ISRAEL JOURNAL OF
VETERINARY MEDICINE
Vol. 56 (3) 2001
WESTERN BLOT ANALYSIS OF STRUCTURAL AND
SOLUBLE PROTEINS OF CONTAGIOUS PUSTULAR
DERMATITIS VIRUS
M. Khatri1, P. Chand2 and S. K. Batra1
1. Department of Veterinary Microbiology
2. Department of Veterinary Pathology, College of Veterinary Sciences, CCS Haryana Agricultural
University, Hisar-125 004, India.
Summary
Western blot analysis was carried out to characterize antigenic proteins associated with purified
contagious pustular dermatitis (CPD) virus and those present in CPD virus-free soluble protein
preparations (SPP). Despite a close similarity in electrophoretic motility in sodium dodecyl sulphatepolyacrylamide gel electrophoresis (SDS-PAGE) between proteins associated with CPD virus and SPP
some differences were detected in Western blot analysis. Western blot analysis revealed that 13 of the
20 virus-associated proteins and 10 of the 16 soluble proteins of SPP were antigenic in nature. Two
(116 Kd and 49 Kd) of the 13 antigenic proteins of CPD virus and two (123 Kd and 47 Kd) of the 10
antigenic proteins of SPP were not detected by SDS-PAGE indicating much better sensitivity of the
Western blot. The 68 Kd, 65 Kd, 49Kd, 38 Kd and 14 Kd proteins of CPD virus and 68 Kd, 53 Kd and
23 Kd proteins of SPP produced strong reactions on Western blot thereby indicating their strong
antigenic nature.
Introduction
Contagious pustular dermatitis (CPD) virus is the type species of the genus parapoxvirus of the family
Poxviridae (1). It causes a highly contagious and eruptive skin disease in sheep and goats that is
transmissible to humans (2). The lesions in naturally infected sheep commonly localize around the
mouth, nose, lips, thighs and udder. Morbidity in this disease may reach 100% (3), but mortality is
usually low in uncomplicated cases (4). Recently this disease has been an important problem in the live
sheep export trade, having resulted in rejection of consignments of sheep shipped from Australia to the
Middle East (5). A live cell culture grown CPD virus vaccine has been shown to be superior to the
traditional vaccines prepared from scab material (6,7) but this also presents a major source of
contaminating virus in the environment. Moreover, several antigenic variants of CPD virus have been
reported (8,9) which may be responsible for vaccination failure. CPD infections can be misdiagnosed
as capripox (sheeppox or goatpox), foot and mouth disease or blue tongue in early stages of the disease.
Serological cross-reactivity between CPD virus and capripox viruses (10,11) further complicates its
specific diagnosis under laboratory conditions but these can be differentiated from each other by
detecting specific antibody response against 32Kd envelope protein of capripox virus (12). Because of
its contagious nature, public health aspects and economic impact of the disease, it is important that
effective procedures based on CPD virus specific protein(s), be developed for its diagnosis and control.
The CPD virus proteins that simulate humoral and cell-mediated immune responses have been well
characterized.
The purpose of this study is to analyze structural and soluble proteins of CPD virus by Western blotting
in order to characterize their antigenic nature.
Materials and Methods
Virus.
Contagious pustular dermatitis (CPD) virus originally isolated from an outbreak of CPD in sheep (13)
and maintained as infected skin scabs was used in the present study. A 10% (w/v) suspension of the
scab tissues was made in PBS (pH 7.2, 0.01M) by triturating in a pestle and mortar. The supernatant
following centrifugation (400g, 20 min at 4 0C) of scab suspension was used as inoculum for
infecting experimental lambs.
Experimental animals and production of scabs.
Four lambs (6-8 month old) of either sex having no previous history of infection either with CPD virus
or sheeppox virus were used in the present study. The animals were kept in the animal house of the
department and fed ad libitum. Two of the lambs were infected by scarification with CPD virus
suspension in the region of axilla, groin and flank after shaving the skin.
Preparation of
hyperimmune
serum.
