Inter- and intra-strain genomic variation in equine herpesvirus type 1 isolates

Summary Restriction enzyme digests of DNA from 22 unselected isolates of EHV-1 were analysed by hybridization with cloned DNA fragments covering the genome. In addition to a small amount of inter-strain variation, heterogeneity within strains was observed, caused by loss of specific restriction endonuclease sites in the DNA of a proportion of the virus particles of any one stock. Fifteen strains demonstrated the same intra-strain variation involving loss of the BamHI L-M site which was shown to lie within coding sequence for the large subunit of ribonucleotide reductase. This particular mutation may therefore be selected for by passage in RK13 cells.     

Purification of equine herpesvirus type 1.

A method for the purification of enveloped infectious equine herpesvirus type 1 (EHV-1) is presented. Virus from cell culture fluids harvested at 48 h post infection was concentrated by sedimentation and partially purified by differential precipitation with ammonium sulphate. The final steps of purification consisted of two cycles of flotation of virus in pre-formed CsCl density gradients. Yields of infectious virus were about 30% (18 to 44%) of that present in starting material. As judged by electron microscopy, mixed radioisotope labelling, and absence of phosphohydrolase, virus preparations possessed a high degree of purity. Sedimentation of EHV-1 into CsCl density gradients resulted in low recovery of infectious virus. Flotation of virus in CsCl gradients, however, was not deleterious to infectivity of viral preparations.     

Structure of the genome of equine herpesvirus type 1

The molecular structure of the genome of equine herpesvirus type 1 (EHV-1) was determined by restriction endonuclease mapping studies. Primary restriction enzyme digestion of purified EHV-1 DNA, either unlabeled, ³²P04 labeled, or [³H]TdR labeled, gave the following cleavage patterns: EcoRI yielded 17 fragments of 23.4 to 1.3 megadaltons (Mdalton); BglII, 16 fragments of 24.5 to 1.0 Mdalton; XbaI, 15 major fragments of 18.6 to 1.7 Mdalton; and BamHI,17 fragments of 13.7 to 2.8 Mdalton. Several fragments were present in 0.5 M amounts while all others were 1.0 M no 0.25 M fragments were detected. Secondary restriction enzyme digestion of these isolated fragments with various enzymes, analysis of terminal fragments using both the methods of λ 5′ exonuclease digestion and end labeling with polynucleotide kinase, and blot hybridization experiments with ³²P-labeled BamHI fragments indicated that this herpesvirus genome is a 92-Mdalton linear, double-stranded DNA molecule and is comprised of two segments designated as L (Long) and S (Short) which are 71.6 and 20.4 Mdalton, respectively. The 0.5 M fragments are located at the ends of the S region, an arrangement which allows the S region to invert relative to the L region; thus, two structural arrangements (isomers) of the genome exist. In addition, areas of heterogeneity were detected at the L terminus, within the S segment, and at a split variable locus in the L region.     

Synthesis and processing of equine herpesvirus type 1 glycoprotein 14

Glycoprotein 14 (gp14) of equine herpesvirus type 1 (EHV-1), the homolog of herpes simplex virus (HSV) glycoprotein B (gB), was investigated employing a panel of monoclonal antibodies to ascertain the regulatory class, rate of synthesis, and type of glycosylation of this polypeptide. Application of immunoprecipitation, Western blot, and SDS-PAGE analysis in conjunction with the use of metabolic inhibitors (cycloheximide, antinomycin D, phosphonoacetic acid, tunicamycin, and monensin), and time-course and pulse-chase experiments revealed the following information: (1) Three gp14-related polypeptides with molecular weights of 138 kilodaltons (K), 77-75K, and 55-53K are present in EHV-1-infected cell extracts. (2) All three species are synthesized in the presence of the DNA synthesis inhibitor phosphonoacetic acid although their synthesis is enhanced by DNA replication, indicative of a beta-gamma class molecule. (3) The 138K species is synthesized first as a precursor of the smaller species of gp14, the 77-75K and 55-53K forms. (4) Use of glycosylation inhibitors and digestion of immunoprecipitated gp14 with endoglycosidases indicate that the primary translation product is a 118K molecule which is cotranslationally glycosylated to the 138K form by the addition of high mannose oligosaccharides. (5) The 77-75K species contains both high mannose and hybrid oligosaccharides while the 55-53K form of gp14 contains some complex oligosaccharides. (6) In the absence of a reducing agent, the 138K polypeptide and a large 145K species are observed in both infected cell extracts and purified virions. Thus, EHV-1 gp14 appears to be synthesized as a large precursor molecule of 138K and is proteolytically cleaved to two smaller forms, 77-75K and 55-53K, which are linked by a disulfide bond(s) to form a 145K complex. This model of gp14 synthesis and maturation is similar to those proposed for a number of HSV gB equivalents found in the Alphaherpesvirnae.     

