Decant and retam the supernatant.
7 Resuspend the precipitate m 10 mL of acetate buffer pH 4 8 per 10 mL of original
serum volume.
8. Centrifuge at 15,300g for 20 min at 10°C
9. Pool the supernatant with that retained in Subheading 3.3.6.
10. Boil the dialysis tubing m 10 mM EDTA for 2 mm m a glass beaker, allow to
cool, and wash thoroughly with dlstllled water
11 Filter treated serum through a Nalgene glass-fiber pre-filter to remove coarse
precipitate and plpet mto the dialysis tubing
12. Dialyze against GBS, mltlally for 4 h at 4”C, followed by a further 16 h at the
same temperature.
13 Check pH of the dialyzed material, if pH is 8 O-8 2, then proceed, if not, then
dialyze for a further period until the desired pH has been achieved
14. Store the purified anti-adenovirus IgG at -7O’C
3.4. Manufacture of Test Antibody-Coated Latex
The method for the production of antibody-coated latex particles has
already been published in detail (IO), as described here; a moditkatlon of
this method is currently used at Microgen Bioproducts Ltd. This provides
hexon-specific polyvalent antibody-coated latex particles produced in Sub-
heading 3.2.
1. Wash the latex particles three times with PBS by centrlfugatlon at SOOOg for 20
min at 4°C
2. Resuspend at a concentration of 0.4% m PBS.
3. Add an equal volume of anti-adenovlrus antibody at 16 mg/mL
4. Shake at room temperature for 2 h
5. Wash coated latex particles three times with PBS by centrifigation at 8000g for 20
mm at 4°C
6. Resuspend to a final concentration of 0.7% in PBS and store at 4°C.
Detection of Adenovirus 5
Table 1
Comparison of Adenovirus Latex Against EM
Adeno latex
+ -
EM results + 53 3a
- lb 79
OThis sample was retested wrth other commercial assays
and was shown to be positive
“On retesting wrth other commercial assays 2 of the 3
samples were also shown to be negatrve
3.5. Manufacture of Control Antibody-Coated Latex
As part of the assay, it is essential that a control latex is always used so that
the test can be correctly evaluated and nonspecific reactions detected. The con-
trol rabbit IgG is derived from normal rabbit serum (NRS) as described m Sub-
heading 3.3. and processed as described m Subheading 3.4.
3.6. Test Procedure
1 Prepare an approximate 10% suspension of the fecal sample by transferring 0.1 g
(0 1 mL) of sample into 1 mL of extraction buffer in a stoppered tube (supplied as
a component of the filter pack). Mix the contents well
2. Allow the reagents to reach room temperature for l-2 min before processing.
3. Remove the stopper and fit integral filter/dropper unit
4. Holding the whole assembly vertically, dispense 1 drop of clear filtrate onto each
of 2 wells on the test slide.
5. Add 1 drop of well-mtxed test latex reagent to one well and 1 drop of control
latex reagent to the other
6. Mix the contents of each well using a separate mixing sttck for each sample,
covering the entire area of the well
7. Gently rock the slide and observe for agglutination for up to 2 min
8. A posrttve result is indtcated by agglutinatron of the test latex reagent wrth no
agglutinatton of the control latex reagent.
9. The result 1s negattve if no agglutination of either the test latex reagent or the
control latex reagent IS observed within the 2-min test period
10. Agglutmatton both in the test and control latex Indicates a nonspecific result and
the sample should be retested.
3.7. Expected Results
To contrast the efficacy of the assay the following results were obtained
when a panel of fecal samples was independently assessed by electron mtcros-
copy (EM) (see Table 1).
6 Tabish, Warnes, and Clark
These results produced a sensitivity of 95% and a specificity of 99% com-
pared to the “Gold standard” EM. Other commercial kits are available from
other companies (see Note 2).
