Equine herpes virus type 1 (EHV-1) belongs to an extensive family of large, genetically complex, and medically important animal viruses. The virion consists of an icosahedral nucleocapsid (T=16) separated from the viral envelope by a proteinaceous tegument layer. Assembly occurs in the nucleus of infected cells where capsids assemble, are packaged with DNA, then bud through the nuclear membrane. Two morphological species of EHV-1 capsids have been distinguished: “lights” which are abortive particles incapable of packaging DNA and “intermediates” which are precursors in the assembly of mature virions. Purified “intermediates” contain an additional proteinP22 (46 kDa), which is not present in “lights” and accounts for ˜10% of the total particle mass. In order to characterize the capsid structures of the two particle types and explore differences between them, we have applied three-dimensional reconstruction techniques to electron micrographs of frozen-hydrated specimens.The Kentucky A strain of EHV-1 was propagated in L-929 cells, and the two types of capsids were obtained as separate fractions from Renografin-76 density gradients. Neither fraction contained any significant amount of DNA. Cryo-electron microscopy of capsids on carbon film substrates was performed as described earlier. Micrographs were recorded at a nominal magnification of x36,000 at 2-3μm underfocus and digitized with a 50/μn step size (˜1.38nm sampling at the specimen). Three-dimensional reconstructions of the two particle types were computed to 4.5nm resolution using modified “common-lines” procedures. Images of 37 “light” and 39 “intermediate” particles were separately combined to compute three-dimensional density distributions.

Background

Alpha-tocopherol (α-TP) supplementation is recommended for the prevention of various equine neuromuscular disorders. Formulations available include RRR-α-TP acetate powder and a more expensive but rapidly water-dispersible liquid RRR-α-TP (WD RRR-α-TP). No cost-effective means of rapidly increasing serum and cerebrospinal fluid (CSF) α-TP with WD RRR-α-TP and then sustaining concentrations with RRR-α-TP acetate has yet been reported.

Objectives

To evaluate serum, CSF and muscle α-TP concentrations in an 8-week dosing regimen in which horses were transitioned from WD RRR-α-TP to RRR-α-TP acetate.

Study design

Non-randomised controlled trial.

Methods

Healthy horses with serum α-TP of <2 μg/mL were divided into three groups and followed for 8 weeks. In the control group (n = 5), no α-TP was administered. In the second group (Group A; n = 7), 5000 IU/day RRR-α-TP acetate was administered. In the third group (Group WD-A; n = 7), doses of 5000 IU/day of WD RRR-α-TP were administered over 3 weeks, followed by a 4-week transition from WD RRR-α-TP to RRR-α-TP acetate, and a final 1 week of treatment with RRR-α-TP acetate. Serum samples were obtained weekly; muscle biopsies were obtained before, at 2.5 weeks and after supplementation. CSF samples were obtained before and after the 8-week period of supplementation.

Results

Serum α-TP increased significantly in Group WD-A at week 1 and remained significantly higher than in Group A and the control group throughout the transition, with inter-individual variation in response. Serum α-TP increased significantly by week 7 in Group A. CSF α-TP increased significantly in Group WD-A only. Muscle α-TP concentrations did not differ significantly across groups. Serum and CSF α-TP were closely correlated (r = 0.675), whereas serum and muscle-α-TP concentrations were not correlated.

Main limitations

The study duration was short and data on pre-transition CSF was lacking.

Conclusions

The administration of 5000 IU/day of water-dispersible RRR-α-TP rapidly increases serum α-TP. Serum and CSF α-TP concentrations are sustained with a gradual transition to 5000 IU/day of RRR-α-TP acetate. Periodic evaluation of serum α-TP concentrations is recommended because responses vary among individuals.

