Tissue damage caused by animal orthopoxviruses cowpox, ectromelia, vaccinia and parapoxvirus ovis in human cornea

Abstract

Editor, Historically, smallpox has been a major cause of blindness (Semba 2003). Testing other orthopoxviruses, we observed that cowpox (CPX), ectromelia (ECT) virus (‘mousepox’) and vaccinia virus (VV) led to tissue damage in human corneal epithelium in vitro. This was also true with Orf virus (Parapoxvirus ovis) from sheep or goats. Human corneal grafts excluded from transplantation were used in this study. VV strain WR was obtained from Dr B. Moss (Bethesda, MD, USA). CPX isolate 81/02 was isolated from an infected cat and ECT isolate MP-1 from diseased mice 1976 in Munich, Germany. Orf virus RVB-065/Burghessler originated from sheep with dermatitis pustulosa in northern Germany. Viruses were grown on RK13 rabbit kidney cells to virus titres of 105–106 plaque-forming units per millilitre. Corneae covered with culture medium were infected by placing 20 μl virus supernatant above the central area followed by incubation for up to 2 weeks. Corneas then were stained with 0.5% trypan blue. Pairs from right and left eyes of the same donors were used for control experiments. Damaged epithelium was visible macroscopically in most circumstances (Fig. 1). Figure 2 shows microscopic magnifications. Using VV cytopathic effects as cell rounding, the fusion and formation of giant cells were observed within 3 days of culture; with CPX and ECT, within 1–2 weeks. The less human-adapted Orf virus grew more slowly but within 2 weeks also led to extended areas of damaged epithelial cells showing typical poxvirus cytoplasmatic inclusions (Fig. 2 base). Interestingly Orf virus did not cause spontaneous cell detachment, indicating the less cytolytic character of this virus. Uninfected controls presented with a smooth epithelial layer (Fig. 2, upper right). Human cornea grafts infected with vaccinia virus (VV), ‘mousepox’/ectromelia (ECT) virus and an uninfected control. Note the extended areas of damaged corneal epithelium as stained by trypan blue. Microscopic views of cornea grafts infected with vaccinia virus (VV), cowpox (CPX) virus, ‘mousepox’/ectromelia (ECT) virus and Parapoxvirus ovis (Orf) from sheep. Cytopathic effects like cell rounding, cell fusion (giant cells) and cell lysis leading to complete destruction of the epithelial layer are seen clearly. The slower growing Orf virus led to extended areas of damaged epithelial cells as stained by trypanblue and the typical poxviral intracytoplasmatic inclusions are seen. Corneal ulcers arise by various ways, mostly mechanical. Infectious agents and the use of contact lenses play key roles in the development of deep ulcers. Ulcerative keratitis is common in tropical climates and agrarian societies, leading to speculation about a connection to animal handling. Animal poxviruses are prevalent in many species, but in most cases are not humanpathogenic or only have limited virulence in humans. CPX transmitted by domestic cats and pet rodents causes skin infection in Europe but may also cause severe disease in immunocompromised patients (Huemer et al. 2007). Ocular involvement is rare and tends to be restricted to the eyelids and conjunctiva (O’Connor et al. 1990). ‘Buffalopox’ virus, a natural infection with VV, still occurs on the Indian subcontinent and VV vaccine strains seem to persist in the environment, as shown by recent transmissions of ‘Catangalo’ virus in Brazil. The prevalence of ECT in wild rodents is largely unknown. Although it causes disease (including conjunctivitis) in mice, human infections have never been reported. Because the course is benign in most cases and diagnostic procedures are difficult, one can assume that animal orthopoxviruses would be largely under-diagnosed if they were not associated with conspicuous skin lesions. Leporipoxviruses cause keratitis and cataracts in rabbits, and keratitis associated with non-specified orthopoxviruses have been observed in mule deer (Williams et al. 1985). Punctuate corneal epithelial erosions have been found with conjunctivitis in patients with molluscum contagiosum eyelid lesions; they have also been described with measles in combination with vitamin A deficiency, leading to corneal ulcerations in African children (Foster & Sommer 1987). Viruses have been suspected in Thygeson’s superficial punctuate keratitis, but so far no association with common human pathogens has been found (Connell et al. 2007). In theory any virus could affect human cornea as long as it has the capability to replicate in those epithelial cells. Because of restricted access of the immune system to the cornea, this scenario seems likely to occur in vivo. Our data suggest that viruses from various animals might cause epithelial erosions in humans and contribute to corneal ulcers. This work was supported by the Austrian Federal Ministry of Health under the agenda of the Austrian Smallpox Preparedness Plan.