Restriction endonuclease analysis of alcelaphine herpesvirus 1 DNA and molecular cloning of virus genomic DNA for potential diagnostic use.

Alcelaphine herpesvirus 1 (AHV-1) genomic DNA was analyzed using restriction enzymes having recognition sequences both low in guanine-cytosine content (BamHI, KpnI, HindIII) and high in guanine-cytosine content (SmaI, AvaI, ApaI). The results from the restriction enzyme analyses along with exonuclease treatment demonstrated that the termini of AHV-1 DNA are likely composed of polyrepetitive sequences high in guanine-cytosine content similar to those found in Herpesvirus saimiri DNA. Cleaving AHV-1 DNA with the restriction enzyme SmaI produced polyrepetitive sequences that appeared as low molecular weight supermolar bands less than 1 kilobase pairs (kb) in size. Analysis of AHV-1 DNA cleaved with HindIII indicated that the polyrepetitive sequences are approximately 29.5 kb in size. The total molecular weight for AHV-1 DNA was determined to be approximately 118 kb. Seventy-five percent of the AHV-1 genome was cloned into the plasmids pAT153 and pBR322. Cloned DNA fragments representing unique sequences of the AHV-1 genome hybridized with AHV-1 genomic DNA but showed no appreciable hybridization with AHV-2 DNA or bovine herpesvirus 1, 2, or 4 DNA.

Application of polymerase chain reaction to detect animals latently infected with agents of malignant catarrhal fever.

Oligonucleotide primers derived from alcelaphine herpesvirus 1 (AHV-1) isolate WC11 DNA, the first identified agent of malignant catarrhal fever (MCF), were used to assay blood lymphocyte DNA using the polymerase chain reaction (PCR). Multiple species of exotic ruminants were examined to determine the suitability of this technique for detecting animals that may be latently infected. To correlate the PCR results with those of serology, serum samples were obtained from each animal concurrently with lymphocyte collection and subjected to an AHV-1 virus-neutralization assay (VNA). A total of 86 MCF-susceptible animals were tested, and the results of the VNA and PCR assays were compared. PCR results were positive for 44 animals. Of these, 13 were positive by VNA. Animals positive by both VNA and PCR were all wildebeest, the asymptomatic carriers of AHV-1, confirming the ability of the primers to amplify AHV-1 sequence. Positive PCR results from species other than wildebeest may represent sequence amplified from viruses related to AHV-1, which may not induce antibodies capable of neutralizing the WC11 isolate used in the VNA. This study demonstrates that PCR is capable of detecting the presence of MCF agents in various populations of captive ruminants prior to the appearance of clinical MCF so that the sources of infection can be more reliably ascertained.

Prevalence of antibodies to alcelaphine herpesvirus-1 and nucleic acid hybridization analysis of viruses isolated from captive exotic ruminants.

A serologic survey was conducted to determine the prevalence of antibodies to alcelaphine herpesvirus-1 (AHV-1) in captive exotic ruminants within the United States. Forty-six percent of the members of the subfamily Alcelaphinae (wildebeest, topi, hartebeest) in the family Bovidae had virus-neutralizing antibody to AHV-1. Other subfamilies of Bovidae with high prevalence of virus-neutralizing antibodies to AHV-1 included Hippotraginae (oryx and addax) and Caprinae (sheep and goats), with prevalence of 45% and 29%, respectively. Herpesviruses that have been isolated from captive exotic ruminant species, including healthy animals and those with clinical malignant catarrhal fever at the Oklahoma City Zoo and the San Diego Zoo/Wild Animal Park, were analyzed by DNA restriction enzyme analysis and blot hybridization. Variation has been detected among the genomes of several malignant catarrhal fever virus isolates obtained from various exotic species of ruminants, using the DNA restriction enzymes BamHI and HindIII. The DNA of these virus isolates is distinct from that of bovine herpesviruses 1, 2, and 4, as demonstrated by restriction enzyme analysis and nucleic acid hybridization. On the basis of restriction enzyme analysis and nucleic acid hybridization data, the DNA from each of the putative alcelaphine herpesvirus isolates examined, except for the topi virus isolate, had a high degree of DNA sequence similarity with the original AHV-1 isolate, WC-11, from a blue wildebeest.

Diagnosis of malignant catarrhal fever by polymerase chain reaction amplification of alcelaphine herpesvirus 1 sequence.

