Detection of ovine herpesvirus 2 major capsid gene transcripts as an indicator of virus replication in shedding sheep and clinically affected animals
Introduction
Ovine herpesvirus 2 (OvHV-2) is a Rhadinovirus in the Gammaherpesvirinae subfamily that causes sheep-associated malignant catarrhal fever (SA-MCF), a severe lymphoproliferative syndrome primarily of ruminant species (Crawford et al., 1999, Plowright, 1990). Although the virus persists subclinically in sheep, its natural reservoir, the disease is frequently fatal in cattle, bison, deer, and certain other species, including pigs (Schultheiss et al., 2000, Loken et al., 1998, Reid, 1992). MCF is a sporadic disease; however, larger outbreaks can occur with significant economic impact, especially in highly disease-susceptible species such as bison and deer (Li et al., 2006a, Berezowski et al., 2005, O’Toole et al., 2002, Tomkins et al., 1997, Orr and Mackintosh, 1988, Beatson et al., 1985). Although the clinical signs and lesions of MCF are well documented (O’Toole et al., 2002, Liggitt and DeMartini, 1980a, Liggitt and DeMartini, 1980b), the research on virus–host interactions and pathogenesis has been constrained by the absence of an in vitro system to propagate the virus as a source of cell-free infectious particles.
Sheep naturally infected with OvHV-2 shed the virus predominantly through nasal secretions; each shedding episode, which occurs more frequently in lambs (6–9 months old) than adults under natural flock conditions, usually lasts less than 24 h (Li et al., 2004). It has been demonstrated that the nasal secretions of sheep experiencing shedding episodes contain infectious OvHV-2 virions (Taus et al., 2005, Kim et al., 2003). Experimental aerosol infection models for OvHV-2 have been recently established in several species (O’Toole et al., 2007, Taus et al., 2006, Taus et al., 2005) and constitute a key step to understanding viral pathogenesis as well as the differences in susceptibility to OvHV-2 among species (Li et al., 2006b). Cattle and bison are clinically susceptible to OvHV-2 and develop a severe clinical disease following natural and/or experimental infection with the virus. The presence of viral DNA in various tissues from cattle and bison with SA-MCF lesions has been demonstrated by PCR (O’Toole et al., 2007, Taus et al., 2006, Li et al., 2005, Simon et al., 2003); however, the virus–host interactions during infection, such as the sites of lytic replication, are still not clear. The goal of this study was to identify sites where OvHV-2 replication occurs in infected sheep during a shedding period, and in cattle and bison with SA-MCF. An RT-PCR targeting the ORF 25, which encodes a structural protein necessary for virus assembly during replication, was developed and used as an indicator for replication in a variety of tissue samples from OvHV-2 infected sheep, cattle, and bison.
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Experimental animals
Fifteen OvHV-2 infected lambs (5–6 months of age), obtained from a flock of sheep maintained under standard husbandry conditions at the U.S. Sheep Experiment Station, Dubois, ID, and an uninfected sheep, obtained from a Washington State University OvHV-2 negative flock, were used in this study. The OvHV-2-free flock was screened monthly by competitive inhibition ELISA and nested-PCR to assure its negative status (Li et al., 2001, Li et al., 1995). All animals were maintained at Washington State
ORF 25 RT-PCR
A one-step RT-PCR targeting 465 bp of the OvHV-2 ORF 25, the gene encoding the major capsid protein, was developed and used to detect its transcripts in total RNA samples obtained from sheep, a bovine calf, and bison tissues. The assay was able to detect a minimum of 12 copies of ORF 25 cDNA per reaction. No ORF 25 transcripts were detected in “minus reverse transcriptase” reactions, “no template” control reactions, or samples obtained from OvHV-2 negative animals.
ORF 25 transcripts in sheep nasal secretion cells
ORF 25 transcripts were only
Discussion
In this study, an RT-PCR to detect transcripts of OvHV-2 ORF 25 was developed and used to identify sites of virus replication in vivo. ORF 25 is a late gene for a viral structural protein, the major capsid protein, and was used as an indicator of viral replication. Evidence for the association between ORF 25 expression and virus replication has been demonstrated in Murine gammaherpesvirus 68, where transcripts of ORF 25 are less abundant when viral replication is inhibited (Ahn et al., 2002).
Acknowledgements
This work was supported by USDA/ARS CWU 5348-32000-024-00D. The authors thank Janice Keller, Lori Fuller, and Shirley Elias for technical assistance, Emma Karel for animal care and handling, and Sue Pritchard for helpful discussions. We also thank Donald P. Knowles for critical review of the manuscript.
Cristina W. Cunha and Donald L. Traul contributed equally to the experimental work for this study.
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