Cryoelectron microscopy and three-dimensional computer reconstruction techniques have been used to compare the structures of two types of DNA-free capsids of equine herpesvirus 1 at a resolution of 4.5 nm. "Light" capsids are abortive, whereas "intermediate" capsids are related to maturable intracellular precursors. Their T = 16 icosahedral outer shells, approximately 125 nm in diameter, are indistinguishable and may be described in terms of three layers of density, totalling 15 nm in thickness. The outermost layer consists of protruding portions of both the hexon and the penton capsomers, rising approximately 5 nm above a midlayer of density. The innermost layer, or "floor," is a 4-nm-thick sheet of virtually continuous density except for the orifices of the channels that traverse each capsomer. Hexon protrusions are distinctly hexagonal in shape, and penton protrusions are pentagonal. The structures of the three kinds of hexons (distinguished according to their positions on the surface lattice) are closely similar but differ somewhat in their respective orientations and in the shapes of their channels. The most prominent features of the midlayer are threefold nodules ("triplexes") at the trigonal lattice points. By analogy with other viral capsids, the triplexes may represent trimers of another capsid protein, possibly VP23 (36 kilodaltons [kDa]) or VP26 (12 kDa). Intermediate capsids differ from light capsids, which are empty, in having one or more internal components. In individual images from which the shell structure has been filtered away, these components are seen to have dimensions of 20 to 30 nm but to lack a visible substructure. This material--which is smeared out in the reconstruction, implying that its distribution is not icosahedrally symmetric or necessarily consistent from particle to particle--consists of aggregates of VP22 (46 kDa). From several lines of evidence, we conclude that this protein is located entirely within the capsid shell. These aggregates may be the remnants of morphogenetic cores retained in capsids interrupted in the process of DNA packaging.

The structures of bovine papillomavirus type 1 (BPV-1) and human papillomavirus type 1 (HPV-1) were determined at 2.5 nm resolution by cryoelectron microscopy and three dimensional image reconstruction techniques. As expected, the reconstructions showed that both viruses consist of a T = 7 icosahedral capsid (approximately 60 nm in diameter) which surrounds a nucleohistone core. The capsid morphologies of the two viruses are nearly indistinguishable. Each capsid consists of a shell layer (approximately 2 nm thick) of nearly continuous density from which capsomers project radially to a maximum height of approximately 5.8 nm. The five-coordinate (pentavalent) and six-coordinate (hexavalent) capsomers both exhibit distinct five-fold axial symmetry as was observed for SV40 and polyoma viruses. Thus, both genera (papilloma and polyoma) of the papovavirus family have now been shown to have the characteristic "all-pentamer" capsid construction. BPV-1 and HPV-1 capsomers consist of a thick (8.6 nm diameter) trunk that broadens distally to form a regular five-pointed, star-shaped head, and proximally to create the shell layer where capsomers associate. A cylindrical channel (approximately 2.8 nm diameter) extends along the axis of each capsomer from the interior of the virus to a point approximately half way to the capsomer surface. Computationally sectioned views of individual capsomers displayed at decreasing radii show that each of the five capsomer subunits (in both pentavalent and hexavalent capsomers) makes a pronounced (30 degrees) left-handed twist just above the outer surface of the capsid shell. Similar views of the reconstructions also clarify the morphology of intercapsomer contacts. For example, they show how hexavalent capsomers coordinate six neighboring capsomers despite the fact that they contain only five subunits. The system of intercapsomer contacts is indistinguishable in BPV-1 and HPV-1, but quite different from that reported for polyoma virus capsids assembled in vitro from the major capsid protein, VP1 (D. M. Salunke, D. L. D. Caspar, and R. L. Garcea. 1989. Biophys. J. 56:887-900). Thus, because both polyoma and papilloma viruses have all-pentamer capsids, it appears that intracapsomer subunit-subunit interactions which stabilize pentameric capsomers are better preserved evolutionarily than those involved in capsomer-capsomer contacts.

The organization of DNA within the HSV-1 capsid has been determined by cryoelectron microscopy and image reconstruction. Purified C-capsids, which are fully packaged, were compared with A-capsids, which are empty. Unlike A-capsids, C-capsids show fine striations and punctate arrays with a spacing of approximately 2.6 nm. The packaged DNA forms a uniformly dense ball, extending radially as far as the inner surface of the icosahedral (T = 16) capsid shell, whose structure is essentially identical in A-capsids and C-capsids. Thus we find no evidence for the inner T = 4 shell previously reported by Schrag et al. to be present in C-capsids. Encapsidated HSV-1 DNA closely resembles that previously visualized in bacteriophages T4 and lambda, thus supporting the idea of a close parallelism between the respective assembly pathways of a major family of animal viruses (the herpesviruses) and a major family of bacterial viruses.