Activities of acyclic nucleoside phosphonates against Orf virus in human and ovine cell monolayers and organotypic ovine raft cultures

Abstract

Orf virus, a member of the Parapoxvirus genus, causes a contagious pustular dermatitis in sheep, goats, and humans. Previous studies have demonstrated the activity of (S)-1-[3-hydroxy-2-(phosphonomethoxy)propyl]cytosine (HPMPC; cidofovir; Vistide) against orf virus in cell culture and humans. We have evaluated a broad range of acyclic nucleoside phosphonates (ANPs) against several orf virus strains in primary lamb keratinocytes (PLKs) and human embryonic lung (HEL) monolayers. HPMPC, (S)-9-[3-hydroxy-2-(phosphonomethoxy)propyl]-2,6- diaminopurine (HPMPDAP), and (R)-9-[3-hydroxy-2-(phosphonomethoxy)propoxy]-2,4-diaminopyrimidine (HPMPO-DAPy) were three of the most active compounds that were subsequently tested in a virus yield assay with PLK and HEL cells by virus titration and DNA quantification. HPMPC, HPMPDAP, and HPMPO-DAPy were evaluated for their activities against orf virus replication in organotypic epithelial raft cultures from differentiated PLK cells. At the highest concentrations (50 and 20 microg/ml), full protection was provided by the three drugs, while at 5 microg/ml, only HPMPDAP and HPMPC offered partial protection. The activities of the three compounds in the raft culture system were confirmed by quantification of infectious virus and viral DNA. These findings provide a rationale for the use of HPMPC and other ANPs in the treatment of orf (contagious ecthyma) in humans and animals.

FIG. 1.

Structures of the compounds tested.

FIG. 2.

Virus yield assay with PLK cells. The effects of several concentrations (0.5, 2, 5, 20, and 50 μg/ml) of HPMPC, HPMPDAP, and HPMPO-DAPy on cell-associated orf virus were determined. The viral growth was measured by virus titration and is expressed as the number of PFU/ml on a log scale. The virus yield was evaluated at 24 h (top panel), 72 h (middle panel), and 96 h (bottom panel) postinfection. At each time point, a virus control (0 μg/ml) and a cell control (CC) were also included.

FIG. 3.

Effects of several concentrations of HPMPC, HPMPDAP, and HPMPO-DAPy on viral DNA concentration measured in the supernatants of PLK infected cells after 24 h (top panel), 72 h (middle panel), and 96 h (bottom panel). Orf virus DNA was quantified by real-time PCR, and the concentration was calculated in reference to those in serial 10-fold dilutions of an internal standard. The DNA concentration was calculated as the mean of three measurements, and the reduction of the viral DNA was expressed as a percentage of the amount of DNA for the untreated control (0 μg/ml). Error bars represent the standard deviations of three measurements for each sample. CC, cell control.

FIG. 4.

Ovine raft cultures. The rafts were infected with orf virus after 6 days of differentiation and treated with various concentrations (50, 20, 5, and 2 μg/ml) of HPMPC. At 11 days of differentiation, the rafts were fixed and stained with hematoxylin-eosin. Magnifications, ×9 and ×36 in the right and left panels, respectively.

FIG. 5.

(Top panel) Orf virus yield from the organotypic cultures is presented on a logarithmic scale; (bottom panel) quantification of orf virus DNA from the rafts, reported as a percentage of the amount of DNA in the infected but untreated raft. Error bars represent the standard deviations of three measurements for each sample. CC, cell control.