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ABSTRACT: The aim of this study was to identify tissues where ovine herpesvirus 2 (OvHV-2) replication occurs in vivo. A reverse-transcriptase PCR targeting the OvHV-2 major capsid protein gene (ORF 25) was developed and the presence of transcripts used as an indicator of virus replication in naturally infected sheep, and cattle and bison with sheep-associated malignant catarrhal fever (SA-MCF). ORF 25 transcripts were detected in 18 of 60 (30%) turbinate, trachea, and lung samples from five sheep experiencing a shedding episode; 12 of the 18 positive samples were turbinates. ORF 25 transcripts were not detected in any other tissue from the shedding sheep (n=55). In contrast, 86 of 102 (84%) samples from clinically affected bovine and bison tissues, including brain, kidney, intestine, and bladder, had ORF 25 transcripts. The data strongly suggest that OvHV-2 replication is localized to the respiratory tract of shedding sheep, predominantly in the turbinate, while it occurs in virtually all tissues of cattle and bison with SA-MCF. These findings represent an important initial step in understanding viral pathogenesis, and in potentially establishing a system for OvHV-2 propagation in vitro.
Virus Research 04/2008; 132(1-2):69-75. · 2.94 Impact Factor
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ABSTRACT: Sheep-associated malignant catarrhal fever (SA-MCF), a frequently fatal disease primarily of certain ruminants, is caused by ovine herpesvirus 2 (OvHV-2). Molecular diagnosis of SA-MCF in affected animals has relied on detection of OvHV-2 DNA using a nested PCR, which has significant potential for amplicon contamination as a routine method in diagnostic laboratories. In this report, a nonnested and a previously developed real-time PCR were validated for detection of OvHV-2 DNA in samples from clinically affected animals. Three sets of blood or tissue samples were collected: 1) 97 samples from 97 naturally affected animals with evidence of clinical SA-MCF; 2) 200 samples from 8 animals with experimentally induced SA-MCF; and 3) 100 samples from 100 animals without any evidence of clinical SA-MCF. Among 97 positive samples defined by nested PCR from clinically affected animals, 95 (98%) were positive by nonnested PCR and 93 (96%) were positive by real-time PCR, respectively. One hundred percent of the samples from the animals with experimentally induced MCF were positive by real-time PCR, while 99% were positive by nonnested PCR. Neither nonnested PCR nor real-time PCR yielded a positive result on any of the 100 nested PCR-negative samples from animals without evidence of clinical MCF. The data confirmed that both nonnested and real-time PCR maintained high specificity and sensitivity for the detection of OvHV-2 DNA in clinical samples.
Journal of veterinary diagnostic investigation: official publication of the American Association of Veterinary Laboratory Diagnosticians, Inc 08/2007; 19(4):405-8. · 1.21 Impact Factor
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ABSTRACT: The rhadinovirus Ovine herpesvirus 2 (OvHV-2) is the causative agent of sheep-associated malignant catarrhal fever. OvHV-2 primarily affects ruminants and has a worldwide distribution. In this study, a composite sequence of OvHV-2 genomic DNA isolated from nasal secretions of sheep experiencing virus-shedding episodes was determined and compared with the sequence of OvHV-2 DNA isolated from a lymphoblastoid cell line derived from a clinically affected cow. The study confirmed the OvHV-2 sequence information determined for the cell line-isolated DNA and showed no apparently significant changes in the OvHV-2 genome during passage through a clinically susceptible species with subsequent maintenance in vitro. Amino acid identity between the predicted open reading frames (ORFs) of the two genomes was 94-100%, except for ORF73, which had an identity of 83%. Polymorphism in ORF73 was due primarily to variability in the G/E-rich repetitive central region of the ORF.