Identification of the envelope surface glycoproteins of equine herpesvirus type 1

The structural polypeptides of purified enveloped virions of the Army 183 strain of equine herpesvirus type 1 (EHV-1) were examined by different analytical techniques to identify the envelope glycoproteins. Glycoproteins were identified by electrophoretic analysis in polyacrylamide slab gels of virus labelled in vivo with [3H]glucosamine or labelled enzymically in vitro with either UDP-[14C]galactose or sodium [3H]borohydride. Fluorograms revealed eleven glycoproteins (mol. wt. 260000, 150000, 138000, 90000, 87000, 65000, 62000, 60000, 50000, 46000, and 24000). These glycoproteins probably correspond to virion protein (VP) 1-2, 9b, 10, 13, 14, 16, 17, 18, 21, 22a and 25 respectively, as designated in two other EHV-1 strains. In addition, a poorly resolved glucosamine-rich region (mol. wt. 250000 to 200000) corresponded to VP 3 to 8. The two isotopic surface labelling methods revealed that all the virus glycoproteins were exposed on the envelope surface.     

Detection of equine herpesvirus type 1 by real time PCR

A real-time PCR assay was developed for detection and quantitation of equid herpesvirus type 1 (EHV-1). The sensitivity of the assay was compared with an established nested-PCR (n-PCR). The real-time PCR detected 1 copy of target DNA, with a sensitivity 1 log higher than gel-based n-PCR. The assay was able to detect specifically EHV-1 DNA in equine tissue samples and there was no cross-amplification of other horse herpesviruses. Real-time PCR was applied to determine EHV-1 load in tissue samples from equine aborted fetuses. The high sensitivity and reproducibility of the EHV-1-specific fluorogenic PCR assay, combined with the wide dynamic range and the high throughput, make this method suitable for diagnostic and research applications.     

Properties of the genome of equine herpesvirus type 3

Methods developed for the isolation and purification of equine herpesvirus type 3 (EHV-3) DNA were shown to yield quantities of intact infectious DNA molecules suitable for characterization by physicochemical, biochemical, and electron microscopic methods. Preparations of EHV-3 DNA were shown by CsCl analytical ultracentrifugation to be comprised of a single species of DNA with a buoyant density of 1.727 g/cm³ which corresponds to a G + C content of 67.9%. Rate velocity centrifugation studies revealed that EHV-3 DNA has a sedimentation coefficient of approximately 55.4 S which corresponds to a molecular weight value of 90 to 100 megadaltons (Md). Intact viral DNA molecules were found to be 96 to 100 Md by electron microscopy, and restriction enzyme analyses with BamHI, EcoRI, and HindIII yielded an average molecular weight of 96.2 Md. Restriction enzyme and blot hybridization methods revealed that: (i) four 0.5 M EcoRI fragments were present, (ii) these four 0.5 M fragments shared significant homology, (iii) three EcoRI terminal fragments were identified and two of these were 0.5 M, and (iv) the two 0.5 M EcoRI end fragments hybridized only to one of the two 1.0 M terminal fragments identified for BamHI or HindIII. These findings indicated that a set of DNA sequences located at one terminus is repeated within the genome and that two populations of molecules exist with regard to the orientation of these repeat sequences. Electron microscopic examination of reannealed EHV-3 DNA molecules revealed structures that contained a single-strand loop equivalent to a duplex molecular weight of 5.3 Md at one end of the molecule contiguous to a double-strand region which terminated in a long single-strand segment. These structures were identical in morphology to those observed for EHV-1 DNA which has an overall genomic structure of an L (long) region covalently linked to an S (short) region; the S region consists of a unique segment (Us) bracketed by inverted repeat sequences (Henry et al., 1981; O'Callaghan et al., 1981). The above findings indicate that the 96.2-Md EHV-3 genome is comprised of an L region of approximately 73–76 Md covalently linked to a 20- to 23-Md S region; the S region contains a 5.3-Md Us segment bounded by 8-Md inverted repeat sequences that allow the S region to invert relative to the fixed L region and the genome to exist in two isomeric arrangements.     

