Experimental studies on equine herpesvirus type 1 infections.

The EHV-1 viruses of fetal origin grew better and had a wider tissue culture host range than those isolated from horses with respiratory diseases. Comparisons of a fetal isolate (F/304) and a respiratory disease isolate (R/NM-3) in partly immune horses showed that the F/304 virus infected horses more readily, grew better in the nasopharynx, was more likely to cause abortion, and was excreted to a greater extent into the environment.     

Detection of equine herpesvirus and differentiation of equine herpesvirus type 1 from type 4 by the polymerase chain reaction.

Although both equine herpesvirus type 1 (EHV-1) and equine herpesvirus type 4 (EHV-4) can be associated with respiratory disease, epizootics caused by EHV-1 are much more serious because the virus can cause abortions and paralysis. It is, therefore, important to identify the type of EHV involved in an outbreak by a test that is quick, sensitive, and reliable. We have adapted the polymerase chain reaction (PCR) to detect and distinguish between EHV-1 and EHV-4 in the same reaction. Primers for PCR were designed from the sequences of the glycoprotein B genes of EHV-1 and EHV-4. The PCR products derived from EHV-1 and EHV-4 were 135 and 326 base pairs, respectively, and could be readily separated by electrophoresis. The identity of the PCR products was confirmed by determining their nucleotide sequence, which agreed with the published sequence of the gB genes. The test could be performed directly on virus pelleted from small volumes (300 microL) of medium in which nasal swabs were transported and did not rely on the presence of infectious virus. The PCR was unaffected by conditions that reduced the infectivity of a virus preparation by 99%. The PCR detected EHV-4 in 5 of 10 nasal mucous samples taken from an outbreak of respiratory disease in race horses. Virus isolation in indicator cells was successful in detecting virus in four of the five samples positive by PCR.     

Nucleocapsid mass and capsomer protein stoichiometry in equine herpesvirus 1: scanning transmission electron microscopic study.

The Brookhaven scanning transmission electron microscope was employed to measure the masses of two nucleocapsid species (of light and intermediate densities) of equine herpesvirus 1. These were found to be 196.7 +/- 9.2 and 229.0 +/- 9.5 megadaltons (MDa), respectively. Biochemical assays showed that neither nucleocapsid contained any significant amount of DNA (less than 0.2% [wt/wt]). Taking into account data on protein composition, we conclude that the difference between their masses is essentially contributed by viral protein 22 (46 kDa), which is an integral component of the maturable intermediate nucleocapsid but not of the abortive light nucleocapsid. In view of earlier ultrastructural information on capsomer symmetry, our mass determinations are consistent only with the 150 hexavalent capsomers being hexamers of the 148-kDa major capsid protein.     

Genomic termini of equine herpesvirus 1.

After cell infection with the equine herpesvirus 1 (EHV-1), the termini of the linear double-stranded DNA genome fuse to form circular forms. To investigate the mechanisms in the generation and cleavage of such replicative-form DNAs, the genomic termini, the fusion of termini from replicative-form molecules, and the junction between the short and long genome segments have been analyzed by restriction mapping, blot hybridizations, cloning, and sequencing. The data suggest that the genome ends are not redundant and that the genomic termini are fused in replicative intermediates via 3' single-base extensions at the termini of the unique long segment (UL) and terminal repeat (TR). Adjacent to the EHV-1 termini are AT and gamma sequence elements highly conserved among different herpesviruses. We propose that both of these sequence elements are important for the cleavage of EHV-1 replicative forms.     

Functional mapping and DNA sequence of an equine herpesvirus 1 origin of replication.

