William C. Lawrence

Latency and reactivation of a thymidine kinase-negative bovine herpesvirus 1 deletion mutant

SummaryA bovine herpesvirus 1 (BHV 1) mutant variant with a deletion in the thymidine kinase (TK) gene was assessed for its ability to establish latency and be reactivated in cattle. After treatment with dexamethasone, reactivated TK− BHV 1 was isolated from one of four cattle that received virus by intravenous inoculation only, and from four of four cattle that received virus by intranasal, intravaginal, and intravenous inoculation. Results prove that TK− BHV 1 will establish latency and can be reactivated in the natural host.

Morphological components of herpesvirus. II. Preservation of virus during negative staining procedures.

The disruption of envelopes and the fragmentation of capsids of equine herpes-virus type I observed in negatively stained samples were attributed to viral dehydration on carbon films during preparation for electron microscopy. Prior fixation of virus with OSO4 or glutaraldehyde and subsequent application of negative stain before drying minimized envelope disruption and virtually eliminated the occurrence of capsomere sheets and broken capsids. This sample procedure significantly improves electron microscopic evaluation of herpesvirus samples.

Relationship of bovine herpesvirus 1 immediate-early, early, and late gene expression to host cellular gene transcription

Bovine herpesvirus 1 (BHV-1) gene expression was examined by RNA blot hybridization using clones representing immediate-early, early, and late genes. An immediate-early protein gene probe hybridized with two transcripts, 3.4 and 5.8 kb, expressed by infected cells in the presence of cycloheximide (CH). During infection of cells without metabolic inhibitors these transcripts were detected as early as 2 hr postinfection (p.i.) and accumulated to 8 hr p.i. The early gene probe, thymidine kinase, hybridized with a 4.3-kb RNA that was detected in the presence of phosphonoacetic acid (PAA), but not in the presence of CH. The late gene probe, glycoprotein III, (gIII) hybridized with a 1.6-kb transcript that was not expressed by infected cells treated with CH and only in very reduced amounts by infected cells treated with PAA. The gIII RNA was not detected until 4 hr p.i. in total cell RNA. Transcripts for the bovine actin and beta-galactosyltransferase genes did not decrease in BHV-1-infected cells until 6 hr p.i., coincident with the increase of BHV-1 DNA and RNA synthesis. Consequently, shutoff of host cell transcription by BHV-1 may be different than what has been described for herpes simplex virus.

Morphological Components of Herpesvirus

Highly purified equine herpesvirus type 1 (EHV-1) and herpes simplex virus type 1 (HSV-1) were observed by electron microscopy in various stages of disintegration. Under the conditions used, envelopes of virions usually were fragmented, and virus capsids collapsed in situ into flattened sheets of capsomeres. A matrix of intercapsomeric fibrils was observed. Evidence suggesting that the capsid hexamer has threefold structural symmetry is presented.

Morphological components of herpesvirus: IV. Ultrastructural features of the envelope and tegument

Highly purified preparations of equine herpesvirus type 1 virions were examined in the electron microscope after preparation by freeze etching, freeze fracturing, critical point drying, and conventional negative staining methods. Virion envelopes were covered with 12- to 18-nm particles and contained intramembranous particles whose distribution was different from those detected in envelopes of herpes simplex virus type 1 ( Rodriguez and DuBois-Dalcq, 1978 ). Observation of virions and deenveloped virus indicated that the tegument was a defined layer of material attached to the capsid, probably at the vertices. Tegument adhered to deenveloped virus, and its morphology was dependent upon the method of preparation for electron microscopy.

Expression of the Genomic Form of the Bovine Viral Diarrhea Virus E2 ORF in a Bovine Herpesvirus-1 Vector

Bovine viral diarrhea virus (BVDV) is a ubiquitous pathogen of cattle with a world-wide distribution. Recently, the possibility of using recombinant virus vectors to immunize cattle against selected BVDV genes has gained widespread interest. Among the virus vectors tested, bovine herpesvirus-1 (BHV1) provides many unique advantages. However, results of recent studies have raised the possibility that the codon usage pattern required for optimal expression in a BHV1-infected cell may be incompatible with the codon usage pattern of BVDV. If true, use of BHV1 to express BVDV proteins would require construction of synthetic BVDV genes that have been modified to resemble the codon pattern of BHV1. To explore this possibility, we constructed a BHV1 recombinant containing the genomic form of the BVDV (NADL) E2 ORF and compared expression of the E2 protein with that of the endogenous BHV1 gD protein. We observed that E2 was expressed at a significant rate compared to that of the gD protein. We conclude that codon usage problems are unlikely to constitute a serious problem for expression of BVDV proteins in BHV1 vectors.

Construction of a viable BHV1 mutant lacking mostof the short unique region

SummaryThe 8.2 kb HindIII K-fragment of bovine herpesvirus 1 (BHV1) lies entirely within the short unique region of the genome and contains all or parts of 7 coding regions. We have probed this fragment for the presence of nonessential genes by a simple, rapid method of insertional mutagenesis. Our analysis indicates that all the genes present in the K-fragment, except the glycoprotein D gene, are nonessential for replication of BHV1 in tissue culture. After inserting a copy of the glycoprotein D gene into the thymidine kinase locus of BHV1 it was possible to delete the entire HindIII K-fragment and the contiguous 0.36 kb O-fragment in one step. The deletion mutant, which contains 7 kb less BHV1 DNA than wt virus and lacks ORF1, US2, the genes for protein kinase, glycoprotein G, glycoprotein I, and most of glycoprotien E was still replication-competent.

Herpesvirus vaccine development: studies of virus morphological components

Interest in the development of herpesvirus vaccines for humans has been intensified by recent evidence linking members of this virus group to human cancer. Of the five human herpesviruses (herpes simplex virus type 1 and 2 [HSV-1, HSV-2], Epstein-Barr virus [EBV], human cytomegalovirus [CMV], varicella-zoster virus [VZV]), EBV and HSV-2 have been associated with neoplastic disease1-14 and all but VZV have been shown to possessin vitro cell transforming capability12, 13, 15-18.