Detection of viral genome in non-neural tissues of cattle experimentally infected with bovine herpesvirus 1

Various bone disorders become manifest as cystic lesions. The differential diagnosis must include benign and malignant tumors and also non-tumorous lesions, such as osteomyelitis. The most important and most frequent types of genuine bone cyst are juvenile bone cyst and aneurysmal bone cyst. When juvenile bone cysts occur in adults they are called solitary bone cysts. Despite intensive research the pathogenesis of bone cysts is still unknown to this day, so that successful causal therapy is impossible. The main problem in the treatment of bone cysts is their high rate of recurrence, rates ranging between 20% and 50% having been cited in the international literature. A critical review of the literature reveals few publications with helpful follow-up results. Most of the publications are case reports, and they frequently merely describe various forms of treatment. More recent reports are mainly concerned with such methods as curettage, steroid injections, and continuous decompression with perforated screws. Until the early 1980s, segmental bone resection was the treatment of choice. Because of its high complication rate it has since been abandoned. In the last analysis, the only well-established method for which long-term results obtained in studies of any size have been published, is curettage of the cyst and grafting with cancellous bone from the iliac crest. In our series, 41 patients were treated with this method, and we recorded a recurrence rate of 17.1%. Complications were rare. The risk of recurrence depended on the age of the patient. A higher recurrence rate must be expected in children under the age of 10 years. For this reason, operative treatment should not be performed until after that age if possible. Newer methods, such as steorid injections and continuous decompression by means of perforated screws, had better results in some studies, but only according to a few authors. Further research is needed to show whether our method will yield good results in the long term when applied in larger patient collectives.     

Concatemeric intermediates of equine herpesvirus type 1 DNA replication contain frequent inversions of adjacent long segments of the viral genome

A reliable reversed-phase high-performance liquid chromatographic method has been developed for the determination of bromocriptine (BCT) in plasma and eye tissues. The BCT and propranolol, added as an internal standard (I.S.), were extracted by a liquid-liquid technique followed by an aqueous back-extraction, allowing injection of an aqueous solvent into a 4-microm Nova-Pak C18 column (150x3.9 mm I.D.). The mobile phase was a mixture of 30 parts of acetonitrile and 70 parts of 0.2% triethylamine (pH 3) at a flow-rate of 1 ml/min. Fluorescence detection was at an excitation wavelength of 330 nm and an emission wavelength of 405 nm. The retention times of I.S. and BCT were 4.1 and 11.6 min, respectively. The calibration curve was linear over the concentration range 0.2-10 microg/l for plasma (r>0.999) and vitreous humour (r>0.997) and 1-50 microg/l for aqueous humour (r>0.985). The limit of quantification was 0.2 microg/l for plasma and vitreous humour using a 1-ml sample and was 1 microg/l for aqueous humour using a 0.2-ml sample. The quality control samples were reproducible with acceptable accuracy and precision. The within-day recovery (n=3) was 100-102% for plasma, 91-106% for aqueous humour and 96-111% for vitreous humour. The between-day recovery (n=9) was 90-114% for plasma, 83-115% for aqueous humour and 90-105% for vitreous humour. The within-day precision (n=3) and the between-day precision (n=9) were 1.7-7.0% and 8.1-13.6%, respectively. No interferences from endogenous substances were observed. Taken together, the above simple, sensitive and reproducible high-performance liquid chromatography assay method was suitable for the determination of BCT in plasma and eye tissues following ocular application of BCT for the therapy of myopia.     

Sequence of two Eco RI fragments from Salmonis herpesvirus 2 and comparison with Ictalurid herpesvirus 1

Two Eco RI fragments from Salmonis herpesvirus 2 (Oncorhynchus masou virus) have been sequenced. Clone pOE 150 coded for a protein homologous to ORF 68 from Ictalurid herpesvirus 1 (channel catfish virus). Clone pOE21 coded for a protein homologous with ORF46 from the same virus. No homology was found with mammalian herpesviruses.     