4. Notes
1 If polyclonal or MAbs are available, the purified antigen should be characterized
usmg both PAGE and immunoblotting. If fusion protems are produced via genetic
engineering techniques, then most protein end products can be visualized used
supplied anttbodies against the tagging protein
2 Commercial assays based on latex agglutination are available from Microgen
Bioproducts (Adenoscreen); Onon, Finland (Adenolex); Bioktt, Spain (Adenogen)
References
1, Hrerholzer, J C (1991) Antigenic relationships among the 47 human adenovnuses
determined m reference horse sera Arch Vzrol 121, 179-l 97
2 Albert, M J (1986) Enteric adenovnuses Arch Vzrol 88, 1-17
3 Cepko, C. L., Whetstone, C. A , and Sharp, P. A. (1983) Adenovnus hexon mono-
clonal antibody that is group specific and potentially useful as a diagnostic reagent.
J Clan. Mcroblol 17(2), 360-364.
4 Grydsuk, J. D., Fortsas, E , Petric, M , and Brown, M. (1996) Common eprtope on pro-
tem VI of enteric adenovuuses from subgenera A and F J Gen Vzrol 77(8), 18 l-l 89
5. Bhan, M. K., Raj, P., Bhandart, N., Svensson, L., Stmtzmg, G., Prasad, A. K.,
Jayashree, S., and Snvastava, R. (1988) Role of enterrc adenovnuses and rotavuuses
m mild and severe acute enterms. Pedzatr Infect Du. J 7(5), 320-323
6 Petrtc, M (1995) Cahcivrruses, astrovnuses and other drarrherc viruses, m Manual of
Clznzcal Mzcroblology, 6th ed. (Murray, P. R , Baron, E. J., Pfaller, M. A , Tenover, F.
C , and Yolken, R. H., eds.), ASM, Washmgton, DC, pp. 1017-1024
7. Noel, J , Mansoor, A., Thaker, U , Herrmann, J., Perron-Henry, D , and Cubitt,
W D. (1994) Identification of adenovnuses m faeces from patients with drar-
rhoea at the Hospitals for Sick Children, London, 1989-l 992 J, Med Vu-01 43(l),
- 84-90
8. Grandlen, M., Pettersson, C. A., Svensson, L., and Uhnoo, I (1987) Latex agglu-
tmatron test for adenovnus dragnosrs m diarrhea1 drsease J. Med Vzrol 23(4),
311-316.
9. Doonan, S. (ed.) (1996) Methods zn Molecular Biology Protein Purlficatron Pro-
tocols. Humana, Totowa, NJ.
10. Sanekata, T., Yoshida, Y , and Okada, H. (1981) Detection of rotavnus m faeces
by latex agglutination J Immunol Methods 41,377-385
2
Alphaviruses
John T. Roehrig, Teresa M. Brown,
Alison J. Johnson, Nick Karabatsos,
Denise A. Martin, Carl J. Mitchell, and Roger S. Nasci
1. Introduction
Alphaviruses are enveloped, positive-stranded RNA viruses that are the etio-
logic agents of severe encephalitis and polyarthritis. These viruses can be
divided into six or seven serocomplexes (1). Four of these serocomplexes-
represented by eastern equine encephalitis (EEE), western equine encepha-
litis (WEE), Venezuelan equine encephalrtts (VEE), and Semliki Forest
viruses+omprise the most medically important alphaviruses. The VEE
serocomplex can be further divided into at least six subtypes (1 to 6), with
subtype 1 having at least five different varieties (1 AB, lC, lD, lE, and
1F ). The importance of VEE virus subtyping 1s that varieties 1AB and 1C
vu-uses cause epidemic/epizootic VEE infection, whereas disease caused
by other VEE viruses is endemic/enzootic. Ross River, Chikungunya,
Mayaro, and Getah viruses are members of the Semliki Forest serocomplex.
Sindbis and Ockelbo viruses are members of the WEE virus serocomplex. A
newly emerging alphavirus, Barmah Forest, may represent a new serocomplex
of alphaviruses.
Laboratory diagnosis of human alphavirus infections has changed greatly
over the last few years. In the past, identification of alphavirus antibody relied
on four tests:h emagglutmation-mhibition, complement fixation, plaque reduc-
tion neutralizatton test, and the indirect fluorescent antibody (IFA) test. Posi-
tive identification using these immunoglobulin M- (IgM-) and IgG-based
assays required a fourfold increase in titer between acute and convalescent
serum samples. A number of very good procedural reviews contam the specif-
ics of these older assays( 2).