Journal of General Virology 02/2007; 88(Pt 1):40-5. · 3.36 Impact Factor
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ABSTRACT: Infection of clinically susceptible ruminants, including domesticated cattle and American bison, with ovine herpesvirus 2 (OvHV-2) can result in the fatal lymphoproliferative and vasculitis syndrome known as malignant catarrhal fever (MCF). A reliable experimental infection model is needed to study the pathogenesis of MCF and to develop effective vaccination strategies to control the disease. An experimental aerosol infection model using sheep, the natural carriers of OvHV-2, has been developed (Taus et al., 2005). Using the protocol and OvHV-2 inoculum established in the previous study, eight calves were nebulized with four different doses of OvHV-2 in nasal secretions from infected sheep. Two control calves were nebulized with nasal secretions from uninfected sheep. Infection status of all calves was monitored using competitive inhibition ELISA, PCR and clinical parameters. Six of eight nebulized calves became infected with OvHV-2. One calf receiving the highest dose of virus developed typical clinical, gross and histological changes of MCF. This study showed that nasal secretions collected from sheep experiencing OvHV-2 shedding episodes were infectious for cattle and capable of inducing MCF. The data also indicate that cattle are relatively resistant to disease following infection. The use of more susceptible species as experimental animal models, such as bison and selected cervid species should be examined.
Veterinary Microbiology 09/2006; 116(1-3):29-36. · 3.33 Impact Factor
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ABSTRACT: American bison (Bison bison) and domestic cattle (Bos taurus and Bos indicus) evolved from a common ancestor 1-1.4 million years ago. Nevertheless, they show dramatic differences in their susceptibility to infectious diseases, including malignant catarrhal fever (MCF). Although bison are highly susceptible to ovine herpesvirus-2 (OvHV-2) associated MCF, about 20% of healthy domesticated and wild bison are positive for OvHV-2 antibody. We are interested in testing the hypothesis that, within the bison population, the polymorphism of major histocompatibility complex (MHC) class II genes influences resistance to MCF. However, since little was known about the MHC class II genes of bison, it was necessary to first characterize class II haplotypes present in Bi. bison (Bibi). Thus, the MHC class II haplotypes carried by 14 bison were characterized by the PCR-based cloning and sequencing of their DRB3, DQA, and DQB alleles. Twelve MHC class II haplotypes were identified in the 14 bison. These haplotypes comprised six previously reported and six new Bibi-DRB3 alleles, along with 11 Bibi-DQA and 10 Bibi-DQB alleles. For each bison class II allele, it was possible to identify closely related cattle sequences. The closest bison and bovine DQA, DQB, and DRB3 alleles, on average, differed by only 1.3, 3.5, and 5.8 amino acids, respectively. Furthermore, bison MHC haplotypes with both nonduplicated and duplicated DQ genes were identified; these haplotypes appear to have originated from the same ancestral haplotypes as orthologous cattle haplotypes.
Immunogenetics 01/2006; 57(11):845-54. · 2.93 Impact Factor
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Hong Li,
Katherine Gailbreath,
Edmund J Flach,
Naomi S Taus,
Jim Cooley,
Janice Keller,
George C Russell,
Donald P Knowles,
David M Haig,
J Lindsay Oaks, Donald L Traul,
Timothy B Crawford
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ABSTRACT: In the course of investigating the malignant catarrhal fever (MCF) subgroup of rhadinoviruses, seven novel rhadinoviruses were identified in a variety of ruminants, including domestic sheep, bighorn sheep, bison, black-tailed deer, mule deer, fallow deer, elk and addax. Based on the DNA polymerase gene sequences, these newly recognized viruses clustered into a second distinct subgroup in ruminants with three members identified previously in cattle, goats and oryx. Phylogenetic analysis revealed that the currently known ruminant rhadinoviruses appear to comprise three distinct genetic lineages: (i) the MCF subgroup, defined by sequence identity and the presence of the 15A antigenic epitope; (ii) a second distinct subgroup, devoid of the 15A epitope, which contains the previously reported bovine lymphotropic herpesvirus and related viruses; and (iii) a third distinct subgroup represented by Bovine herpesvirus 4. Comparison of phylogenetic trees between the rhadinoviruses and their corresponding hosts further supports the gammaherpesvirus and host co-evolution theory.
