Ovine Herpesvirus 2 Glycoprotein B Complementation Restores Infectivity to a Bovine Herpesvirus 4 gB-Null Mutant.

Ovine herpesvirus 2 (OvHV-2) and bovine herpesvirus 4 (BoHV-4) are gamma herpesviruses that belong to the genera Macavirus and Rhadinovirus, respectively. As with all herpesviruses, both OvHV-2 and BoHV-4 express glycoprotein B (gB), which plays an essential role in the infection of host cells. In that context, it has been demonstrated that a BoHV-4 gB-null mutant is unable to infect host cells. In this study, we used homologous recombination to insert OvHV-2 ORF 8, encoding gB, into the BoHV-4 gB-null mutant genome, creating a chimeric BoHV-4 virus carrying and expressing OvHV-2 gB (BoHV-4∆gB/OvHV-2-gB) that was infectious and able to replicate in vitro. We then evaluated BoHV-4∆gB/OvHV-2-gB as a potential vaccine candidate for sheep-associated malignant catarrhal fever (SA-MCF), a fatal disease of ungulates caused by OvHV-2. Using rabbits as a laboratory model for MCF, we assessed the safety, immunogenicity, and efficacy of BoHV-4∆gB/OvHV-2-gB in an immunization/challenge trial. The results showed that while BoHV-4∆gB/OvHV-2-gB was safe and induced OvHV-2 gB-specific humoral immune responses, immunization conferred only 28.5% protection upon challenge with OvHV-2. Therefore, future studies should focus on alternative strategies to express OvHV-2 proteins to develop an effective vaccine against SA-MCF.

A Vaccine Targeting Ovine Herpesvirus 2 Glycoprotein B Protects against Sheep-Associated Malignant Catarrhal Fever.

Malignant catarrhal fever (MCF) is a complex and often fatal disease of ungulates. Effective vaccines are needed to avoid MCF outbreaks and mitigate losses. This study aimed to evaluate a sheep-associated MCF (SA-MCF) vaccine candidate targeting ovine herpesvirus 2 (OvHV-2) glycoprotein B (gB). Rabbits were used as a laboratory animal model to test the safety, immunogenicity, and protective efficacy of a chimeric virus consisting of a recombinant, non-pathogenic strain of alcelaphine herpesvirus-1 encoding OvHV-2 ORF8 to express gB (AlHV-1∆ORF73/OvHV-2-ORF8). Viral-vectored immunizations were performed by using the AlHV-1∆ORF73/OvHV-2-ORF8 chimera alone or as a DNA prime (OvHV-2-ORF8)-virus boost regimen. The viral vector was inoculated by intravenous or intramuscular routes and the DNA was delivered by intradermal shots using a gene gun. The vaccine candidates were deemed safe as no clinical signs were observed following any of the immunizations. Anti-OvHV-2 gB antibodies with neutralizing activity were induced by all immunogens. At three weeks post-final immunization, all animals were challenged intranasally with a lethal dose of OvHV-2. MCF protection rates ranging from 66.7% to 71.4% were observed in vaccinated rabbits, while all mock-vaccinated animals developed the disease. The significant protective efficacy obtained with the vaccine platforms tested in this study encourages further trials in relevant livestock species, such as cattle and bison.

X-Linked Hypohidrotic Ectodermal Dysplasia in Crossbred Beef Cattle Due to a Large Deletion in EDA.

