Characterization of the Genome of Equine Herpesvirus 1 Subtype 2

Cullinane, Ann A (1986) Characterization of the Genome of Equine Herpesvirus 1 Subtype 2. PhD thesis, University of Glasgow.

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Equine herpesvirus type-1 (EHV-1), a member of the Alphaherpesvirinae. is a major cause of abortion and respiratory disease in horses worldwide. It is also associated with a neurological syndrome, neonatal foal disease and more rarely, coital exanthema. Collectively, these diseases represent a significant economic loss to the thoroughbred industry each year. Two antigenically and genetically distinct subtypes of EHV-1 exist. They can be unequivocally differentiated by restriction endonuclease analysis of their DNAs. Molecular epizootiological studies in America and Australia indicate that both subtypes of EHV-1 are respiratory pathogens with the potential to cause abortion, but only subtype 1 has been associated with abortion storms and the neurological form of the disease. Most of the molecular data published concern the subtype 1 virus. At the onset of this project the genome of EHV-1 subtype 2 was totally uncharacterized. This was probably due in part to the original misconception that it was identical to EHV-1 subtype 1 and in part to the relative difficulty of growing this virus to a high titre in tissue culture. The purpose of this work was to determine whether the two subtypes of EHV-1 shared a common genome structure, to construct the first restriction endonuclease maps for EHV-1 subtype 2 and to investigate the homology between this virus and other members of the Alphaherpesvirinae by molecular hybridization and DNA sequence analysis. Electron microscopy of EHV-1 subtype 2 DNA which had been denatured and self-annealed indicated that a sequence of 11kbp approximately at one genome terminus is repeated in inverse orientation at one internal site. The inverted repeats were shown to be separated by a unique sequence of approximately 13kbp. The presence of repeated sequence within the EHV-1 subtype 2 genome was confirmed by hybridization studies using DNA probes isolated from virion DMA. A library of plasmid clones containing Ram HI fragments representing approximately 75% of the genome was prepared, and the clones were then used to derive Bam HI and Eco RI restriction endonuclease maps for EHV-1 subtype 2 DNA. The results show that the EHV-1 subtype 2 genome consists of two segments, L (111kbp) and S (35kbp). The S component consists of a unique sequence (Us; 9.6kpb -16kbp) flanked by inverted repeats (TRs and 9.5kbp - 12.7kbp). Published data indicate that the EHV-1 subtype 1 genome has a similar structure. However, the maps for the two subtypes are quite different. Eco RI and Bam HI cleave within the TRs/IRs , and so it was not possible to determine whether Us inverts relative to the L region, as it does in subtype 1. HSV-1 DNA fragments containing coding sequences for genes which have been shown previously to be well-conserved in the alphaherpesviruses were hybridized to EHV-1 subtype 2 DMA. Thus, the regions of the EHV-1 subtype 2 genome homologous to probes for the HSV-1 genes encoding the ribonucleotide reductase, the major capsid protein, the major DNA-binding protein and the immediate early protein VmwIE175 were identified. Cloned DNA fragments of EHV-1 subtype 2 were used in comparative hybridization experiments to further determine the extent and distribution of homologous sequences in the genomes of both subtypes of EHV-1 and HSV-1. Regions of detectable homology are arranged colinearly along the genomes suggesting that the three viruses share a common gene arrangement. These results imply that it should be possible to predict the locations of most EHV-1 subtype 2 genes on the basis of our existing knowledge of HSV-1 gene location and function. Published data indicate that the S segment is the least related region in the genomes of several members of the Alphaherpesvirinae and that the TRs /Us and IRs /Us junctions have altered in location, relative to adjacent genes, during evolution. To elucidate the nature of the genes near the EHV-1 subtype 2 TRs/Us junction, the DNA sequence of a 4.57kbp Bam HI fragment was determined using the Sanger chain terminating dideoxynucleotide method. The junction was located within a 100bp region by using several M13 clones in a hybridization study, indicating that TRs/IRs and Us are approximately 10.9kbp and 13.1kbp in size respectively. The G+C content of the TRs portion of the fragment is approximately 17% greater that that of the Us portion. An 8bp sequence is tandemly repeated within the TRs. Analysis of the sequence showed that Bam HI 1 contains two complete open reading frames and the parts of two others. The amino acid sequences of predicted EHV-1 subtype 2 proteins were compared with those coded by the S segments of VZV and HSV-1. Homologues of the four EHV-1 genes were detected in both HSV-1 and VZV. The EHV-1 genes and the TRs/Us junction have an arrangement intermediate between that of their HSV-1 and VZV counterparts. One of the EHV-1 subtype 2 genes apparently encodes a glycoprotein.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Keywords: Virology, Veterinary science
Date of Award: 1986
Depositing User: Enlighten Team
Unique ID: glathesis:1986-77441
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 14 Jan 2020 11:53
Last Modified: 14 Jan 2020 11:53

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