Molecular Diagnosis of Alcelaphine Herpesvirus (Malignant Catarrhal Fever) Infections by Nested Amplification of Viral DNA in Bovine Blood Buffy Coat Specimens

Abstract

A fragment of alcelaphine herpesvirus-1 (AHV-1; malignant catarrhal fever) DNA was subcloned into pUC 18 and sequenced. The subclone hybridized strongly to AHV-1 DNA, weakly to alcelaphine herpesvirus-2 (AHV-2) DNA, and not at all to DNA from bovine herpesvirus-1 (BHV-1; infectious bovine rhinotracheitis [IBR] virus), bovine herpesvirus-2 (BHV-2; bovine herpes mamillitis [BHM] virus), and bovine herpesvirus-4 (BHV-4; isolate DN599). A 2-stage (nested) polymerase chain reaction (PCR) diagnostic test was devised based on a portion of the subcloned AHV-1 DNA sequence. First and second stage amplified AHV-1 DNA targets were 487 and 172 base pairs (bp) in length, respectively. Unique Pvu II and Stu I restriction endonuclease cleavage sites confirmed the identity of amplified AHV-1 DNA. Five AHV-1 and 2 AHV-2 isolates were identically and specifically PCR positive. BHV-1, BHV-2, and BHV-4 viruses were negative by the same procedure. As little as 0.01 TCID50 AHV-1 was detected using the nested amplification procedure. Simple methods of buffy coat isolation from bovine blood were employed to prepare specimens for PCR. An AHV-1-infected calf was PCR positive from 3 to 77 days postinoculation (PI), with rising seroconversion first noted 14 days PI. The AHV-1 DNA sequence was 62% homologous to a portion of the Epstein-Barr virus genome. The nested PCR procedure may improve the viral diagnosis of clinical and subclinical alcelaphine herpesvirus infections.

PCR detection of the sheep-associated agent of malignant catarrhal fever

“…A PCR for the detection of AHV-1 DNA sequence [41], has been reported for the diagnosis of AHV infections [13,14]. The sensitivity of this nested PCR was reported to be 0.01 TCIDs0 of infective AHV-1 virus [ 14]. The apparent high sensitivity of detection most probably was due to the presence of virus particles which failed to replicate in tissue culture.…”

“…The high degree of sensitivity afforded by the PCR has provided the technology for the detection of rare DNA sequences and has been used extensively for viral detection and diagnosis. A PCR for the detection of AHV-1 DNA sequence [41], has been reported for the diagnosis of AHV infections [13,14]. The sensitivity of this nested PCR was reported to be 0.01 TCIDs0 of infective AHV-1 virus [ 14].…”

“…The PCR has been applied to the detection of other herpesviruses and a PCR test has been described for the detection of AHV-1 [13,14]. A 2-stage nested amplification reaction, utilising different primer pairs from the same AHV-1 cloned sequence, subsequently was shown to amplify both AHV-1 and AHV-2 isolates [ 14].…”

Malignant Catarrhal Fever-Like Disease in Barbary Red Deer (Cervus elaphus barbarus) Naturally Infected with a Virus Resembling Alcelaphine Herpesvirus 2

“…The latter has been isolated several times from clinically normal topi or Cape hartebeest, members of the bovid subfamily Alcelaphinae (30,38,44,45), but experimental infections of cattle produce no discernible effects; moreover, inoculation of cattle with AlHV-2 does not elicit antibodies protective against subsequent AlHV-1 challenge (30). AlHV-2 can be detected by AlHV-2-specific PCR as well as by a PCR that will amplify both AlHV-1 and AlHV-2 (14,17,29) and by CI-ELISA (19).…”

“…These viruses are now included with alcelaphine herpesvirus 1 (AlHV-1) and ovine herpesvirus 2 (OvHV-2) as members of the MCF virus group with known pathogenic potential. AlHV-1, first described and linked to MCF in 1960 (31), can presently be detected by PCR (14,17,29) or by serologic methods (17,(20)(21)(22) in clinical MCF cases or herd screening. However, unambiguous identification of AlHV-1 may require sequencing of PCR-amplified DNA.…”