Hyperimmune
serum against
CPD virus was
prepared in two
recovered lambs
which were used
to raise virus
scab stock. Each
lamb was
injected
intramuscularly
with 1 ml of 10%
scab suspension
homogenized
with equal
volume of
Freund's
complete
adjuvant. Lambs
were boosted
twice at two
weeks interval
with 1 ml of
virus scab
suspension
homogenized
with 1 ml of
Freund's
incomplete
adjuvant. Lambs
were test bled 10
days after the last
injection. Serum
was separated
and stored at 200C till further
use.
Purification of
CPD Virus from
Fig. 1. Electronmicrograph to show several contagious pustular dermatitis virus
particles in different planes with a tendency to form aggregates.(X21500)
scabs.
The CPD virus
was purified by
sucrose density
gradient
centrifugation
method as
described by
Joklik (14). The
purified virus
after pelleting
was suspended in
0.001M TrisHCl, EDTA
buffer (pH 9).
The purity of
virus preparation
was determined
by transmission
electron
microscopy
examination (Fig.
1).
CPD Virus soluble proteins.
Soluble proteins of CPD virus were prepared by sonicating (2 min) and centrifugation (2000g for 30
min at 40C) of 10% skin scab suspension. The supernatant was ultracentrifuged
(100,000g 40C) in a Beckman centrifuge for 1hour using a 36% sucrose cushion in a SW-28 rotor.
The supernatant following centrifugation was collected and concentrated to 1/5th of its original volume
by counter-dialysis against 20%(w/v) solution of PEG-6000 and designated as soluble protein
preparation (SPP).
Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS -PAGE).
The proteins of purified CPD virus preparation and SPP were resolved on a 12% (w/v) acrylamide gel
(15). Electrophoresis was carried out at a constant voltage (120 V) until tracking dye front
(Bromophenol blue) moved to the bottom of the gel. The gels were stained with Coomassie brilliant
blue. Molecular weight of each protein band was calculated with reference to a standard curve derived
from the migration pattern of standard molecular weight markers (16).
Western blotting.
The electrophoretic transfer of polyacrylamide gel resolved proteins to the nitrocellulose membrane
was carried out by semi-dry electroblotting as described (17) using Nova Blot Electrophoretic transfer
unit (Pharmacia). The unoccupied sites on the nitrocellulose membrane were blocked with blotto (PBS,
pH 7.2 containing 0.1% Tween-20 and 5% (w/v) non-fat-milk powder). The nitrocellulose membrane
was then incubated with CPD hyperimmune serum (1:50 in blotto) at 37 0C for 1 hour followed by
washing three times with PBS-tween 20. The membrane was then incubated at 370C for 1 hour in
donkey antisheep immunoglobulinG (IgG) horseradish peroxidase conjugate (Sigma Chem, Co.,
1:1500 in blotto). The membrane was then washed as above and incubated in freshly prepared substrate
solution (10 mg diaminobenzidine tetrahydrochloride in 50 ml PBS containing 50 µl of 30 % H 2O2)
for 3-4 min for colour development.
Results
The lambs inoculated by scarification with 10% skin scab suspension
developed skin lesions similar to that observed in CPD virus infection
of sheep. The lesions were apparent on day 4 post-infection in the
form of reddening and swelling around the site of inoculation. They
progressed into small vesicles over next 2 days and after pustular stage
turned into scabs by 10-12 days post-infection (Fig. 2). The scabs
started peeling by day 16 pi. All the lambs infected experimentally
showed similar pattern of development of lesions and clinical signs.
The protein profile of purified CPD virus and SPP as detected by
SDS-PAGE is shown in Figure 3 and Table I. Eighteen proteins of
various molecular weights ranging between 125 Kd and 14 Kd were
detected in purified CPD virus preparation. The protein bands of 68
Kd, 53 Kd, 43 Kd and 21 Kd were thick and deeply stained. The SPP
resolved into 14 proteins of which the 92 Kd, 68 Kd, 53 Kd and 21 Kd
proteins were deeply stained. Similar electrophoretic mobility in SDSPAGE was observed between 11 proteins of the CPD virus and SPP.