The genome of equine herpesvirus 1 (EHV-1) defective interfering (DI) particle DNA originates from discrete regions within the standard (STD) EHV-1 genome: the left terminus (0.0 to 0.04 map units) and the inverted repeats (0.78 to 0.79 and 0.83 to 0.87 map units of the internal inverted repeat; 0.91 to 0.95 and 0.99 to 1.00 map units of the terminal inverted repeat). Since DI DNA must contain cis-acting DNA sequences, such as replication origins, which cannot be supplied in trans by the STD EHV-1 virus, regions of the EHV-1 genome shown to be in DI DNA were assayed for the presence of a viral origin of DNA replication. Specifically, STD EHV-1 DNA fragments encompassing the genomic regions present in DI particle DNA were inserted into the vector pAT153, and individual clones were tested by transfection assays for the ability to support the amplification and replication of plasmid DNA in EHV-1-infected cells. The Sma-1 subfragment of the internal inverted repeat sequence (0.83 to 0.85 map units) was shown to contain origin of replication activity. Subcloning and BAL 31 deletion analysis of the 2.35-kilobase-pair (kbp) Sma-1 fragment delineated a 200-bp fragment that contained origin activity. The origin activities of all EHV-1 clones which were positive by the transfection assay were confirmed by methylation analysis by using the methylation-sensitive restriction enzymes DpnI and MboI. DNA sequencing of the 200-bp fragment which contained an EHV-1 origin of replication indicated that this region has significant homology to previously characterized origins of replication of human herpesviruses. Furthermore, comparison of known origin sequences demonstrated that a 9-bp sequence, CGTTCGCAC, which is conserved among all origins of replication of human lytic herpesviruses and which is contained within the 18-bp region in herpes simplex virus type 1 origins shown by others to be protected by an origin-binding protein (P. Elias, M. E. O'Donnell, E. S. Mocarski, and I. R. Lehman, Proc. Natl. Acad. Sci. USA 83:6322-6326) is also conserved across species in the EHV-1 origin of replication.     

Isolation and partial characterization of equine herpesvirus type 1 in Czechia

Equine herpesvirus type 1 was determined as the etiological cause of an abortion storm in Czechia in 2003 after the virus strain was isolated from aborted fetus and identified by serological means and by PCR technique. Cloning and sequencing of the glycoprotein D confirmed the identity of the isolates and showed molecular relationships to known EHV-1 strains. Comparison of glycoprotein D sequences with corresponding sequence of EHV-1 reference strains (Kentucky-A and Ab1) revealed high nucleotide homology. The Czech isolate of EHV-1 virus does not differ significantly from the Ab1 strain regarding the glycoprotein D gene and does not bear the frameshift in the 3' terminus which occurs in the Kentucky-A strain.     

Biochemical transformation of deoxythymidine kinase-deficient mouse cells with UV-irradiated equine herpesvirus type 1.

A line of 3T3 mouse cells lacking deoxythymidine kinase (dTK-) was stably transformed to the dTK+ phenotype after exposure to UV-irradiated equine herpesvirus type 1 (EHV-1). Biochemical transformants were isolated in a system selective for the dTK+ phenotype (Eagle minimal essential medium containing 10(-4) M hypoxanthine, 6 X 10(-7) M aminopterin, and 2 X 10(-5) M deoxythymidine). Transformation was accompanied by the acquisition of a dTK activity with immunological, electrophoretic, and biochemical characteristics identical to those of the dTK induced by EHV-1 during productive infection. The transformed cells have been maintained in selective culture medium for more than 50 passages and have retained the capacity to express EHV-1--specific antigens. Spontaneous release of infectious virus has not been detected in the transformed lines, and the the cells were not oncogenic for athymic nude mice. In contrast to normal dTk+ 3T3 cells, EHV-1 transformants were unable to grow in the presence of arabinosylthymine, a drug selectively phosphorylated by herpesvirus-coded dTK's. These results indicate that a portion of the EHV-1 genome is able to persist in the transformed cells for many generations and be expressed as an enzymatically active viral gene product.     

Electron microscopic study of alpha-actinin.

Electron microscopic studies of the structure of purified α-actinin alone and in complex with F-actin have determined the molecular shape and size of this protein. α-Actinin molecules represent rods of about 300 Å in length and about 20 Å in diameter.     

Genetic relatedness of equine herpesvirus types 1 and 3.

The genetic relatedness of two types of equine herpesviruses (EHVs), 1 (EHV-1) and 3 (EHV-3), was determined by DNA-DNA reassociation kinetics. Denatured, labeled viral DNA probe was allowed to reassociate in the presence or absence of the second unlabeled viral DNA. The initial rate of reassociation of either labeled viral DNA was increased by the presence of the heterologous viral DNA to an extent indicating only 2 to 5% homology between the two EHV genomes. Moreover, labeled RNA extracted from EHV-3-infected cells hybridized to filter-immobilized EHV-1 DNA only 2 to 3 percent as efficiently as to the homologous EHV-3 DNA. These results demonstrate that the genital (EHV-3) and nongenital (EHV-1) types of EHVs exhibit very little genetic homology.     