Physical mapping of the genomic heterogeneity of isolates of equine herpesvirus 2 (equine cytomegalovirus)

Summary TheBamHI,EcoRI, andHindIII physical maps of the genomes of 14 isolates of equine herpesvirus 2 (EHV 2) were determined by Southern blot analysis using DNA fragments of a previously mapped EHV 2 strain 86/67. No two isolates had identical maps for all 3 enzymes, the number of differing cleavage sites between pairs of isolates varying from 3 to 21. Overall 75 cleavage sites were mapped, of which 40 were variable. Cleavage sites occurred throughout the genome, including within the terminal repeat regions. Additionally, fragment length polymorphisms, independent of cleavage site loss or gain, were mapped to 5 regions of the genome, 4 of which occurred within the terminal repeat regions.     

Structure of the genome of equine herpesvirus type 3

Restriction endonuclease mapping studies were performed to determine the molecular structure of the genome of equine herpesvirus type 3 (EHV-3). Purified EHV-3 DNA, either unlabeled or 32P-labeled, was analyzed using the restriction enzymes BamHI, BclI, BglII, EcoRI, and HindIII. The findings that four 0.5 M (molar) fragments were present, that two of these were terminal fragments, and that all 0.5 M fragments contained homologous DNA sequences as judged by DNA hybridization analyses indicated that DNA sequences located at one terminus are repeated within the molecule and that two populations of molecules exist with regard to the arrangement of this pair of shared sequences. Mapping of BamHI, BclI, BglII, EcoRI, and HindIII fragments by double digestion of intact EHV-3 DNA, reciprocal digestion of isolated restriction enzyme fragments, and blot hybridization experiments revealed that the EHV-3 genome is a linear, double-stranded DNA molecule with a molecular size of 96.2 +/- 0.48 MDa and is comprised of two covalently linked segments, designated L (long) and S (short). The S region is approximately 22.9 MDa in size and consists of a unique segment (Us) of approximately 5.8 MDa bracketed by 8.5 MDa inverted repeat sequences that allow the S region to invert relative to the fixed L region which is approximately 73.3 MDa in size and consists only of unique sequences. Thus, these data confirm that EHV-3 DNA exists in two isomeric forms and has a molecular structure similar to that of the genomes of EHV-1 (B. E. Henry, S. A. Robinson, S. A. Dauenhauer, S. S. Atherton, G. S. Hayward, and D. J. O'Callaghan, Virology 115, 97-114, 1981; D. J. O'Callaghan, G. A. Gentry, and C. C. Randall, "The Herpesvirus," Vol. 2, pp. 215-318, Plenum, New York, 1983; D. J. O'Callaghan, B. E. Henry, J. H. Wharton, S. A. Dauenhauer, R. B. Vance, J. Staczek, and R. A. Robinson, "Developments in Molecular Virology," Vol. 1, pp. 387-418, Nijhoff, The Hague, 1981; W. T. Ruyechan, S. A. Dauenhauer, and D. J. O'Callaghan, J. Virol., 42, 297-300, 1982), pseudorabies virus (W. Stevely, J. Virol., 22, 232-234, 1977; T. Ben-Porat, F. J. Rixon, and M. L. Blankenship, Virology, 95, 285-294, 1979), varicella-zoster virus (A. M. Dumas, J. L. Geelen, M. W. Weststrate, P. Wertheim, and J. Van Der Noordaa, J. Virol., 39, 390-400, 1981; S. E. Straus, H. S. Aulakh, W. T. Ruyechan, J. Hay, T. A. Casey, G. F. Vande Woude, J. Owens, and H. A. Smith, J. Virol., 40, 516-525, 1981.(ABSTRACT TRUNCATED AT 400 WORDS)     

Prevalence of equine herpesvirus type 1 latency detected by polymerase chain reaction

