Introduction   Materials and Methods   Results   Discussion

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. 

Introduction   Materials and Methods   Results   Discussion

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 40C) 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 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 Tris-HCl, EDTA buffer (pH 9). The purity of virus preparation was determined by transmission electron microscopy examination (Fig. 1).

Fig. 1. Electronmicrograph to show several contagious pustular dermatitis virus particles in different planes with a tendency to form aggregates.(X21500)

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 370C 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 % H2O2) for 3-4 min for colour development.

Introduction   Materials and Methods   Results   Discussion

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 SDS-PAGE was observed between 11 proteins of the CPD virus and SPP.	Fig. 2. Scabs formation on the wool-free skin in the axilla of  a lamb experimentally infected with contagious pustular dermatitis virus.
It was observed that out of the 18 proteins detected in CPD virus on SDS-PAGE gel, 11 reacted in Western blot analysis (Fig. 4). In addition, two proteins (116 Kd, 49 Kd) not detected in Coomassie blue stained gel, were found to react on Western blot. The 49 Kd protein produced an oblique immunostaining signal on Western blot. The 68 Kd, 65 Kd, 38 Kd, 14 Kd and the obliquely running 49 Kd proteins were found to react strongly with hyperimmune serum. Similarly 10 proteins of the SPP reacted with CPD virus hyperimmune serum, of which two, 123 Kd and 47 Kd proteins, were not detected on stained SDS-PAGE gel. Amongst the soluble proteins, the 68 Kd, 53 Kd and 23 Kd proteins gave strong signals on Western blot. Despite a high similarity in electrophoretic mobility in SDS-PAGE and reactions in Western blot between the proteins of CPD virus and SPP, some differences were observed. For example, 7 proteins of CPD virus (125 Kd, 107 Kd, 81 Kd, 65 Kd, 60 Kd, 35 Kd and 29 Kd), four 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 proteins of the SPP (152 Kd, 110 Kd and 85 Kd) were not detected in purified CPD virus preparation. A 43 Kd protein present in CPD virus on SDS-PAGE gel was not detected on Western blot, but a protein of similar molecular weight (43 Kd) was detected in SDS-PAGE gel as well as on Western blot of the SPP. The 14 Kd protein was detected in stained SDS-PAGE gel and Western blot of CPD virus was detected only in SDS-PAGE gel of the SPP. The 23 Kd protein was detected in both the preparations by both the techniques but a stronger immunostaining signal was produced by the SPP.
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; Ovalbumin- 45 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 dermatitis virus and soluble protein preparations. Lane 1-Soluble protein preparation from uninfected sheep skin; Lane 2-Purified contagious pustular dermatitis virus; Lane 3- Soluble protein preparation from contagious pustular dermatitis virus infected sheep skin.

Introduction   Materials and Methods   Results   Discussion

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 non-structural 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.

Introduction   Materials and Methods   Results   Discussion

References

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17. Kyhse-Andersen, J.: Electroblotting of multiple gels: A simple apparatus without buffer tank for rapid transfer of proteins from polyacylamide to nitrocellulose. J. Biochem Biophy. Methods, 10: 203-209, 1984.

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19. Philbey, A. W., Petersen, R. K., McLonn, M. O. and Chin, J.: Development of a recombinant subunit vaccine against scabby mouth virus. Proceedings of Fourth International Congress for Sheep Veterinarians 1997 (Australian Sheep Veterinary Society, New South Wales, Australia): 303-304, 1997.

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Table 1. SDS-PAGE profile and Western blot analysis of purified CPD virus structural proteins and soluble protein preparation (SPP).

* Molecular weights are expressed in Kilodalton (Kd).

Introduction   Materials and Methods   Results   Discussion