From Methods m Molecular Medmne, Vol 12 Diagnosbc Wology Protocols
EdRed by J R Stephenson and A Warnes 0 Humana Press Inc , Totowa, NJ
7
8 Roehrig et al.
With the advent of solid-phase antibody-binding assays, such as enzyme-
linked immunosorbent assay (ELISA), the diagnostic algorithm for identifica-
tion of viral activity has changed. Rapid serologic assayss uch as IgM-capture
ELISA (MAC-ELISA) are now employed early in infection (3). In many cases,
a positive MAC-ELISA with an acute serum sample precludes the need for
testing of a convalescent serum sample. Early m infection, IgM antibody is
more serocomplex specific, while later in infectton, IgG antibody is more
serocomplex crossreactive. Inclusion of monoclonal antibodies (MAbs) with
defined virus specificities in these solid-phase assaysh as allowed for a level of
standardization that was not previously possible. All tests described in this
chapter are equally applicable to all alphaviruses.
Virus isolation and identification have also been useful in defining viral
agents in serum, cerebrospinal fluid (CSF), or mosquito vectors. Although vnus
isolation still depends upon growth of an unknown virus in cell culture or neo-
natal mice, virus identification has also been greatly facilitated by the avatl-
ability of virus-specific MAbs for use m IFA assays. Similarly, MAbs with
avidities sufficiently high to allow for specific binding to virus antigens in a
complex protein mixture (e.g., mosquito pool suspensions) have enhanced our
ability to rapidly identify virus agents in situ. Although polymerase chain reac-
tion has been developed to identify a number of viral agents, such tests have
not yet been developed for routine rapid identification of alphaviruses in the
clinical setting.
2. Materials
2.1. General ELISA Materials List
The following materials list is employed m all subsequent ELISA procedures:
1. 96-Well Immulon 2 microtiter plates (Dynatech Industries, Inc., Chantilly, VA).
2 Carbonate-bicarbonate (PH 9.6) coating buffer: 1.59 g of Na$Os, 2.93 g NaHCOs
in 1 L of distilled water (4).
3. Phosphate-buffered saline (PBS): BBL FTA buffered saline (9.23 g/L, Becton
Dickinson, Cockeysville, MD)
4. Blocking buffer: 5% skim milk, 0.5% Tween-20 m PBS.
5. Rinse buffer: 0.05% Tween-20 in PBS.
6. ELISA plate-reader.
7. Refrigerator.
8. Humid incubator, 37°C.
2.2. Antigen Detection ELlSA in Virus-infected Mosquitoes
1. Grinding apparatus( Ten Broeck homogenizerso r mortars and pestles).
2. Microcentrifuge with accompanying 1.5-mL microcentrifuge tubes
3 Probe sonicator.
Alphaviruses 9
4 BA- 1 dduent. 1 X cell culture medium M 199,0.05 MTris-HCl, 1% bovme serum
albumm, 0.35 g/L NaHCO,, final pH 7.6. Filter sterilize
5. Lysls buffer: 5% Tween-20 in PBS
6. Substrate: 3-3’,5-5’-tetra-methyl benztdme (TMB) Commercial source: TMB-
ELISA reagent (Gibco-BRL, Gaithersburg, MD)
7. Stopping reagent: 1 N H,SO,.
8 Positive control antigen (sucklmg mouse bram [SMB] antigen of either EEE
virus strain NJ-60, or WEE virus strain Fleming) Procedures for preparation of
SMB antigens have been previously described (2)
9. Capture antibody* Murine MAb, lA4B-6, for EEE vnus or 2A3D-5, for WEE
virus ($6).
10. Detector antibody: Murine MAb, lB5C-3, conjugated to horseradish peroxtdase
(HRP) for EEE virus, or 2BlC-6, conjugated to HRP for WEE virus (5,6).
