Katherine L. Gailbreath

Evidence of three new members of malignant catarrhal fever virus group in muskox (Ovibos moschatus), Nubian ibex (Capra nubiana), and gemsbok (Oryx gazella).

Six members of the malignant catarrhal fever (MCF) virus group of ruminant rhadinoviruses have been identified to date. Four of these viruses are clearly associated with clinical disease: alcelaphine herpesvirus 1 (AlHV-1) carried by wildebeest (Connochaetes spp.); ovine herpesvirus 2 (OvHV-2), ubiquitous in domestic sheep; caprine herpesvirus 2 (CpHV-2), endemic in domestic goats; and the virus of unknown origin found causing classic MCF in white-tailed deer (Odocoileus virginianus; MCFV-WTD). Using serology and polymerase chain reaction with degenerate primers targeting a portion of the herpesviral DNA polymerase gene, evidence of three previously unrecognized rhadinoviruses in the MCF virus group was found in muskox (Ovibos moschatus), Nubian ibex (Capra nubiana), and gemsbok (South African oryx, Oryx gazella), respectively. Based on sequence alignment, the viral sequence in the muskox is most closely related to MCFV-WTD (81.5% sequence identity) and that in the Nubian ibex is closest to CpHV-2 (89.3% identity). The viral sequence in the gemsbok is most closely related to AlHV-1 (85.1% identity). No evidence of disease association with these viruses has been found.

Herpesviral Inclusion Body Disease in Owls and Falcons is Caused by the Pigeon Herpesvirus (Columbid herpesvirus 1).

A herpesviral disease of Rock Pigeons (Columba livia), called “inclusion body disease” or “inclusion body hepatitis,” was first described in the 1940s. The disease involves hepatic and splenic necrosis with associated intranuclear inclusion bodies and occurs primarily in young squabs. A similar herpesviral disease occurs in falcons and owls. Serologic and restriction endonuclease digestion studies indicate that herpesviruses from pigeons, falcons, and owls are very closely related and that most reported cases of disease in falcons and owls involve prior documented or possible ingestion of pigeons. These findings led to the hypothesis that an endemic herpesvirus of pigeons may be causing disease in falcons and owls. In order to test this hypothesis, we sequenced a fragment of the herpesviral DNA polymerase gene from naturally infected owls, falcons, and pigeons with inclusion body disease collected between 1991 and 2006. Sequences from all three sources were almost identical, and we therefore propose that the usual agent of inclusion body hepatitis in owls and falcons is columbid herpesvirus 1.

Experimental infection of rabbits with ovine herpesvirus 2 from sheep nasal secretions

Malignant catarrhal fever (MCF) is a generally fatal disease that primarily occurs in ruminants and is caused by a group of gammaherpesviruses. Outside of Africa MCF is mainly caused by ovine herpesvirus 2 (OvHV-2) which is carried subclinically by sheep. Cell-free virus is present in nasal secretions of shedding sheep and aerosol is the primary mode of transmission. Although OvHV-2 has never been propagated in vitro, experimental infection involving intranasal nebulization with nasal secretions from shedding sheep has been used to induce MCF in cattle and bison. This method of inoculation has never been tested in rabbits, which are the primary small animal model. The objectives of this study were to determine whether rabbits become infected with OvHV-2 after intranasal nebulization with cell-free virus from sheep nasal secretions and whether they develop MCF with consistent gross and histologic lesions. Five of eight rabbits became infected, showed clinical signs and developed histologic lesions typical of MCF including multisystemic vasculitis and perivascular lymphoid accumulation. These lesions are similar to those reported in rabbits infected by intravenous injection with tissues from clinically affected animals containing cell-associated virus. Viral DNA and mRNA transcripts of a structural viral protein were present in tissues from affected rabbits suggesting that viral replication occurred, although the significance in terms of pathogenesis is unknown. This work demonstrates that OvHV-2 infection of rabbits by intranasal nebulization is a potentially useful model that mimics the natural route of infection and may be used to study viral replication and pathogenesis.

Ovine herpesvirus 2 replicates initially in the lung of experimentally infected sheep.

