Quantitative Fluorogenic PCR Assay for Measuring Ovine Herpesvirus 2 Replication in Sheep

Abstract

A fluorogenic PCR specific for ovine herpesvirus 2 (OvHV-2) DNA was developed and compared to a previously established conventional seminested PCR. Testing of a total of 152 blood samples from both positive and negative animals revealed that the results of both assays corresponded to each other in 100% of the cases. A second fluorogenic PCR for genomic sheep DNA was required to normalize the quantity of viral DNA in the sample. Separate standard curves had to be constructed for each PCR. The analytical sensitivity of the new PCRs ranged between at least 10 copies and sometimes even 1 copy of target DNA per reaction mixture. In dilution series of the target DNAs, linear decreases of the signals were observed over 7 orders of magnitude. Thus, it was possible to calculate the amounts of viral DNA in relation to the amounts of cellular DNA by normalizing the absolute quantity of OvHV-2 DNA with the amount of genomic sheep DNA. By this technique, it was possible for the first time to quantitatively characterize the course of OvHV-2 replication in naturally infected sheep.Malignant catarrhal fever (MCF) is a sporadic but usually fatal infectious disease of cattle and other ruminant species (8,9,10,12). Ovine herpesvirus 2 (OvHV-2) is believed to be the etiologic agent of the sheep-associated form of MCF (SA-MCF) that occurs almost worldwide. The agent of the wildebeest-associated form of MCF, alcelaphine herpesvirus 1 (AlcHV-1), was readily isolated and propagated in cell culture (4, 13). In contrast, the isolation of OvHV-2 in cell cultures has not been reported (5), while our own attempts remained unsuccessful (unpublished data). The establishment of a conventional PCR for the detection of OvHV-2 DNA was therefore important (2, 3). This conventional PCR was applied for detection of OvHV-2 in vivo. It turned out that the natural reservoir of this virus is sheep. Among cattle, only samples from animals suffering from MCF and some rare survivors of MCF (9, 10) react positively in this PCR (U. U. Müller-Doblies, J. Egli, H. Li, U. Braun, and M. Ackermann, in press). On the basis of these observations, PCR was established as the new "gold standard" for the diagnosis of MCF in cattle (11). Thus, without being able to grow the virus in cell culture, the technical requirements for tracing of this agent in a qualitative manner had been developed.However, to get a better insight into the pathogenesis and replication of OvHV-2, it is essential to trace the agent in a quantitative manner along the time axis of a natural infection.Conventional quantitative PCRs are time-consuming and carry a high risk of false-positive results due to contamination, especially when a high sample throughput is required. Therefore, a fluorogenic PCR was developed for quantitative determination of OvHV-2 DNA in samples obtained from sheep. The assay makes use of a dually labeled fluorogenic probe, which is designed to hybridize to the sequence between the primers (User's Manual, ABI PRISM 7700 Sequence Detection System, Perkin-Elmer Applied Biosyste...

Naturally Occurring Sheep-Associated Malignant Catarrhal Fever in North American Pigs

“…Peripheral blood samples from both clinically normal New York pigs (one of which was positive for MCF viral antibody) were negative by OvHV-2 PCR. By realtime PCR, 7,17 targeting the same region of open reading frame 75, OvHV-2 DNA levels in the spleen, lymph node, and lung were detected as 89,500, 119,000, and 382,000 copies per 2 mg of tissue DNA, respectively. In the Kentucky case, a 238-bp DNA fragment was also amplified by nested PCR from the tissue samples (intestine and the pooled tissues) of the clinically affected pig.…”

Experimental Transmission of Ovine Herpesvirus-2 in Sheep

“…Though we did not attempt other transmission routes in this study, there have been several reports that no OvHV-2 DNA was detected in the urinary bladder [2], urine or faeces [7,8]. Consequently, it is suggested that nasal secretions play an active role in transmission of the virus among sheep.…”

Transactivator Protein BICP0 of Bovine Herpesvirus 1 (BHV-1) Is Blocked by Prostaglandin D2(PGD2), Which Points to a Mechanism for PGD2-Mediated Inhibition of BHV-1 Replication

“…The glycoprotein gB gene was targeted for amplification of 97 nucleotides from the viral DNA by the TaqMan technique. The experimental conditions were as described previously (23), with the following exceptions. The sequences of the oligonucleotides used were as follows: 5Ј-TGT GGA CCT AAA CCT CAC GGT-3Ј (forward primer), 5Ј-GTA GTC GAG CAG ACC CGT GTC-3Ј (reverse primer), and 5Ј-FAM-AGG ACC GCG AGT TCT TGC CGC-TAMRA-3Ј (probe).…”