DNA condensation with polyamines. II. Electron microscopic studies

Approximately 75% of the wheat and rye genomes consist of repeated sequence DNA. Three-quarters of the non-repeated or few copy sequences in wheat are less than 1000 base-pairs long, whilst in rye approximately half of the non-repeated or few copy sequences are in this size class. Most of the remaining non-repeated or few copy sequences appear to be a few thousand base-pairs long.In this paper a somewhat novel approach has been used to quantitatively analyse the linear organisation of the large proportion of repeated sequence DNA as well as the non-repeated DNA in the wheat and rye genomes. Repeated sequences in the genomes of oats, barley, wheat and rye have been used as probes to distinguish and isolate four different groups of repeated sequences and their neighbouring sequences from the wheat and rye genomes. Radioactively labelled wheat or rye DNA fragments ranging from 200 to over 9000 nucleotides long were incubated separately with large excesses of denatured unlabelled oats, barley, wheat and rye DNAs to C_ot values which enable all the repeated sequences of the unlabelled DNA to renature. The following parameters were then determined from the proportions of total labelled DNA in fragments which had at least partially renatured. (1) The proportions of the repeated sequences in the labelled DNAs that were able to hybridise to each unlabelled DNA; (2) the mean distance apart of the hybridising sequences on the longer labelled fragments; and (3) the proportion of the genome in which the hybridising sequences were concentrated. Analysis of these results, together with those of separate experiments designed to quantitatively estimate the nature of sequences unable to reanneal with the repeated sequences of each of the probe DNAs, have enabled schematic maps to be drawn which show how the repeated and non-repeated sequences are arranged in the wheat and rye genomes.Both genomes are constructed from millions of relatively short sequences, most of them considerably shorter than 3000 base-pairs. This structure was recognised because adjacent sequences can be distinguished by their frequency of repetition (i.e. repeated or non-repeated) or by their evolutionary origin. Approximately 40 to 45% of the wheat genome and 30 to 35% of the rye genome consists of short non-repeated sequences interspersed between short repeated sequences. Approximately 50% of the wheat genome and 60% of the rye genome consists of tandemly arranged repeated sequences of different evolutionary origins. It is postulated that much of this complex repeated sequence DNA could have arisen from amplification of compound sequences, each containing repeated and non-repeated sequence DNA.Short repeated sequences with a number average length of around 200 base-pairs and which occupy about 20% of the wheat and rye genomes are related to repeated sequences also found in oats and barley. They are concentrated in 60 to 70% of the wheat and rye genomes, being interspersed with different short repeated sequences and a significant proportion of the short non-repeated sequences.Rye chromosomes contain more DNA than wheat chromosomes. This is principally, but not entirely, due to additional repeated sequence DNA. Many quantitative changes appear to have occurred in both genomes, possibly affecting most families of repeated sequences, since wheat and rye diverged from a common ancestor. Both species contain species-specific repeated sequences (24% of rye genome; 16% of wheat genome) but a large proportion of these are closely interspersed with repeated sequences found in both genomes.     

Electron microscopic visualization of restriction sites on DNA molecules.

DNA molecules were adsorbed to a polylysine-treated carbon film and digested directly on the film by restriction enzymes. After washing the film with 1 M NaCl, 0.4% Kodak Photo-Flo and 9% formamide, each cleavage site introduced was visualized as a gap under the electron microscope. By measuring the gapped positions on linear DNA molecules induced by other enzymes, a single EcoRI site on a lambda dv1 molecule and three HinHI sites on an fd1RF molecule were mapped at the positions expected from the cleavage maps, respectively. This electron-microscopic procedure may be useful for the construction of a cleavage map.     

Electron microscopic studies of bacteriophage M13 DNA replication.