X-linked hypohidrotic ectodermal dysplasia-1 (ECTD1) in people results in a spectrum of abnormalities, most importantly hypotrichosis, anodontia/oligodontia, and absent or defective ectodermally derived glands. Five Red Angus-Simmental calves born over a 6-year period demonstrated severe hypotrichosis and were diagnosed as affected with ECTD1-like syndrome. Two died of severe pneumonia within a week of birth. The skin of three affected calves revealed a predominance of histologically unremarkable small-caliber hair follicles. Larger follicles (>50 µm) containing medullated hairs (including guard and tactile hairs) were largely restricted to the muzzle, chin, tail, eyelids, tragus and distal portions of the limbs and tail. The mean histological density of hair follicles in flank skin of two affected calves was slightly greater than that in two unaffected calves. One affected calf was examined postmortem at 10 days of age to better characterize systemic lesions. Nasolabial, intranasal and tracheobronchial mucosal glands were absent, whereas olfactory glands were unaffected. Mandibular incisor teeth were absent. Premolar teeth were unerupted and widely spaced. Other than oligodontia, histological changes in teeth were modest, featuring multifocal disorganization of ameloblasts, new bone formation in dental alveoli, and small aggregates of osteodentin and cementum at the margins of the enamel organ. A 52,780 base pair deletion spanning six out of eight coding exons of EDA and all of AWAT2 was identified. Partial deletion of the EDA gene is the presumed basis for the reported X-chromosomal recessive inherited genodermatosis.

OvHV-2 Glycoprotein B Delivered by a Recombinant BoHV-4 Is Immunogenic and Induces Partial Protection against Sheep-Associated Malignant Catarrhal Fever in a Rabbit Model.

An efficacious vaccine for sheep-associated malignant catarrhal fever (SA-MCF) is important for the livestock industry. Research towards SA-MCF vaccine development is hindered by the absence of culture systems to propagate the causative agent, ovine herpesvirus-2 (OvHV-2), which means its genome cannot be experimentally modified to generate an attenuated vaccine strain. Alternative approaches for vaccine development are needed to deliver OvHV-2 antigens. Bovine herpesvirus 4 (BoHV-4) has been evaluated as a vaccine vector for several viral antigens with promising results. In this study, we genetically engineered BoHV-4 to express OvHV-2 glycoprotein B (gB) and evaluated its efficacy as an SA-MCF vaccine using a rabbit model. The construction of a viable recombinant virus (BoHV-4-AΔTK-OvHV-2-gB) and confirmation of OvHV-2 gB expression were performed in vitro. The immunization of rabbits with BoHV-4-AΔTK-OvHV-2-gB elicited strong humoral responses to OvHV-2 gB, including neutralizing antibodies. Following intra-nasal challenge with a lethal dose of OvHV-2, 42.9% of the OvHV-2 gB vaccinated rabbits were protected against SA-MCF, while all rabbits in the mock-vaccinated group succumbed to SA-MCF. Overall, OvHV-2 gB delivered by the recombinant BoHV-4 was immunogenic and partly protective against SA-MCF in rabbits. These are promising results towards an SA-MCF vaccine; however, improvements are needed to increase protection rates.

In Situ Hybridization for Localization of Ovine Herpesvirus 2, the Agent of Sheep-Associated Malignant Catarrhal Fever, in Formalin-Fixed Tissues.

A constraint on understanding the pathogenesis of malignant catarrhal fever (MCF) is the limited number of tools to localize infected cells. The amount of detectable virus, visualized in the past either by immunohistochemistry or in situ hybridization (ISH), has been modest in fixed or frozen tissues. This complicates our understanding of the widespread lymphoid proliferation, epithelial necrosis/apoptosis, and arteritis-phlebitis that characterize MCF. In this work, we developed a probe-based in situ hybridization assay targeting 2 ovine herpesvirus 2 (OvHV-2) genes, as well as their respective transcripts, in formalin-fixed tissues. Using this approach, OvHV-2 nucleic acids were detected in lymphocytes in MCF-affected animals following both natural infection (American bison and domestic cattle) and experimental infection (American bison, rabbits, and pigs). The probe did not cross-react with 4 closely related gammaherpesviruses that also cause MCF: alcelaphine herpesvirus 1, alcelaphine herpesvirus 2, caprine herpesvirus 2, and ibex-MCF virus (MCFV). No signal was detected in control tissues negative for OvHV-2. ISH will be of value in analyzing the natural progression of OvHV-2 infection in time-course studies following experimental infection and in addressing the pathogenesis of MCF.