Summary.  In this study, an improved polymerase chain reaction (PCR) was used for detection of DNA of latent EHV-1 strains from several sources. Three pairs of oligonucleotide primers spanning fragments of 333 bp, 226 bp and 268 bp of the thymidine kinase (tk) gene, and one primer pair spanning 225 bp of the glycoprotein C (gC) gene were used in specific amplifications. Primers for EHV-4 PCR were also designed. Restriction digests with TaqI confirmed the identity of tk PCR fragments from EHV-1. The sensitivity to detect PCR products was further improved by visualisation in silver-stained acrylamide gels. PCR assays were applied to 267 samples including pools of tissue, peripheral blood leukocytes (PBL) and nasal swabs of archived, farms and abattoir specimens from a total of 116 animals. The EHV-1 DNA was found in 88% of the analysed samples. The prevalence of the EHV-1 latent or persistent form in adult horses was similar to others reports but found higher than previously described in foetuses and young foals. EHV-4 latency was not detected in the Brazilian studied specimens. Received September 13, 1999/Accepted April 11, 2000     

Detection of equine herpesvirus type 1 using a real-time polymerase chain reaction

Equid herpesvirus 1 (EHV1) is a major disease of equids worldwide causing considerable losses to the horse industry. A variety of techniques, including PCR have been used to diagnose EHV1. Some of these PCRs were used in combination with other techniques such as restriction enzyme analysis (REA) or hybridisation, making them cumbersome for routine diagnostic testing and increasing the chances of cross-contamination. Furthermore, they involve the use of suspected carcinogens such as ethidium bromide and ultraviolet light. In this paper, we describe a real-time PCR, which uses minor groove-binding probe (MGB) technology for the diagnosis of EHV1. This technique does not require post-PCR manipulations thereby reducing the risk of cross-contamination. Most importantly, the technique is specific; it was able to differentiate EHV1 from the closely related member of the Alphaherpesvirinae, equid herpesvirus 4 (EHV4). It was not reactive with common opportunistic pathogens such as Escherichia coli, Klebsiella oxytoca, Pseudomonas aeruginosa and Enterobacter agglomerans often involved in abortion. Similarly, it did not react with equine pathogens such as Streptococcus equi, Streptococcus equisimilis, Streptococcus zooepidemicus, Taylorella equigenitalis and Rhodococcus equi, which also cause abortion. The results obtained with this technique agreed with results from published PCR methods. The assay was sensitive enough to detect EHV1 sequences in paraffin-embedded tissues and clinical samples. When compared to virus isolation, the test was more sensitive. This test will be useful for the routine diagnosis of EHV1 based on its specificity, sensitivity, ease of performance and rapidity.     

Biological and biochemical properties of defective interfering particles of equine herpesvirus type 1.

Defective interfering (DI) particles of equine herpesvirus type 1(EHV-1) were purposely generated in an in vitro system of L-M cells by repeated high-multiplicity, serial, undiluted passage. Quantitation of infectious virus revealed a definite cyclic pattern which increased in magnitude with continued passage; additional experiments indicated that these fluctuations in virus titer were due to the presence of a population of DI particles as judged by interference assays. Attempts to separate standard and defective EHV-1 were unsuccessful. Analysis of DNA isolated from mixed populations of these virions revealed the presence of a high-density (H) variant DNA (? = 1.724 g/cm³) in addition to standard EHV-1 DNA (? = 1.716 g/cm³). Furthermore, it was found that the relative amount of this H-DNA in each passage corresponded very closely to the fluctuations in infectious virus titer. Sedimentation velocity studies of DNA isolated from populations of virions rich in H-DNA (>99%) indicated that the variant genomes were the same size as the standard EHV-1 genome (50–55 S). Comparisons of purified virion populations from 17 high-multiplicity passages with regard to particle counts, relative amount of H-DNA, and infectious virus titer indicated that the relative interference capacity of EHV-1 DI particles increased significantly with continued passage. Although the factor(s) responsible for the increased interference activity is unknown, DNA-DNA hybridization analyses of selected passages rich in H-DNA indicated that the genomes of EHV-1 DI particles became genetically less complex with passage and contained significant amounts of reiterated sequences. The possible mechanisms of the evolution of EHV-1 DI particles and their role in the interference process are discussed.     