We derived sequence information from cloned HindIII fragment "D" of alcelaphine herpesvirus 1 strain WC11, an agent of malignant catarrhal fever (MCF). Based on this sequence, oligonucleotide primers were selected and synthesized for use in a polymerase chain reaction amplification assay. These primers were used to test samples of total nucleic acids isolated from multiple tissues taken from an Indian gaur (Bos guarus gaurus) at the San Diego Wild Animal Park in San Diego, California (USA) which had clinical signs of a natural infection of MCF. Six of eight tissue samples examined had amplifiable sequences present. A nucleic acid probe complementary to the sequence of the original clone between the primer sites also was synthesized and used to confirm the identity of the amplified viral sequences, thus providing a diagnosis of MCF at the molecular level.

Alcelaphine herpesviruses 1 and 2 SDS-PAGE analysis of virion polypeptides, restriction endonuclease analysis of genomic DNA and virus replication restriction in different cell types.

Herpesviruses have been isolated from white-tailed, white-bearded and blue wildebeest, as well as from Jimela topi and Cape hartebeest. These animals are members of the sub-family Alcelaphinae of the family Bovidae. Viruses isolated from wildebeest cause malignant catarrhal fever (MCF) in susceptible ruminant species. Alcelaphine herpesviruses (AHV) isolated from wildebeest replicate in both fetal aoudad sheep kidney (FAK) cells and bovine embryonic lung (BEL) cells. However, virus isolates from topi and hartebeest, which have not been linked to clinical MCF, replicate only in FAK cells. Buoyant density analysis by analytical ultracentrifugation, restriction endonuclease analysis and blot hybridization of virus genomic DNA from both alcelaphine herpesviruses as well as from bovine herpesviruses 1, 2, and 4 demonstrate that there are two types of alcelaphine herpesviruses, each distinct and different from the other bovine herpesviruses. Genomic size of both alcelaphine herpesviruses, estimated from DNA restriction fragments, is approximately 110 kilobase pairs. Alcelaphine herpesvirus DNA resembles Herpesvirus saimiri DNA during equilibrium sedimentation in that the majority of the DNA bands as a light (L) fraction with a minor heavy (H) component. Polyacrylamide gel analysis of virion proteins indicates that both viruses have distinct patterns, each consisting of 36 polypeptides ranging in molecular weight from 12,000 to 275,000. Virus isolates from wildebeest have been designated AHV-1, while viruses isolated from topi and hartebeest have been designated AHV-2.

Identification and analysis of an alcelaphine herpesvirus 1 (AHV-1) cDNA clone expressing a fusion protein recognized by AHV-1-neutralizing antisera.

Rabbit antiserum to psoralen-inactivated alcelaphine herpesvirus 1 (AHV-1) virions was shown to react specifically with AHV-1-infected cells by indirect immunofluorescence. Western blot analysis using this antiserum identified a 15-kD virion protein that was also detected in infected-cell proteins between 12 and 144 h p.i., and a 37-kD protein present in infected cells between 24 and 120 h p.i. A cDNA library was constructed using mRNA obtained from AHV-1-infected fetal mouflon sheep kidney (FMSK) cells at 48 h p.i., when infected-cell proteins detected by antiserum were in abundance. Screening of the library with the rabbit anti-AHV-1 serum identified several positive clones. Southern blot analysis showed that one clone, designated 8'a, hybridized to a 4.4 kb HindIII fragment of AHV-1 DNA. This AHV-1 cDNA clone expressed a fusion protein that was recognized by serum from a naturally and asymptomatically infected white-bearded wildebeest (Connochaetes taurinus albojubatus). The insert was sequenced and found to contain 833 bp. A search of the GenBank database for related sequences revealed greater than 40% homology to several other gammaherpesviruses: herpesvirus saimiri, cottontail herpesvirus, and Epstein-Barr virus.

Naturally occurring fatal herpes simplex virus 1 infection in a family of white-faced saki monkeys (Pithecia pithecia pithecia).

A family of three white-faced saki monkeys (Pithecia pithecia pithecia) died 48-96 hours after the onset of anorexia, nasal discharge, pyrexia and oral ulceration. One animal also had clonic seizures. Lesions found post-mortem consisted of oral and esophageal ulcers, hepatic and intestinal necrosis, meningoencephalitis and sporadic neuronal necrosis. Intranuclear inclusion bodies and syncytial cells were present in oral lesions and affected areas of liver. Herpes simplex virus 1 (HSV-1) was identified as the etiology of disease by virus isolation, polymerase chain reaction, or in situ hybridization in all three animals. Immunohistochemistry for detection of apoptotic DNA and activated caspase-3 showed significant levels of apoptosis in oral and liver lesions and occasional apoptotic neurons in the brain. These findings demonstrate the vulnerability of white-faced saki monkeys to HSV-1 and provide initial insight into the pathogenesis of fatal HSV-1-induced disease, indicating that apoptosis plays a significant role in cell death.