Cross-Reactivity of Neutralizing Antibodies among Malignant Catarrhal Fever Viruses.

Some members of the gamma herpesvirus genus Macavirus are maintained in nature as subclinical infections in well-adapted ungulate hosts. Transmission of these viruses to poorly adapted hosts, such as American bison and cattle, can result in the frequently fatal disease malignant catarrhal fever (MCF). Based on phylogenetic analysis, the MCF viruses (MCFV) cluster into two subgroups corresponding to the reservoir hosts' subfamilies: Alcelaphinae/Hippotraginae and Caprinae. Antibody cross-reactivity among MCFVs has been demonstrated using techniques such as enzyme linked immunosorbent and immunofluorescence assays. However, minimal information is available as to whether virus neutralizing antibodies generated against one MCFV cross react with other members of the genus. This study tested the neutralizing activity of serum and plasma from select MCFV-infected reservoir hosts against alcelaphine herpesvirus 1 (AlHV-1) and ovine herpesvirus 2 (OvHV-2). Neutralizing antibody activity against AlHV-1 was detected in samples from infected hosts in the Alcelaphinae and Hippotraginae subfamilies, but not from hosts in the Caprinae subfamily. OvHV-2 neutralizing activity was demonstrated in samples from goats (Caprinae) but not from wildebeest (Alcelaphinae). These results show that neutralizing antibody cross reactivity is present to MCFVs within a virus subgroup but not between subgroups. This information is important for diagnosing infection with MCFVs and in the development of vaccines against MCF.

Antibodies to ovine herpesvirus 2 glycoproteins decrease virus infectivity and prevent malignant catarrhal fever in rabbits.

Ovine herpesvirus-2 (OvHV-2) is the etiological agent of sheep-associated malignant catarrhal fever (SA-MCF), a fatal lymphoproliferative disease of many species in the order Artiodactyla. Development of a vaccine is critical to prevent mortality. Because OvHV-2 has not been cultured in vitro, SA-MCF research is hindered by the lack of in vitro tools to study viral constituents and specific host immune responses. As an alternative, in this study the neutralizing activity of antibodies against OvHV-2 glycoproteins gB and gH/gL was evaluated in vivo using rabbits. OvHV-2-specific antibodies were developed in rabbits by immunization using biolistic delivery of plasmids expressing the genes of interest. A lethal dose of OvHV-2 was incubated with the antisera and then nebulized into rabbits. Virus neutralization was assessed by measuring infection parameters associated with the virus infectious dose. Anti-gB or anti-gH/gL antibodies alone blocked infection in five out of six rabbits (83%), while a combination of anti-gB and anti-gH/gL antibodies protected all six rabbits (100%) from infection. These results indicate that antibodies to OvHV-2 gB and gH/gL are capable of neutralizing virions, and consequently, reduce virus infectivity and prevent SA-MCF in rabbits. Thus, OvHV-2 gB and gH/gL are suitable targets to be tested in a SA-MCF vaccine aimed at stimulating neutralizing antibody responses.

Malignant catarrhal fever in American bison (Bison bison) experimentally infected with alcelaphine herpesvirus 2.

Malignant catarrhal fever (MCF), due to ovine herpesvirus 2 (OvHV-2), causes appreciable death loss in ranched bison (Bison bison) throughout North America. No vaccine exists to protect animals from disease. Since OvHV-2 has not been propagated in vitro, one strategy to develop a modified live vaccine is to use a closely related, non-pathogenic member of the malignant catarrhal fever virus family as a vector expressing potentially protective OvHV-2 epitopes. To date, no controlled experimental challenge studies with alcelaphine herpesvirus 2 (AlHV-2) derived from topi (Damaliscus lunatus jimela) have been reported The unique or light DNA segment of the AlHV-2 genome was sequenced and annotated and the virus was tested for its ability to infect and induce disease in American bison. Yearling bison were inoculated intranasally (n=4) or intramuscularly (n=3) with 2 × 10(-4.7) TCID50 of AlHV-2, and monitored for infection and the development of disease. Six inoculated bison became infected with AlHV-2. Two of the six animals developed clinical signs and had gross and histological lesions consistent with terminal MCF, which differed in distribution from those in bison with MCF due to OvHV-2. One other animal developed minor clinical signs and had gross and histological pulmonary lesions consistent with early (pre-clinical) stages of MCF. Unmodified low cell culture passage AlHV-2 derived from topi is an unsuitable vaccine vector for the prevention of MCF. However, the annotated genome might be useful in identifying genes which could be deleted to potentially attenuate the virus for bison.

