Viral vascular endothelial growth factor plays a critical role in orf virus infection

Abstract

Infection by the parapoxvirus orf virus causes proliferative skin lesions in which extensive capillary proliferation and dilation are prominent histological features. This infective phenotype may be linked to a unique virus-encoded factor, a distinctive new member of the vascular endothelial growth factor (VEGF) family of molecules. We constructed a recombinant orf virus in which the VEGF-like gene was disrupted and show that inactivation of this gene resulted in the loss of three VEGF activities expressed by the parent virus: mitogenesis of vascular endothelial cells, induction of vascular permeability, and activation of VEGF receptor 2. We used the recombinant orf virus to assess the contribution of the viral VEGF to the vascular response seen during orf virus infection of skin. Our results demonstrate that the viral VEGF, while recognizing a unique profile of the known VEGF receptors (receptor 2 and neuropilin 1), is able to stimulate a striking proliferation of blood vessels in the dermis underlying the site of infection. Furthermore, the data demonstrate that the viral VEGF participates in promoting a distinctive pattern of epidermal proliferation. Loss of a functional viral VEGF resulted in lesions with markedly reduced clinical indications of infection. However, viral replication in the early stages of infection was not impaired, and only at later times did it appear that replication of the recombinant virus might be reduced.

FIG. 1

Characterization of the orf virus recombinant, ovVEGF-Δ. (A) Schematic diagram showing the location of the VEGF gene (hatched bar) within a 1.45-kb SmaI-SphI fragment of the orf virus genome (26). The region between the marked AvrII and BsmI sites was deleted from the WT VEGF gene during the construction of ovVEGF-Δ. This region was replaced with the gpt and lacZ genes under the control of an orf virus early promoter (PE1) and a late promoter (PF1), respectively. The box representing lacZ is interrupted to indicate that it is not drawn to scale. The locations of four BamHI sites are marked with B. The distances between these sites, from left to right, are 812, 111, and 5,414 bp. Arrowheads indicate the locations of PCR primers (see below). Sequences denoted as bars were included in the plasmid (pVEGF-Δ) used in the construction of ovVEGF-Δ. Horizontal lines extending from these boxes represent adjacent orf virus genome sequences not included in pVEGF-Δ. (B) BamHI restriction endonuclease cleavage fragments derived from ovVEGF-Δ and WT genomic DNAs were separated on a 0.7% agarose gel (left panel). The 5.4-kb BamHI fragment derived from ovVEGF-Δ is indicated by an arrowhead. The fragments were Southern blotted and hybridized with the lacZ probe (right panel). (C) HindIII restriction endonuclease cleavage fragments derived from ovVEGF-Δ and WT genomic DNAs were separated on a 0.7% agarose gel (left panel). The HindIII fragment unique to ovVEGF-Δ is indicated by an arrowhead. The fragments were Southern blotted and probed with a plasmid containing the 1.45-kb SmaI-SphI fragment shown in panel A (right panel). (D) PCR products derived from WT and ovVEGF-Δ genomic DNAs were electrophoresed on a 1.3% agarose gel and visualized with ethidium bromide. The primer pairs used for PCR amplifications are indicated by arrowheads in panel A, and the bars below those arrowheads indicate the predicted sizes of the PCR products. Template DNA used in the reactions was WT DNA (even-numbered lanes) or ovVEGF-Δ DNA (odd-numbered lanes). Lane 1, low-DNA-mass ladder (Gibco BRL); bands of 1,200, 800, 400, and 200 bp are shown. Lanes 2 and 3, primers gf1 and gf2. Lanes 4 and 5, primers gf1 and gpt. Lanes 6 and 7, primers lac and gf2. Lanes 8 and 9, primers lac and out.

FIG. 2

VEGF activity is not produced by ovVEGF-Δ. CM was prepared from WT-, ovVEGF-Δ-, vaccinia virus (VV)-, and mock-infected cells. (A) Mitogenic activity of CM for endothelial cells. HMVEC were seeded at 10⁴ cells per well in medium supplemented (10%) with CM from the indicated source (LT cells were used), and cell numbers were determined after 72 h. Values are the mean ± standard deviation (SD) of triplicate experiments. (B) Activation of VEGFR-2 by CM. Ba/F3 cells expressing chimeric VEGFR-2 were resuspended in dilutions of CM from virus-infected BT cells (WT or ovVEGF-Δ) and from COS-7 cells transiently expressing mouse VEGF₁₆₄ (VEGF). Cells were incubated for 48 h, and DNA synthesis was quantified by measuring ³H-thymidine incorporation and β counting. Values are the mean ± SD of duplicate readings. (C) Induction of vascular permeability by CM (Miles assay). Guinea pigs were injected intracardially with Evans blue dye. CM (from LT cells) and 5 ng of purified mouse VEGF₁₆₄ (VEGF) were injected intradermally. After 20 min, the skin was excised and photographed (top), the dye was eluted in formamide, and the absorbance at 620 nm (OD/620 nm) was recorded (bottom). Values are the mean ± SD of duplicate injections.

FIG. 3

Histopathological analysis of the role of VEGF-ORFV_NZ2 in infection of sheep. Scarified skin was inoculated with WT or ovVEGF-Δ (VEGFΔ) or mock infected. (A) Gross pathology. Representative lesions at 2, 6, 10, and 14 days (d) p.i., shown at approximately half life size. All lesions are from the same animal (101). (B) Histological comparison of WT-infected (panels 1 and 2), ovVEGF-Δ-infected (panels 3 and 4), and mock-infected (panels 5 and 6) lesions at 10 days p.i. (magnification, ×50). Panels 1, 3, and 5 show representative sections stained with hematoxylin and eosin (H + E), while panels 2, 4, and 6 show the adjacent sections from the same biopsy stained with peroxidase-conjugated anti-von Willebrand factor antibody (anti-vWF) and 3,3′-diaminobenzamine to reveal endothelial cells. The pustule (P), epidermis (E), and dermis (D) are indicated where appropriate, and arrows indicate representative blood vessels highlighted by the antibody staining.

FIG. 4

Vascularization of orf virus lesions. Scarified skin was inoculated with WT (black), ovVEGF-Δ (hatched bars), and PBS (grey bars). Five-millimeter punch biopsies were taken from separate lesions of each animal at days 2, 6, 10, and 14 p.i. The dermal areal fraction reacting with anti-von Willebrand factor antibody was calculated from the analysis of three equidistant sections as outlined in Materials and Methods. Values are the mean ± standard deviation. The asterisk indicates that no data were obtained from this biopsy, but regression methods were used to derive an estimate of 0.24; this value was used in subsequent analyses.

FIG. 5

Effect of inactivation of VEGF-ORFV_NZ2 on virus yield in infected sheep. Scarified skin was inoculated with WT (black bars) and ovVEGF-Δ (hatched bars). Three-millimeter punch biopsies were taken from separate lesions of each animal at days 2, 6, 10, and 14 p.i. Samples were processed and viral titers were determined as described in Materials and Methods. Values are the mean ± standard deviation of duplicate titrations. Asterisks indicate that the viral titer was less than 5 PFU per sample.