Fig. 2. Scabs formation on the wool-free skin in t
experimentally infected with contagious pustular d
It was observed that out of the 18 proteins detected in CPD virus on SDS-PAGE gel, 11 reacted in Western blot analysis (Fig.
proteins (116 Kd, 49 Kd) not detected in Coomassie blue stained gel, were found to react on Western blot. The 49 Kd protein p
immunostaining signal on Western blot. The 68 Kd, 65 Kd, 38 Kd, 14 Kd and the obliquely running 49 Kd proteins were found
with hyperimmune serum. Similarly 10 proteins of the SPP reacted with CPD virus hyperimmune serum, of which two, 123 Kd
proteins, were not detected on stained SDS-PAGE gel. Amongst the soluble proteins, the 68 Kd, 53 Kd and 23 Kd proteins gav
Western blot. Despite a high similarity in electrophoretic mobility in SDS-PAGE and reactions in Western blot between the pr
and SPP, some differences were observed. For example, 7 proteins of CPD virus (125 Kd, 107 Kd, 81 Kd, 65 Kd, 60 Kd, 35 K
of which (81 Kd, 65 Kd, 60 Kd, 29 Kd) were antigenic but were not detected in SDS-PAGE gel of the SPP. Likewise, three pro
(152 Kd, 110 Kd and 85 Kd) were not detected in purified CPD virus preparation. A 43 Kd protein present in CPD virus on SD
not detected on Western blot, but a protein of similar molecular weight (43 Kd) was detected in SDS-PAGE gel as well as on W
SPP. The 14 Kd protein was detected in stained SDS-PAGE gel and Western blot of CPD virus was detected only in SDS-PAG
The 23 Kd protein was detected in both the preparations by both the techniques but a stronger immunostaining signal was prod
Fig. 3. SDS-PAGE profile of proteins of purified contagious pustular
dermatitis virus and soluble protein preparation. Lane 1- Molecular
weight marker; (Myosin-205 Kd; b-galactosidase- 116 Kd;
Phosphorylase B- 97 Kd; Fructose-6-phosphate kinase- 84 Kd; Bovine
serum albumin- 66 Kd; Glutamic dehydrogenase- 55 Kd; Ovalbumin45 Kd; Glyceraldehyde-3 phosphate dehydrogenase- 36 Kd;
Trypsinogen- 24 Kd; Trypsin inhibitor-20 Kd). Lane 2 soluble protein
preparation (SPP) from uninfected lamb skin. Lane 3- Purified
contagious pustular dermatitis virus. Lane 4- Soluble protein
preparation from contagious pustular dermatitis virus infected skin.
Fig. 4. Western blot of purified contagious pustular d
soluble protein preparations. Lane 1-Soluble protein p
uninfected sheep skin; Lane 2-Purified contagious pu
virus; Lane 3- Soluble protein preparation from conta
dermatitis virus infected sheep skin.
Discussion
The proteins synthesized by pox viruses, including CPD virus, during their cytoplasmic multiplication
can be divided into structural and non-structural proteins. The non-structural proteins and structural
proteins synthesized in excess can be obtained by disrupting the infected cell and ultracentrifuging the
lysate to remove virus particles. This separation allows analysis of virus-associated structural proteins,
non-structural proteins and the soluble structural proteins. In the present study skin scabs collected
from CPD virus infected lambs were used to purify CPD virus and obtain SPP. The SPP was assumed
to contain the non-structural proteins and the soluble structural proteins which were synthesized in
excess during CPD virus multiplication. The CPD virus preparation was resolved into 18 proteins by
SDS-PAGE. Taken into consideration that the method used for purification of CPD virus (14) yielded
sufficiently pure viral preparation, the 18 proteins detected by SDS-PAGE could be defined as the
structural proteins and 11 of these were identified antigenically by Western blotting. The oblique
pattern of banding of 49 Kd antigenic protein (Fig. 4) cannot be explained though it was repeatedly
observed during the study. The SPP was separated into 14 proteins by SDS-PAGE and 11 of these
were co-migrating with the 11 structural proteins. Assuming that all the 14 proteins of SPP were
encoded by the virus genome then 3 of these could be considered non-structural soluble proteins and 11
co-migrating proteins as structural soluble proteins. Out of the 14 proteins of SPP, 8 were antigenic.