Properties and functions of feline herpesvirus type 1 glycoproteins

Feline herpesvirus type 1 (FHV-1) is a causative agent of feline viral rhinotracheitis and belongs to the subfamily Alphaherpesvirinae of the family Herpesviridae. Since first isolated in 1958 by Crandell and Maurer, FHV-1 is distributed worldwide and is the most clinically significant agent for respiratory infections in cats. In this review, we describe the recent findings with properties and functions of FHV-1 glycoproteins, especially hemagglutinins.     

Novel entry pathway of bovine herpesvirus 1 and 5

Herpesviruses enter cells by a yet poorly understood mechanism. We visualized the crucial steps of the entry pathway of bovine herpesvirus 1 (BHV-1) and BHV-5 by transmission and scanning electron microscopy, employing cryotechniques that include time monitoring, ultrarapid freezing, and freeze substitution of cultured cells inoculated with virus. A key step in the entry pathway of both BHV-1 and BHV-5 is a unique fusion of the outer phospholipid layer of the viral envelope with the inner layer of the plasma membrane and vice versa resulting in "crossing" of the fused membranes and in partial insertion of the viral envelope into the plasma membrane. The fusion area is proposed to function as an axis for driving the virus particle into an invagination that is concomitantly formed close to the fusion site. The virus particle enters the cytoplasm through the opened tip of the invagination, and the viral envelope defuses from the plasma membrane. There is strong evidence that the intact virus particle is then transported to the nuclear region.     

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.     

Drugs in salmonid aquaculture--a review

In contrast to mammalian therapeutics, the use of pharmaceutical substances is rather limited in fish. It is basically restricted to anaesthetic agents and anti-infective agents for parasitic and microbial diseases. Anaesthetic agents are used primarily in fish farm and laboratory settings to provide analgesia and immobilization of fish for minor procedures. The anti-infective agents are used for controlling diseases and the choice of drug depends on efficacy, ease of application, human safety, target animal safety including stress to the fish, environmental impact, regulatory approval, costs, and implications for marketing the fish. In this article, the major drugs used in salmonids in North America and Europe will be reviewed and some insight into future directions for drug development and use for the salmonid industry will be introduced. The mechanisms of action, pharmacokinetics, side effects, and uses of the drugs are emphasized.     

The DNA sequence of equine herpesvirus 2

The complete DNA sequence of equine herpesvirus 2 (EHV-2) strain 86/67 was determined. The genome is 184,427 bp in size and has a base composition of 57.5% G + C. Unusually for a herpesvirus, about a third of the sequence distributed in several large blocks appears not to encode proteins. The 79 open reading frames that were identified as probably polypeptide-coding are predicted to encode 77 distinct proteins. Amino acid sequence comparisons confirmed that EHV-2 is a gamma-herpesvirus that is genetically collinear with herpesvirus saimiri (HVS; a gamma 2-herpesvirus) and Epstein-Barr virus (EBV; a gamma 1-herpesvirus), with a closer relationship to the former. Moreover, EHV-2 specifies eight proteins that have counterparts in HVS but not in EBV and only a single protein that has a homologue in EBV but not in HVS (EBV BCRF1, which encodes an interleukin 10-like protein). EHV-2 also encodes three potential G protein-coupled receptors, one with a counterpart in HVS that is specific for alpha chemokines, another with a counterpart in human cytomegalovirus (a beta-herpesvirus), which is specific for beta chemokines, and a third that is assigned more tentatively and lacks detectable counterparts in other herpesviruses.     