Intracellular forms of M13 phage DNA isolated after infection of Escherichia coli with wild-type phage have been studied by electron microscopy and ultracentrifugation. The data indicate the involvement of rolling-circle intermediates in single-stranded DNA synthesis. In addition to single-stranded circular DNA, we observed covalently closed and nicked replicative-form (RF) DNAs, dimer RF DNAs, concatenated RF DNAs, RF DNAs with single-stranded tails (theta, rolling circles), and, occasionally, RF DNAs with theta structures. The tails in theta molecules are always single stranded and are never longer than the DNA from mature phage; the proportion of theta to other RF molecules does not change significantly with time after infection. The origin of single-stranded DNA synthesis has been mapped by electron microscopy at a unique location on RF DNA by use of partial denaturation mapping and restriction endonuclease digestion. This location is between gene IV and gene II, and synthesis proceeds in a counterclockwise direction on the conventional genetic map.     

Electron microscopic study of troponin

Troponin and its components or fragments were observed in an electron microscope by the use of the rotary shadowing technique. In freshly prepared troponin with low viscosity, globular particles were mainly observed. The size of the long axis of the particles was 13.2 +/- 1.3 nm and the size perpendicular to the long axis was 9.5 +/- 1.2 nm. The mean axial ratio was 1.4 +/- 0.3. Most of the particles observed in a stored troponin preparation, having a higher viscosity than that of fresh troponin, had a globular head with a thin tail, with the total length of 25.4 +/- 1.4 nm (head-tail type particles). The axial size of the globular portion was 8.3 +/- 1.2 nm and the tail length was 17.1 +/- 1.6 nm. Observation of various particles during the transitional stages indicated that, in the globular particles, the tail region of head-tail type particle was associated along the globular head region. Troponin T was a filamentous particle with 16.9 +/- 1.5 nm length. The 26K fragment of troponin T, which was devoid of the N-terminal 45 residues from troponin T, was a filamentous particle with the length of 14.4 +/- 1.3 nm. Troponin T1, one of two chymotryptic subfragments of troponin T, was a filamentous particle of 11.6 +/- 1.4 nm length. Troponin C.T in the presence of Ca2+ was a particle with a globular head (7 nm in size) and a tail of about 17 nm length. The Fab fragment of anti-troponin T1 formed regular transverse striations along the thin filament of rabbit skeletal muscle with a 38 nm period.(ABSTRACT TRUNCATED AT 250 WORDS)     

Electron microscopic study of DNA compactization under the effect of putrescine]

DNA-putrescine complexes were studied by electron-microscopy with the use of protein-free method. The latter gives the opportunity to investigate the interaction of DNA molecules spread on the surface layer of hypophase and the polyamine molecules in the thick layer of hypophase. Polyamine concentration varied from 5 x 10(-4) mM to 5 x 10(-1) mM. Under the low concentration of putrescine the complexes are represented by agglomerations of kinked knobbed fibres 10 to 20 nm thick, consisting of several fibres of duplex DNA. Upon increasing of putrescine concentration from 5 x 10(-4) to 1.5 x 10(-1) mM, the fibres become more thick (up to 25 nm), highly twisted and have the appearance of cylinders. Very often in the composition of complexes, it is possible to encounter the circular structures, which were formed at the expense of intermolecular interaction of different parts of the complex. The circular structures can serve as "embryos" of toroids of different sizes, that is of different degree of saturation with DNA and putrescine. At the concentration of putrescine 5 x 10(-1) mM the complexes have the appearance of toroids and structures on the basis of toroids, cylinders. The scheme of possible transitions of fibres of various thickness is proposed. The regularities of the compactization process, stimulated by polyamines, don't depend on the degree of compactization (the thickness of compacting fibre), that is they are similar for duplex DNA and for the fibres 25 nm thick, consisting of dozens of DNA molecules.     

Regulatory function of the equine herpesvirus 1 ICP27 gene product.