Fibroblasts express OvHV-2 capsid protein in vasculitis lesions of American bison (Bison bison) with experimental sheep-associated malignant catarrhal fever.

American bison (Bison bison) are particularly susceptible to developing fatal sheep-associated malignant catarrhal fever (SA-MCF) caused by ovine herpesvirus-2 (OvHV-2), a γ-herpesvirus in the Macavirus genus. This generally fatal disease is characterized by lymphoproliferation, vasculitis, and mucosal ulceration in American bison, domestic cattle (Bos taurus), and other clinically susceptible species which are considered non-adapted, dead-end hosts. The pathogenesis and cellular tropism of OvHV-2 infection have not been fully defined. An earlier study detected OvHV-2 open reading frame 25 (ORF25) transcripts encoding the viral major capsid protein in tissues of bison with SA-MCF, and levels of viral transcript expression positively correlated with lesion severity. To further define the cellular tropism and replication of OvHV-2 infection in vascular lesions of bison, immunofluorescence studies were performed to identify cell type(s) expressing ORF25 protein within tissues. Cytoplasmic and not nuclear ORF25 protein was demonstrated in predominantly perivascular fibroblasts in six bison with experimentally-induced SA-MCF, and there was no evidence of immunoreactivity in vascular endothelium, smooth muscle, or infiltrating leukocytes. The cytoplasmic distribution of viral major capsid protein suggests that viral replication in perivascular fibroblasts may be abortive in this dead-end host. These findings provide a novel foundation for defining the pathogenesis of vasculitis in non-adapted hosts with SA-MCF.

Are rabbits a suitable model to study sheep-associated malignant catarrhal fever in susceptible hosts?

Sheep-associated malignant catarrhal fever (SA-MCF), caused by ovine herpesvirus 2 (OvHV-2), is an often fatal syndrome affecting mainly ruminants. SA-MCF pathogenesis and vaccine studies rely solely on live animals, since OvHV-2 has not been successfully propagated in vitro. Thus, the identification of a laboratory animal model is desirable and necessary to accelerate the identification of virus-host interactions that lead to disease. Rabbits are susceptible to infection with OvHV-2 and the disease can be reliably induced experimentally; however, the viral dynamics and host immune responses in the context of SA-MCF development in rabbits have not yet been evaluated. We addressed these knowledge gaps by experimentally infecting rabbits with OvHV-2 and monitoring viral and host infection parameters. Following intranasal nebulization of OvHV-2 in rabbits, the virus transiently replicates in the lungs inducing only subtle local inflammatory responses; the virus then disseminates systemically and increased levels of viral DNA and transcripts can be detected in multiple tissues as disease develops. The severity of lesions was shown to increase with both viral DNA copy number and expression levels of ORF25, ORF50 and ORF73. The events observed in rabbits following OvHV-2 infection occurred in the same fashion previously reported in bison, a natural clinically susceptible host. The results of this study in conjunction with previous reports demonstrate that rabbits are a valuable model for SA-MCF pathogenesis and vaccine studies.

Development of an in vivo system to measure antibody-blocking of ovine herpesvirus 2 entry.