11 Polyclonal control antibodies: Procedures for producing murine polyclonal
anttvirus antibodies for use in the inhibition assay have been previously
described (2).
2.3. IgM-Capture ELBA (MAC-ELBA)
1 Previously tttered goat antihuman IgM capture antibody (Cappel Labs, Organon
Tekmka, Durham, NC)
2 Previously tttered vn-us and control SMB antigens (2).
3. Prevtously tttered HRP-conjugated MAb detector, 2A2C-3 (5)
4 Known-posmve human serum or CSF samples reactive with test viruses to serve
as positive controls.
5 Known-negative human serum or CSF samples to serve as negative controls
2.4. IgG ELlSA
1 Capture antibody* Murme MAb, EEE lA4B-6 (5)
2. Previously titered virus and control SMB antigens (2).
3. Detecting antibody: Goat antthuman IgG (Fc-spectfic)-alkaline phosphatase (AP)
coqugate (Jackson Immunochemtcals, West Grove, PA).
4. Known-positive human serum samples reactive wtth test vu-uses to serve as posi-
tive controls.
5. Known-negative serum samples to serve as negative controls
6. Substrate: 3 mg/mL Sigma 104 m 1 MTris-HCl, pH 8.0 (Sigma, St. LOUIS, MO).
7. 3 MNaOH (120 g in 1 L water) to stop reaction.
2.5. IFA Assay
1. Unconjugated MAbs of various specificity (Table 1).
2. Fluoresceinated anttmouse antibody (Jackson Imrnunochemtcals).
3 Sodium azide (as preservative).
4. Penictllin-streptomycin,
5. PBS.
6. Counterstain: Trypan blue diluted 1:4000 in PBS.
IO Roehrig et al.
Table 1
Monoclonal Antibodies Useful in Alphaviruses Serology
MAb Specificity Serologic reactivity Ref.
VlruS
lA2B-10 VEE peptlde E2 All VEE except TC-83 7
and subtype 6
5B4D-6 VEE TC-83 E2 TC-83 specific 8
3B4C-4 VEE TC-83 E2 VEE IAB, lC, lD, 2 8
lA3A-9 VEE TC-83 E2 VEElAB,lC,lD,lE,lF 9
lAlB-9 VEE Mena 2 E2 VEE lD,lE,lF,3 10
lA3B-7 VEE TC-83 E2 VEE complex 10
2BlC-6 WEE McMlllan El WEE specific 6
2A6C-7 WEE McMillan El WEE complex 6
2A3D-5 WEE McMlllan El WEE complex 6
2D4- 1 HJ Original E2 HJ specific 11
49 Sindbls Ar339 E2 Smdbls specific 12
lB5C-3 EEE NJ-60 El North American EEE 5
lBlC-4 EEE BeAn 122 El EEE complex 5
UM5 1 Semhkl Forest E2 Semhkl Forest specific 13
2A2C-3 WEE McMillan El All alphaviruses 6
lA4B-6 EEE NJ60 El All alphaviruses 5
7. Mounting solution: Aqua-mount (Lerner Labs, Pittsburgh, PA)
8. High-quality fluorescence mlcroscope with eplfluorescence interference filters
and a tungsten light source
9. Twelve-spot IFA slides (Erie Scientific Co., Portsmouth, NH)
10. Coverslips (Cornmg Co , Coming, NY).
3. Methods
3.1. Antigen Detection ELISA in Virus-Infected Mosquitoes
Monitoring levels of virus m vector mosquitoes allows for rapld assessment
of disease threat. The antigen-capture ELISA does not require use of expensive
isolation techniques such as cell culture or animal moculatlon. The test 1s not
as sensitive as virus isolation m cell culture; however, if mosquito pools are
kept below 25 individuals, the test has appropriate sensitivity to detect virus at
levels necessary for alphavirus transmission (25.0 log,, PFU).
3. I. 1. Mosquito Pool Preparation
1. Using sterile procedures, triturate pools of suspect mosquitoes (25 mosquitoes/
pool or less) m 1.5 mL BA-1 buffer by standard protocol. We have used two
methods. Method one employs homogenization m Ten Broeck tissue homog-