The UL3 protein of equine herpesvirus 1 (EHV-1) KyA strain is a homolog of the ICP27 alpha regulatory protein of herpes simplex virus type 1 (HSV-1) and the ORF 4 protein of varicella-zoster virus. To characterize the regulatory function of the UL3 gene product, a UL3 gene expression vector (pSVUL3) and a vector expressing a truncated version of the UL3 gene (pSVUL3P) were generated. These effector plasmids, in combination with an EHV-1 immediate-early (IE) gene expression vector (pSVIE) and chimeric EHV-1 promoter-chloramphenicol acetyltransferase (CAT) reporter constructs, were used in transient transfection assays. These assays demonstrated that the EHV-1 UL3 gene product is a regulatory protein that can independently trans activate the EHV-1 IE promoter; however, this effect can be inhibited by the repressive function of the IE gene product on the IE promoter (R. H. Smith, G. B. Caughman, and D. J. O'Callaghan, J. Virol. 66:936-945, 1992). In the presence of the IE gene product, the UL3 gene product significantly augments gene expression directed by the promoters of three EHV-1 early genes (thymidine kinase; IR4, which is the homolog of HSV-1 ICP22; and UL3 [ICP27]) and the promoter of the EHV-1 late gene IR5, which is the homolog of HSV-1 US10. Sequences located at nucleotides -123 to +20 of the UL3 promoter harbor a TATA box, SP1 binding site, CAAT box, and octamer binding site and, when linked to the CAT reporter gene, are trans activated to maximal levels by the pSVIE construct in transient expression assays. Results from CAT assays also suggest that the first 11 amino acids of the UL3 protein are not essential for the regulatory function of the UL3 gene product.     

Electron microscopic study of Bacillus subtilis protoplast fusion.

When protoplasts derived from sporulating cells of Bacillus subtilis were fused by exposure to polyethylene glycol (PEG) and fixed immediately thereafter, protoplasts with two enclosed prespores could be seen by electron microscope. The number of fusion events was greatly increased, and multiply fused protoplasts appeared, when the PEG-treated suspension was diluted in hypertonic broth and reincubated before fixation. This post-PEG incubation effect is taken to indicate a fusion mechanism of two steps: a short, PEG-dependent step of membrane activation, followed by a slow, metabolism-requiring step completing fusion. When prespore-bearing protoplasts from two genetically different strains were mixed and fused, the extent of fusion could also be followed by counting clones of recombinant bacteria. Maximal from the start, their number (1% of each parent type protoplast present) was unaffected by post-PEG incubation. Fusion in this case is apparently completed after plating on the wall-regeneration medium. After optimal post-PEG incubation, the majority of the protoplasts were seen to participate in fusion, and the cytological fusion observed, corrected for wall-regeneration frequency, accounted quantitatively for the prototrophic bacteria eventually recovered. These results are in good agreement with those obtained independently by Sanchez-Rivas and Garro (J. Bacteriol. 137:1340--1345, 1979).     

Electron microscopic analysis of partially replicated bacteriophage T7 DNA.

Partially replicated bacteriophage T7 DNA was isolated from Escherichia coli infected with UV-irradiated T7 bacteriophage and was analyzed by electron microscopy. The analysis determined the distribution of eye forms and forks in the partially replicated molecules. Eye forms and forks in unit length molecules were aligned with respect to the left end of the T7 genome, and segments were scored for replication in each molecule. The resulting histogram showed that only the left 25 to 30% of the molecules was replicated. Several different origins of DNA replication were used to initiate replication in the UV-irradiated experiments in which 32P-labeled progeny DNA from UV-irradiated phage was annealed with ordered restriction fragments of T7 DNA (K. B. Burck and R. C. Miller, Jr., Proc. Natl. Acad. Sci. U.S.A. 75:6144--6148, 1978). Both analyses support partial-replica hypotheses (N. A. Barricelli and A. H. Doermann, Virology 13:460--476, 1961; Doermann et al., J. Cell. comp. Physiol. 45[Suppl.]:51--74, 1955) as an explanation for the distribution of marker rescue frequencies during cross-reactivation; i.e., replication proceeds in a bidirectional manner from an origin to a site of UV damage, and those regions of the genome which replicate most efficiently are rescued most efficiently by a coinfecting phage. In addition, photoreactivation studies support the hypothesis that thymine dimers are the major UV damage blocking cross-reactivation in the right end of the T7 genome.     

An improved cosmid vector for the cloning of equine herpesvirus DNA

We have modified the commercial cosmid vector, triple helix vector (THV), such that I-Sce-I restriction endonuclease sites flank the cloning site. I-Sce-I is a rare-cutting endonuclease which recognizes an 18-bp sequence. It does not restrict the genome of either of the equine herpesvirus 1 or 4 (EHV-1 and EHV-4) strains we have cosmid cloned. Thus, cosmid- cloned EHV fragments can be excised intact from the vector by I-Sce-I digestion, facilitating production of large overlapping EHV fragments for use in transfections to produce recombinant virus.