Ovine herpesvirus 2 (OvHV-2), the causative agent of sheep-associated malignant catarrhal fever (SA-MCF), has never been propagated in vitro. Thus, an alternative to in vitro virus neutralization is needed to assess neutralizing antibody activity to OvHV-2 in SA-MCF vaccine development. An in vivo system in sheep and rabbits was evaluated to determine whether it could be used to assess the ability of antibodies to block OvHV-2 at the entry site by mixing virus and anti-OvHV-2 serum before challenge by intranasal nebulization. A dose of OvHV-2 (10(6) viral DNA copies) incubated with sheep sera (1:4 final dilution) at 37°C for 1 hr was delivered by intranasal nebulization to sheep and rabbits. All sheep became infected, but the positive serum reduced viral infectivity by approximately 1000 fold based on delayed detection of viral DNA and seroconversion as compared to the negative control group, which received virus treated with negative sheep serum. All rabbits that received the virus mixed with the positive sheep serum, either with or without complement, were protected from the infection while all rabbits in the control groups developed SA-MCF. The data indicate that this type of in vivo system, sheep or rabbits, can be used to assess antibody's ability to block OvHV-2 entry, which is a significant tool for the analysis of protective antibody responses to the virus.

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.

Experimental induction of malignant catarrhal fever in pigs with ovine herpesvirus 2 by intranasal nebulization.

Malignant catarrhal fever (MCF), a frequently fatal herpesviral disease primarily of ruminant species, has been sporadically reported in pigs. All cases of naturally occurring porcine MCF reported to date have been linked to ovine herpesvirus 2 (OvHV-2), a gammaherpesvirus in the genus Macavirus carried by sheep. Experimental induction of MCF by aerosolization of the virus in nasal secretions collected from infected sheep has been successful in bison, cattle and rabbits. The goals of this study were to determine the susceptibility of pigs to MCF following experimental intranasal inoculation of OvHV-2, and to characterize the disease. Twelve pigs in four groups were nebulized with 10(5), 10(6), 10(7), or 10(8) DNA copies of OvHV-2 from sheep nasal secretions. Three control pigs were nebulized with nasal secretions from uninfected sheep. Three additional pigs were inoculated intravenously with 10(7) DNA copies of OvHV-2 to evaluate this route of infection with cell-free virus. Seven of twelve intranasally challenged pigs became infected with OvHV-2. Five of these seven, all in higher dose groups, developed MCF. Lesions resembled those reported in natural cases of porcine MCF. The most striking and consistent histological lesions were in trachea, lung, kidney and brain. These comprised mucopurulent tracheitis, interstitial pneumonia, necrotizing arteritis-periarteritis, and nonpurulent meningoencephalitis. No infection was established in the intravenously challenged or control groups. The study showed that MCF can be experimentally induced in pigs by aerosol challenge using sheep nasal secretions containing OvHV-2. Domestic pigs are a natural clinically susceptible host for sheep-associated MCF. They represent a useful, cost-effective model for MCF research.

Characterization of ovine herpesvirus 2-induced malignant catarrhal fever in rabbits.

Malignant catarrhal fever (MCF) is a frequently fatal lymphoproliferative disease syndrome primarily of ruminant species, caused by gammaherpesviruses in the genus Macavirus. Ovine herpesvirus 2 (OvHV-2), carried by sheep, causes sheep-associated MCF worldwide, while Alcelaphine herpesvirus 1 (AlHV-1), carried by wildebeest, causes wildebeest-associated MCF, mainly in Africa. Diseases in rabbits can be induced by both viruses, which are clinically and pathologically similar; however, recent studies revealed different expression of viral genes associated with latency or lytic replication during clinical disease between the two viruses. In this study, we further characterized experimentally induced MCF in rabbits by nebulization with OvHV-2 from sheep nasal secretions to elucidate the course of viral replication, along with in vivo incorporation of 5-Bromo-2'-Deoxyuridine (BrdU), to evaluate lymphoproliferation. All six rabbits nebulized with OvHV-2 developed MCF between 24 and 29 days post infection. OvHV-2 DNA levels in peripheral blood leukocytes (PBL) remained undetectable during the incubation period and increased dramatically a few days before onset of clinical signs. During the clinical stage, we found that predominantly lytic gene expression was detected in PBL and tissues, and both T and B cells were proliferating. The data showed that the viral gene expression profile and lymphoproliferation in rabbits with OvHV-2 induced MCF were different from that in rabbits with AlHV-1 induced MCF, suggesting that OvHV-2 and AlHV-1 may play a different role in MCF pathogenesis.