Ovine herpesvirus 2 (OvHV-2), a rhadinovirus in the subfamily Gammaherpesvirinae, is the causative agent of sheep-associated malignant catarrhal fever (SA-MCF), a frequently fatal lymphoproliferative disease primarily of ruminants worldwide. Inability to propagate the virus in vitro has made it difficult to study OvHV-2 replication. Aerosol inoculation of sheep with OvHV-2 from nasal secretions collected from naturally infected sheep during shedding episodes results in infection of naive sheep, providing an excellent system to study OvHV-2 initial replication in the natural host. In this study, we showed that OvHV-2 delivered through the nasal route by nebulization resulted in infection in all lambs, but no infection was established in any lambs after intravenous or intraperitoneal injection. In nebulized lambs, while it was not detected initially in any other tissues, OvHV-2 DNA became detectable in the lung at 3 days post-infection (p.i.), increased to about 900 copies per 50 ng DNA at 5 days p.i., reached peak levels ( approximately 7500 copies) at 7 days p.i., and then declined to an average of 800 copies at 9 days p.i. Transcripts of OvHV-2 open reading frame 25 (coding for the capsid protein), an indicator of virus replication, were only detected in lung tissues, and were positively correlated with OvHV-2 DNA levels in the lungs. In addition, selected immune response genes were also highly expressed in the lung at 5 and 7 days p.i. The data indicate that lung is the primary replication site for OvHV-2 during initial infection in sheep and suggest that viral replication is promptly controlled by a host defence mechanism.

Ovine herpesvirus 2 infection in American bison: virus and host dynamics in the development of sheep-associated malignant catarrhal fever.

Ovine herpesvirus 2 (OvHV-2) is a gammaherpesvirus that causes sheep-associated malignant catarrhal fever (SA-MCF), a frequently fatal disease mainly of ruminants. This study was designed to define virus-host dynamics following experimental OvHV-2 infection in bison. A transient peak in viral DNA accompanied by the presence of OvHV-2 ORF25, ORF50 and ORF73 transcripts was observed in lungs only from 9 to 12 days post-inoculation (DPI), suggesting occurrence of viral replication. This initial viral replication was associated with only a subtle increase in transcription of inflammation related genes in lungs and tracheal bronchial lymph nodes, while the level of expression of the majority of immune genes measured remained comparable to uninfected animals. Increasing viral load was observed in the blood and peripheral tissues at 16 and 21 DPI, respectively, indicating systemic viral dissemination. Clinical signs of MCF were observed between 28 and 35 DPI and the severity of lesions increased as disease progressed. Lesion scores were positively correlated with expression levels of ORF25, suggesting a contribution of viral replication in the pathogenesis of SA-MCF. Viral transcripts were observed in all tissues examined from 23 DPI to the end of the experiment at 35 DPI and expression levels of ORF25 were significantly higher in clinically infected animals as compared to pre-clinical stage. The data from this study provide a predictable viral-host interaction time course to test hypotheses concerning disease pathogenesis as well as mitigation of SA-MCF in susceptible species.

Sheep (Ovis aries) airway epithelial cells support ovine herpesvirus 2 lytic replication in vivo

Ovine herpesvirus 2 (OvHV-2) is the causative agent of sheep-associated malignant catarrhal fever (SA-MCF), a frequently fatal disease of some members of the order Artiodactyla. OvHV-2 is carried as a lifelong subclinical infection in sheep (Ovis aries). To date OvHV-2 has not been propagated in vitro and this has hampered studies of viral pathogenesis and efforts to develop a vaccine to protect animals from SA-MCF. Lytic OvHV-2 replication occurs in the lungs of experimentally infected sheep at early times post-inoculation (PI) and in the nasal cavities of naturally infected sheep during virus shedding episodes. Identification of specific cell types supporting lytic virus replication in vivo provides information that can be used in the development of an in vitro propagation system for the virus. Using fluorescence immunohistochemical techniques, we identified lytically infected alveolar epithelial cells in the lungs of sheep early during infection. Lytically infected epithelial cells were also detected in samples of nasal secretions collected from naturally infected sheep during episodes of virus shedding. This is the first reported identification in the natural reservoir species of specific cell types that support OvHV-2 lytic replication in vivo.

Experimental nebulization of American bison (Bison bison) with low doses of ovine herpesvirus 2 from sheep nasal secretions

Malignant catarrhal fever (MCF), caused by ovine herpesvirus 2 (OvHV-2), is an important cause of mortality in ranched American bison and domestic cattle in North America. Previous studies showed that bison can be infected by intranasal nebulization with sheep nasal secretions containing OvHV-2 and provided preliminary information on viral doses required for infection and disease progression. The goals of this study were to establish optimal minimal infectious and minimal lethal doses of OvHV-2 by the intranasal route in bison, evaluate the influence of dose on incubation period and other clinical parameters and determine if bison seropositive for antibody against MCF-group viruses are resistant to developing MCF after intranasal challenge. In this study, the minimal infectious dose and minimal lethal dose overlap, suggesting that experimental production of subclinically infected bison is impractical. Dose is inversely related to both incubation period and the period between nebulization and first detection of >1000 OvHV-2 DNA copies/500 ng total DNA in peripheral blood leukocytes. Interestingly, all of the bison seropositive for anti-MCF-group viral antibody prior to inoculation died of MCF after nebulization. We conclude that previous exposure to an MCF-group virus does not necessarily provide resistance to OvHV-2-induced MCF in bison.