Journal of General Virology 12/2005; 86(Pt 11):3021-6. · 3.36 Impact Factor
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ABSTRACT: Alcelaphine herpesvirus-1 (AlHV-1) is a rhadinovirus that causes malignant catarrhal fever in certain ruminant species and is an important pathogen in Africa and other areas where carrier species and clinically susceptible ruminants intermingle. In this study, a real-time PCR for AlHV-1 DNA was developed and compared to an established nested PCR. The nested PCR amplifies a region of the AlHV-1 gene coding for a transactivator protein (ORF 50), while the real-time PCR assay targets the AlHV-1 gene coding for a tegument protein (ORF 3). The real-time PCR assay reproducibly detected 10 copies of target DNA. In a dilution series of the target DNA there was linearity of the assay across 8 orders of magnitude (10(1)-10(9) copies). The nested PCR was more sensitive (approximately with 1 log) than the real-time PCR. The assay specifically amplified samples containing only AlHV-1, but not other common herpesviruses of cattle. In conclusion, we have developed a rapid, relatively sensitive, and reliable real-time PCR assay specific for AlHV-1. Similar to the real-time PCR for Ovine herpesvirus-2, this assay should prove useful for differential diagnostics of clinical MCF and for research to better define the epidemiology of AlHV-1 in wildebeest as well as in animals with wildebeest-associated MCF.
Journal of Virological Methods 12/2005; 129(2):186-90. · 2.01 Impact Factor
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ABSTRACT: Ovine herpesvirus 2 (OvHV-2) is the causative agent of sheep-associated malignant catarrhal fever in clinically susceptible ruminants, including cattle, bison and deer. Studies of OvHV-2 have been hampered by the lack of an in vitro propagation system. Here, the use of nasal secretions collected from OvHV-2-infected sheep experiencing intense virus shedding episodes as a source of infectious virus for experimental animal infections was examined. OvHV-2 uninfected sheep were nebulized with nasal secretions containing approximately 10(8) to 10(1) copies of OvHV-2 DNA. The time to detectable viral DNA in peripheral blood leukocytes (7-12 days post-infection) and virus-specific antibody in plasma (9-32 days post-infection) varied with the dose of inocula administered. Here, the use of nasal secretions as a source of infectious OvHV-2 was defined and the minimum infectious dose of a pool of nasal secretions that can be used in further studies of viral pathogenesis and vaccine development was determined.
Journal of General Virology 04/2005; 86(Pt 3):575-9. · 3.36 Impact Factor
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ABSTRACT: Ovine herpesvirus 2 (OvHV-2), the major causative agent of malignant catarrhal fever in ruminant species worldwide, has never been propagated in vitro. Using real-time PCR, a striking, short-lived, peak of viral DNA, ranging from 10(5) to over 10(8) copies/2 microg of DNA, was detected in nasal secretions from over 60.7% of adolescent sheep (n = 56) at some point during the period from 6 to 9 months of age. In contrast, only about 18% of adult sheep (n = 33) experienced a shedding episode during the study period. The general pattern of the appearance of viral DNA in nasal secretions was a dramatic rise and subsequent fall within 24 to 36 h, implying a single cycle of viral replication. These episodes occurred sporadically and infrequently, but over the 3-month period most of the 56 lambs (33, or 60.7%) experienced at least one episode. No corresponding fluctuations in DNA levels were found in either peripheral blood leukocytes or plasma. In a DNase protection assay, complete, enveloped OvHV-2 virions were demonstrated in the nasal secretions of all sheep examined during the time when they were experiencing an intense shedding episode. OvHV-2 infectivity in nasal secretions was also demonstrated by aerosolization of the secretions into OvHV-2-negative sheep. The data herein show that nasal shedding is the major mode of OvHV-2 transmission among domestic sheep and that adolescents represent the highest risk group for transmission.
Journal of Clinical Microbiology 01/2005; 42(12):5558-64. · 4.15 Impact Factor