CD8(+)/perforin(+)/WC1(-) gammadelta T cells, not CD8(+) alphabeta T cells, infiltrate vasculitis lesions of American bison (Bison bison) with experimental sheep-associated malignant catarrhal fever.

Sheep-associated malignant catarrhal fever (SA-MCF) caused by ovine herpesvirus-2 (OvHV-2), a gamma-herpesvirus in the Macavirus genus, is a fatal disease associated with lymphoproliferation, lymphocytic vasculitis, and mucosal ulceration in clinically susceptible species. SA-MCF is an important threat to American bison (Bison bison) due to their high susceptibility to this disease. Currently, the pathogenesis of disease in SA-MCF is poorly understood, and the immunophenotype of lymphocytes that infiltrate the vascular lesions of bison and cattle with SA-MCF has been only partially defined. Previous single-color immunohistochemistry studies have demonstrated that CD8(+) cells and CD4(+) cells predominate within vascular infiltrates in cattle and bison. The CD8(+) cells detected in the vascular lesions of cattle and bison were assumed to be cytotoxic alphabeta T lymphocytes. However, polychromatic immunophenotyping analyses in this study showed that CD8(+)/perforin(+) gammadelta T cells, CD4(+)/perforin(-) alphabeta T cells, and B cells infiltrate vascular lesions in the urinary bladder, kidney, and liver of six bison with experimentally-induced SA-MCF. CD8(+) alphabeta T cells and WC1(+) gammadelta T cell cells were only infrequently and inconsistently identified. This study confirmed our hypothesis that the predominant CD8(+) lymphocytes infiltrating the vascular lesions of bison with SA-MCF are cytotoxic lymphocytes of the innate immune system, not CD8(+) alphabeta T cells. Results of the present study support the previous suggestions that MCF is fundamentally a disease of immune dysregulation.

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.

Long distance spread of malignant catarrhal fever virus from feedlot lambs to ranch bison.

Malignant catarrhal fever (MCF) caused by OvHV-2 occurred in ranch bison herds separated by significant distances from feedlot lambs. Mortality rates correlated with distances: 17.5%, 6.1%, and 0.43% at approximately 1.6, 4.2, and 5.1 km, respectively. The study further defines the importance of distance of species separation for MCF control.

Detection of ovine herpesvirus 2 major capsid gene transcripts as an indicator of virus replication in shedding sheep and clinically affected animals.

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.

Validation of nonnested and real-time PCR for diagnosis of sheep-associated malignant catarrhal fever in clinical samples.

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.

Infection of guinea pigs with vesicular stomatitis New Jersey virus Transmitted by Culicoides sonorensis (Diptera: Ceratopogonidae).

Intrathoracically inoculated Culicoides sonorensis Wirth & Jones were capable of transmitting vesicular stomatitis New Jersey virus (family Rhabdoviridae, genus Vesiculovirus, VSNJV) during blood feeding on the abdomen of six guinea pigs. None of the guinea pigs infected in this manner developed clinical signs of vesicular stomatitis despite seroconversion for VSNJV. Guinea pigs infected by intradermal inoculations of VSNJV in the abdomen also failed to develop clinical signs of vesicular stomatitis. Three guinea pigs given intradermal inoculations of VSNJV in the foot pad developed lesions typical of vesicular stomatitis. Transmission by the bite of C. sonorensis may have facilitated guinea pig infection with VSNJV because a single infected C. sonorensis caused seroconversion and all guinea pigs infected by insect bite seroconverted compared with 50% of the guinea pigs infected by intradermal inoculation with a higher titer VSNJV inoculum. The role of C. sonorensis in the transmission of VSNJV is discussed.