Deletion of the Chemokine Binding Protein Gene from the Parapoxvirus Orf Virus Reduces Virulence and Pathogenesis in Sheep

Abstract

Orf virus (ORFV) is the type species of the Parapoxvirus genus of the family Poxviridae and infects sheep and goats, often around the mouth, resulting in acute pustular skin lesions. ORFV encodes several secreted immunomodulators including a broad-spectrum chemokine binding protein (CBP). Chemokines are a large family of secreted chemotactic proteins that activate and regulate inflammation induced leukocyte recruitment to sites of infection. In this study we investigated the role of CBP in vivo in the context of ORFV infection of sheep. The CBP gene was deleted from ORFV strain NZ7 and infections of sheep used to investigate the effect of CBP on pathogenesis. Animals were either infected with the wild type (wt) virus, CBP-knockout virus or revertant strains. Sheep were infected by scarification on the wool-less area of the hind legs at various doses of virus. The deletion of the CBP gene severely attenuated the virus, as only few papules formed when animals were infected with the CBP-knock-out virus at the highest dose (10⁷ p.f.u). In contrast, large pustular lesions formed on almost all animals infected with the wt and revertant strains at 10⁷ p.f.u. The lesions for the CBP-knock-out virus resolved approximately 5-6 days p.i, at a dose of 10⁷ pfu whereas in animals infected with the wt and revertants at this dose, lesions began to resolve at approximately 10 days p.i. Few pustules developed at the lowest dose of 10³ p.f.u. for all viruses. Immunohistochemistry of biopsy skin-tissue from pustules showed that the CBP-knockout virus replicated in all animals at the highest dose and was localized to the skin epithelium while haematoxylin and eosin staining showed histological features of the CBP-knockout virus typical of the parent virus with acanthosis, elongated rete ridges and orthokeratotic hyperkeratosis. MHC-II immunohistochemistry analysis for monocytes and dendritic cells showed greater staining within the papillary dermis of the CBP-knock-out virus compared with the revertant viruses, however this was not the case with the wt where staining was similar. Our results show that the CBP gene encodes a secreted immunodulator that has a critical role in virulence and pathogenesis.

Keywords: Orf virus; chemokine binding protein; pathogenesis; poxvirus; virulence; virus immune modulation.

Figure 1

CBP_NZ7 is a broad-spectrum chemokine binding protein. The chemokines shown were incubated with increasing amounts of either chemokine binding protein ORFV strain NZ7 (CBP_NZ7) or CBP_NZ2. Unbound chemokine was then detected using a capture ELISA. The results are presented as the total chemokine detected. High levels of chemokine indicate no interference in the detection of the chemokine (no chemokine binding) where as low levels of chemokine indicates interference with detection (positive chemokine binding).

Figure 2

CBP_NZ7 inhibits LPS-induced recruitment of inflammatory monocytes and dendritic cells (DC) into skin. Bone marrow cells from eGFP donor mice were transfused intravenously into recipient mice 24 h before intradermal injection of LPS (1 μg per site) with and without CBP_NZ7. In addition each mouse received injections of CBP only and PBS for a total of four injections per mouse (each mouse within the group received LPS only, LPS + CBP, CBP only, PBS only and n = 3 animals per group for one experiment). Twenty-four hour later, the recruitment of eGFP/Gr1⁺/CD11b⁺ cells and eGFP/CD11c⁺/MHCII⁺ cells into the skin were analyzed. The bar represents the mean ± SD of 3 combined experiments (biological replicates) where n = 9 mice. Asterisks indicate results that are significantly different (p < 0.01; paired Students t-test).

Figure 3

ORFV lesion clinical scores of sheep infected with wt and recombinant viruses. Infection with a dose of 10⁷ p.f.u. virus of each wt, CBP knock-out virus (ko) CBP revertant virus and CBP_FLAG revertant virus. There were 6 animals per group and the clinical scores are shown for each animal from day 3 to day 18 p.i. at which time the lesions in all animals had resolved (detached scab).

Figure 4

The mean ORFV lesion clinical scores of sheep infected with wt and recombinant viruses. The mean clinical score for each group infected with a dose of 10⁷ p.f.u. is shown. Asterisks indicate where the CBP-ko virus is significantly different to wt and revertants (p < 0.05, Mann-Whitney test).

Figure 5

Lesions of ORFV infected sheep at day 6 p.i., dose 10⁷ p.f.u. virus.

Figure 6

ORFV lesion clinical scores of sheep from infection with three different doses of virus (10³, 10⁵, and a 10⁷ p.f.u.) of wt, CBP-ko, CBP-revertant and CBP_FLAG- revertant at day 6 p.i. There were 6 animals per group and the clinical scores are shown for each animal. The bar represents the mean clinical score for each group and asterisks indicate where the CBP-ko virus is significantly different to wt and revertants (p < 0.05, Mann-Whitney test).

Figure 7

Histological sections from punch biopsies of virus-infected animals were stained by H & E or by immunohistochemical staining for the ORFV envelope protein F1L. (A) Day 4 p.i. at a dose of 10⁷ p.f.u. wt virus. Adjacent sections were stained by H & E and ORFV F1L antigen. In addition ant-FIL staining was performed for the mock-infected (PBS) control. Staining for ORFV infected tissue is seen localized to the epidermis and in epidermal cells of the hair follicle. (B) Typical F1L staining in animals infected with the CBP knock-out virus at a dose of 10⁷ p.f.u. virus at day 6 p.i. The biopsies were taken from papules/pustules that had developed along the virus-inoculated scratch line. The images were taken at 4X magnification.

Figure 8

Histopathology of ORFV infected skin tissue. H & E stained sections from biopsy tissue at day 4 p.i., dose 10⁷ of p.f.u. virus. (A) mock-infected (PBS) skin (B) CBP_FLAG -revertant showing histological features of infected tissue (C,D) histological features of CBP-knockout virus (E) CBP-revertant virus infected tissue showing parakeratosis and necrotic keratinocytes (F) wt infected tissue showing hyperkeratosis. The images were taken at 40X magnification.

Figure 9

MHC-II staining of ORFV infected skin tissue and mock infected. Biopsy tissue day 4 p.i., dose 10⁷ p.f.u. virus. Adjacent sections for H & E (a) and MHC-II staining (b) are shown: (1) mock infected (PBS), (2) wt, (3) CBP-knock-out virus, (4) CBP revertant virus. MHC-II positive cells (green); cell nuclei (DAPI) (blue); non-specific staining of red blood cells (red). The images were taken at 40X magnification.

Figure 10

MHC-II staining in the epidermis and papillary dermis. The percent area for MHC-II staining was determined using Image J Fiji. Biopsy tissue day 4 p.i., dose 10⁷ p.f.u. virus and for mock infected (PBS). The data was analyzed by One Way ANOVA plus Sidoks test where N = 6 for wt, CBP-knockout (KO), CBP-FLAG revertant, and CBP revertant, that is one biopsy taken from each infected animal. N = 22 for mock infected (biopsies taken from animals across all 4 groups).

Figure 11

Serological analysis of anti-ORFV antibodies in infected sheep. Sheep sera from animals infected with either ORFV wt or CBP-knock-out virus (ko) or CBP-revertant virus (rev) or CBP_FLAG revertant (rev-F) were tested for antibodies against ORFV antigen (A) or the ORFV scaffold protein ORFV075 (B) at 0 and 18 days p.i. by ELISA. Shown are the serum antibody absorbance values for each animal (serum dilution 1/400) and the mean serum antibody absorbance values for each group. P values determined by Mann-Whitney test.