Analysis of equine herpesvirus 2 strain variation using monoclonal antibodies to glyucoprotein B.

The antigenic relationships of four genomically divergent strains of equine herpesvirus 2 (EHV2.86/67, EHV2.5FN, EHV2.141 and EHV2.T-2) and equine herpesvirus 5 (EHV5) were examined in ELISA using a panel of EHV2.86/67 gB-specific MAbs. EHV2.86/67 and EHV2.5FN were shown to be more similar to each other than to EHV2.T-2, EHV2.141 or EHV5. Seven of nine EHV2.86/67 gB specific MAbs tested in serum neutralisation assays were shown to neutralise EHV2.86/67 and EHV2.5FN but not EHV2.141, EHV2.T-2 or EHV5. The complete nucleotide and deduced amino acid sequences of EHV2.86/67, EHV2.5FN, EHV2.141 and EHV2.T-2 gB were compared and contrasted with each other and with EHV5 gB. The four EHV2 strains were 94-96% similar at the amino acid level and variability in amino acid sequence mapped to three mains sites designated I, II and III. By contrast, the four EHV2 strains were 77-79% similar to EHV5 gB at the amino acid level. The epitope of these seven gB specific neutralising MAbs has been previously mapped to amino acids 29-74 of EHV2 gB and examination of the deduced amino acid sequence of the four sequenced strains localised the epitope of the seven MAbs to amino acids 30 to 49 located within Site I. Six other divergent strains of EHV2 were examined for variability at Site I using DNA sequencing. Examination of the deduced amino acid sequences of all ten EHV2 strains tested indicated, that based on the epitope of the neutralising MAbs the EHV2 strains formed two distinct antigenic groups, EHV2.86/67-like and EHV2.141-like. EHV5 gB showed divergence from all of the EHV2 gB sequences between amino acids 29-74.

Identification, sequence analysis and characterisation of equine herpesvirus 5 glycoprotein B.

The complete nucleotide sequence of the gammaherpesvirus equine herpesvirus 5 (EHV5) glycoprotein B (gB) was determined and the deduced amino acid sequence compared with that of the second equine gammaherpesvirus EHV2. EHV5 gB is an 870 amino acid protein and is 79% similar and 66% identical with EHV2 gB at the amino acid level. EHV5 gB like EHV2 gB is a disulphide linked heterodimer with subunits of 92 and 68 kDa. EHV5 gB is an integral membrane glycoprotein containing only N-linked oligosaccharides and contains a putative endoproteolytic cleavages site at amino acids 422-485. The EHV5 gB amino acid sequence showed greatest homology with other members of the Rhadinovirus genus of the subfamily Gammaherpesvirinae. Alignment of EHV5 gB sequence with the gB sequence of seven other gammaherpesviruses showed conservation of 10 cysteine residues as well as conservation of three predicted sites of N-linked glycosylation; the highest degree of conservation of the predicted sites of N-linked glycosylation was observed between EHV5 and the other members of the Rhadinovirus genus. Phylogenetic analysis confirmed EHV2 and EHV5 were most closely related to each other and equally distant from other members of the Rhadinovirus genus included in the analysis.

Genetic content of a 20.9 kb segment of human herpesvirus 6B strain Z29 spanning the homologs of human herpesvirus 6A genes U40-57 and containing the origin of DNA replication.

A continuous 20.9 kb sequence from human herpesvirus 6 variant B (HHV-6B) strain Z29 (GenBank accession number L16947) is genetically colinear with a discrete segment of the human cytomegalovirus (HCMV) UL region and with HHV-6 variant A (HHV-6A). Short nucleotide sequence determinations at multiple sites within an 8.5 kb region immediately 3' to the 20.9 kb contig revealed additional colinearity between HHV-6B, HCMV and HHV-6A. Homology studies with the predicted peptide sequences from 11 complete and 12 partial HHV-6B open reading frames (ORFs) revealed that most encode proteins conserved to varying degrees in all previously sequenced primate herpesviruses. HHV-6B homologs were identified for the HSV-1 ICP18.5, ICP8, UL52, UL24, UL25 and major capsid protein. Several HHV-6B proteins had limited amino acid similarity to their positional homologs in other herpesviruses. Each gene identified is highly homologous to its HHV-6A counterpart, including two unique HHV-6 genes predicted to encode membrane-associated glycoproteins. However, two regions of substantial divergence were noted, one spanning the origin of replication and the other encoding one of the putative HHV-6-specific glycoprotein genes. Substitutions in the latter region lead to predicted differences in reading frames and protein lengths among HHV-6 isolates.

Comparison of a 20 kb region of human herpesvirus 6B with other human beta herpesviruses reveals conserved replication genes and adjacent divergent open reading frames.