Biological properties of equine herpesvirus type 1 DNA: transfectivity and transforming capacity.

A requirement for the classic complement pathway in opsonization of group B streptococci was observed by using both a chemiluminescence and a radiolabeled bacterial uptake technique. The classic pathway increased levels of opsonization for types Ia and II stock and wild strains and for some type III wild strains. In contrast, other type III wild strains and the type III stock strain had accelerated kinetics of uptake in the presence of an intact classic pathway, but the level of opsonization was unchanged from that with antibody alone. We could not demonstrate a significant role for the alternative pathway in opsonizing stock or wild strains of group B streptococci. Futhermore, electrophoretic and complement consumption analysis by hemolytic titration failed to reveal alternative pathway activation by the majority of strains of this group. Therapy aimed at supplying opsonins for these organisms will require the presence of type-specific antibody.     

Properties of an equine herpesvirus 1 mutant devoid of the internal inverted repeat sequence of the genomic short region

The 150 kbp genome of equine herpesvirus-1 (EHV-1) is composed of a unique long (UL) region and a unique short (Us) segment, which is flanked by identical internal and terminal repeat (IR and TR) sequences of 12.7 kbp. We constructed an EHV-1 lacking the entire IR (vL11ΔIR) and showed that the IR is dispensable for EHV-1 replication but that the vL11ΔIR exhibits a smaller plaque size and delayed growth kinetics. Western blot analyses of cells infected with vL11ΔIR showed that the synthesis of viral proteins encoded by the immediate-early, early, and late genes was reduced at immediate-early and early times, but by late stages of replication reached wild type levels. Intranasal infection of CBA mice revealed that the vL11ΔIR was significantly attenuated as mice infected with the vL11ΔIR showed a reduced lung viral titer and greater ability to survive infection compared to mice infected with parental or revertant virus.     

Analysis of the in vitro translation products of the equine herpesvirus type 1 immediate early mRNA

Equine herpesvirus type 1 (EHV-1) gene expression is coordinately regulated in an alpha, beta, gamma fashion. Viral alpha gene products include a 6.0-kb immediate early (IE) mRNA species (W. L. Gray et al., 1987, Virology 158, 79-87) and at least four closely related IE polypeptides (IEPs) (G.B. Caughman et al., 1985, Virology 145, 49-61). In this report, we describe results obtained from a series of in vitro translation experiments which were performed in an effort to characterize the IEPs and identify the mechanism by which individual IE protein species are generated. Our data indicate that a family of IEPs is generated in vitro from the 6.0-kb mRNA size class and that these IEPs correspond in overall size and antigenicity to those synthesized in infected cells. Using time-course/pulse-chase analyses, we show that production of three of the major IEPs [IE1' (193 kDa), IE3' (166 kDa), and IE4' (130 kDA)] occurs concomitantly, that none of these protein species can be chased completely into another, and that at least two additional minor species appear to be processed following synthesis. Finally, we show that the 6.0-kb mRNA species isolated during early or late stages of the infection cycle can be translated to yield all of the major IE proteins, indicating that production of the family of IEPs is not dependent upon accumulation of the IE mRNA which occurs during a cycloheximide blocked infection cycle. The implications of these findings are discussed as they relate to the origin and production of the IEPs both in vivo and in vitro.     

Sequence and organization of southern bean mosaic virus genomic RNA

The genomic RNA sequence of the cowpea strain of southern bean mosaic virus (SBMV-C) has been determined. The genome is 4194 nucleotides in length and has four open reading frames. A 5' proximal open frame, from base 49 to base 603, corresponds to the length of the P4 proteins translated in cell-free extracts from full-length and smaller virion RNA. The largest open frame extends from base 570 to base 3437 and encodes the two largest proteins translated in cell-free extracts from full-length virion RNA. Segments of this open reading frame's predicted amino acid sequence resemble those of known viral RNA polymerases, ATP-binding proteins, and viral genome-linked proteins. A third open frame extends from base 1895 to base 2380 and has not been correlated with an in vitro translation product. The fourth open reading frame is located in the 3' terminal region of the genome extending from base 3217 to base 4053. This frame encodes the SBMV capsid protein which is translated from subgenomic, virion RNA.