Though it was likely that SPP could contain some host cell-derived proteins as one of the proteins (53
Kd) in the normal skin preparation did not cross-react in Western blot analysis, the 7 antigenic proteins
which were identified by Western blotting, were virus specific. Interestingly, one (85 kd) of the 3 nonstructural soluble proteins and 7 of the 11 structural soluble proteins were antigenic. However, it was
not possible to ascertain using hyperimmune serum that the co-migrating antigenic proteins in the two
preparations were identical. Monospecific serum or monoclonal antibody should be used to identify the
identical proteins in two different preparations. The remaining two non-structural soluble proteins and
4 structural soluble proteins may be either virus encoded proteins or of host cell origin. However, the
later possibility is less likely as a Western blot of the normal skin extract did not reveal any of these
proteins.
Taken together the results of the SDS-PAGE and Western blotting a total of 20 proteins were found to
be associated with purified CPD virus and 16 proteins in the SPP (Table I). These results suggested that
18 of the 20 virus associated proteins and 14 of the 16 proteins of SPP were present in amounts
detectable by SDS-PAGE. The amount of the 2 proteins in each of the two preparations was too low to
be detected by SDS-PAGE but was sufficient to produce detectable signals on Western blot. The
Western blot technique has been shown to be able to detect 1 ng antigenic protein transferred onto
nitrocellulose membrane (18). As the SDS-PAGE detects proteins in the range of 100 ng to 500 ng (18)
the Western blot technique seems to be 100 to 500 times more sensitive than the SDS-PAGE. This was
perhaps the reason why 2 proteins of CPD virus and 2 proteins of SPP were detected by Western
blotting and not detected by Coomassie blue staining of the SDS-PAGE gels.
A subunit vaccine capable of inducing protective immunity against CPD virus infections has been
advocated for vaccinating sheep destined for export (19). To achieve this goal it will be necessary to
identify antigenic proteins and to clone genes that express proteins capable of inducing a protective
immune response in sheep. This approach has been shown to be successful to prepare an effective
subunit recombinant vaccine against goatpox virus infection (20). The antigenic analysis of CPD virus
proteins as carried out in the present study would be useful to further evaluate these antigenic proteins
for developing a suitable subunit vaccine. Moreover, the potential of the SPP containing antigenic
proteins could be explored for use as immunogens against CPD virus infection in the live sheep export
industry to avoid transportation of live CPD virus from the exporting country.
Acknowledgement
Authors are thankful to the University authorities and the Director, Centre of Advanced Studies,
Department of Veterinary Microbiology for providing necessary facilities. The financial help awarded
by the Indian Council of Agricultural Research in the form of Junior Research Fellowship to M. Khatri
is thankfully acknowledged.
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Table 1. SDS-PAGE profile and Western blot analysis of purified CPD virus structural proteins and
soluble protein preparation (SPP).
Sr No
Normal skin*
Purified CPD virus*
SDS-PAGE Western blot SDS-PAGE
1
4
5
Western blot SDS-PAGE Western blot
205
2
3
Soluble Protein
Preparation*
152
150
125
123
6
117
7
116
8
110
9
107
10
100
11
97
12
95
13
92
100
92
14
15
81
16
81
81
68
68
17
65
65
65
65
18
60
60
60
60
19
53
53
53
53
20
50
21
92
92
85
85
68
68
53
53
49
22
47
23
43
24
42
25
38
38
26
43
38
38
35
27
34
28
32
29
30
32
30
29
31
27
32
38
43
32
29
27
24
33
23
23
23
23
34
21
21
21
21
14
14
14
18
13
14
35
20
36
18
37
16
38
Total
19
3
10
* Molecular weights are expressed in Kilodalton (Kd).