The sequence of a 20.15 kb region from human herpesvirus 6 variant B (HHV-6B) strain Z29 is described (GenBank accession number L14772). Determinations of protein homologies for seventeen predicted gene products revealed HHV-6B homologs of six proteins well-conserved both in genetic context and amino acid sequence throughout the alpha-, beta-, and gammaherpesvirus subfamilies. These include proteins involved in viral DNA replication, packaging and nucleotide metabolism, and conserved proteins of undefined function. The close evolutionary relationship of the human betaherpesviruses, HHV-6B, HHV-6A, HHV-7 and human cytomegalovirus (HCMV) was confirmed by identification of several protein sequences encoded only by these viruses, including homologs of the HCMV early phosphoprotein family and a series of HCMV open reading frames predicted to encode glycoprotein exons. Homologs of essential HSV-1 replication proteins, UL8 and UL9, were also identified. Downstream from the conserved replication locus, each betaherpesvirus contains a region of divergent, small open reading frames. The evolution of this region and its potential use in the development of a viral vector system are discussed.

Restriction endonuclease mapping and molecular cloning of the human herpesvirus 6 variant B strain Z29 genome.

Human herpesvirus 6(HHV-6) variants A and B differ in cell tropism, reactivity with monoclonal antibodies, restriction endonuclease profiles, and epidemiology. Nonetheless, comparative nucleotide and amino acid sequences from several genes indicate that the viruses are very highly conserved genetically, The B variant is the major etiologic agent of exanthem subitum and is frequently isolated from children with febrile illness; no disease has been etiologically associated with HHV-6A. One HHV-6A strain has been cloned and sequenced, but similar information and reagents are not available for HHV-6B. We report here the determination of maps of the restriction endonuclease cleavage sites for BamHI, C1aI, HindIII, KpnI, and Sa1I, and the cloning in plasmids and bacteriophages of fragments representing over 95% of the HHV-6B strain Z29 [HHV-6B(Z29)] genome. Hybridization experiments and orientation of several blocks of nucleotide sequence information onto the genomic map indicate that HHV-6A and HHV-6B genomes are colinear.

Avian cardiac tropomyosin gene produces tissue-specific isoforms through alternative RNA splicing.

We have isolated a quail cardiac tropomyosin gene which encodes three distinct isoforms through the use of alternative exon splicing. Characterization of cDNA clones produced by this gene indicate that the gene encodes a unique 284 amino acid cardiac tropomyosin isoform, along with a 248 amino acid cytoskeletal and 284 amino acid smooth muscle isoforms. Northern analyses indicate that the gene is primarily expressed in cardiac muscle, with only minor expression of the cytoskeletal and smooth muscle transcripts.

Properties of the human herpesvirus 6 strain Z29 genome: G + C content, length, and presence of variable-length directly repeated terminal sequence elements.

We have studied the structure of the human herpesvirus 6 (HHV-6) genome. The density of genomic DNA is approximately 1.702 g/cm3 as determined by isopycnic density gradient centrifugation, from which a mean G + C content of 43% was calculated. The genomic termini were examined by exonuclease digestion and DNA/DNA hybridization; relative molarities of restriction fragments were determined by quantitative densitometry. The results indicate that the HHV-6(Z29) genome has two unique termini and consists of a long unique segment bounded by a directly repeated sequence element found in one copy at each end of the genome. We estimated the length of the genome by pulsed-field gel electrophoresis and by summation of restriction endonuclease fragment lengths. We observed two forms of HHV-6(Z29) DNA of approximately 162 and 168 kb in length. The length heterogeneity was localized within the terminal repeat element, each copy of which is approximately 10.1 kb in length in the shorter form of the genome and 13.2 kb in length in the longer form of the genome.

Avian tropomyosin gene expression.

Sequence analysis of overlapping fragments from a quail genomic library has revealed a tropomyosin gene consisting of 13 exons spaced over about 18 kilobase pairs of DNA. Skeletal muscle and smooth muscle transcripts share the same 5' untranslated sequence and may initiate from the same promoter. However, the regions encoding amino acids 39-80 and 258-284 are specific to each muscle type. The two sets of exons encoding these regions undergo mutually exclusive alternative splicing in a tissue-specific manner as determined by Northern blots and S1-nuclease protection. Similarly, the 3' ends of the transcripts are different in skeletal muscle and smooth muscle, and each contains two polyadenylation signals which appear to be utilized in vivo. The avian alpha-tropomyosin gene is not expressed in cardiac muscle. The sequence of the gene shows great homology with other muscle-specific tropomyosins and includes a region homologous to the amino terminus of nonmuscle tropomyosins.