The protease and the assembly protein of Kaposi's sarcoma-associated herpesvirus (human herpesvirus 8)

A genomic clone encoding the protease (Pr) and the assembly protein (AP) of Kaposi's sarcoma-associated herpesvirus (KSHV) (also called human herpesvirus 8) has been isolated and sequenced. As with other herpesviruses, the Pr and AP coding regions are present within a single long open reading frame. The mature KSHV Pr and AP polypeptides are predicted to contain 230 and 283 residues, respectively. The amino acid sequence of KSHV Pr has 56% identity with that of herpesvirus salmiri, the most similar virus by phylogenetic comparison. Pr is expressed in infected human cells as a late viral gene product, as suggested by RNA analysis of KSHV-infected BCBL-1 cells. Expression of the Pr domain in Escherichia coli yields an enzymatically active species, as determined by cleavage of synthetic peptide substrates, while an active-site mutant of this same domain yields minimal proteolytic activity. Sequence comparisons with human cytomegalovirus (HCMV) Pr permitted the identification of the catalytic residues, Ser114, His46, and His134, based on the known structure of the HCMV enzyme. The amino acid sequences of the release site of KSHV Pr (Tyr-Leu-Lys-Ala*Ser-Leu-Ile-Pro) and the maturation site (Arg-Leu-Glu-Ala*Ser-Ser-Arg-Ser) show that the extended substrate binding pocket differs from that of other members of the family. The conservation of amino acids known to be involved in the dimer interface region of HCMV Pr suggests that KSHV Pr assembles in a similar fashion. These features of the viral protease provide opportunities to develop specific inhibitors of its enzymatic activity.     

A double-strand break in a herpesvirus genome stimulates targeted homologous recombination with exogenous, cloned viral sequences

Cassette mutagenesis was used to produce a library of mutations at the interface of the N- and C-terminal helices of Saccharomyces cerevisiae iso-1-cytochrome c. The library is random and comprises > 98% mutations. Over 11,000 candidates were assayed for function by selecting for the ability of yeast, with the mutated gene as their sole cytochrome c source, to grow on nonfermentable carbon sources. We estimate that approximately 0.5% of the 160,000 total amino acid combinations at these four residues result in a functional cytochrome c. Significant correlations are found between the phenotype of yeast harboring the alleles and both the Dayhoff mutation matrix and transfer free energies (cyclohexane-to-water and n-octanol-to-water). Similar correlations are observed with respect to growth rate. Finally, sequences that are consistent with function follow a binary amino acid pattern.     

Structural and functional analysis of bovine herpesvirus 1 minor glycoproteins

Topoisomerases are enzymes that catalyse the transient breakage and rejoining of either one (topo I) or two (topo II) DNA strands, to allow one strand to pass through another and prevent unresolvable tangles during processes such as DNA replication. A number of important clinical antitumour agents act through inhibition of topo II enzymes, while some topo I inhibitors appear likely to enter clinical use. Although these chemicals do not covalently interact with DNA, they have strong mutagenic potential, generally causing events at the level of the chromosome rather than that of the gene. Most are recombinogens, may affect gene expression and can also lead to aneuploidy through effects on chromosome segregation. Most topo I and topo II inhibitors primarily cause mutagenic events associated with the replication fork. However, at least in mitotic chromosomes, topo II enzymes are located at the base of chromosome loops, and topo II inhibitors may facilitate subunit exchanges, leading to major deletions and illegitimate recombinational events. There is evidence that programmed cell death provides an alternative pathway to mutagenesis following treatment by either topo I or topo II inhibitors. The final fate of the cell will result from a balance between these two processes.     

Transcriptional control of the equine herpesvirus 1 immediate early gene

Transient expression assays measuring induction of an equine herpesvirus 1 (EHV-1) immediate early (IE) promoter construct, pIE beta gal, were used to examine trans-induction of the IE gene (IR1) promoter by superinfection with intact EHV-1 (Kentucky A strain), uv-inactivated EHV-1, or EHV-1 stocks highly enriched for defective interfering particles (DIPs), and by cotransfection with plasmids containing EHV-1 DNA fragments. Our results confirm reports by others in that the IE promoter can be induced by both intact and uv-inactivated EHV-1 and provide evidence that DIP-rich virus stocks also possess transinducing activity. Furthermore, IE promoter activity was induced by cotransfection of the promoter construct with either of two plasmids containing restriction enzyme DNA fragments that span ORF12, the putative EHV-1 homolog of the herpes simplex virus 1 (HSV-1) alpha-trans-inducing factor (alpha-TIF) gene, UL48, or by cotransfection with an isolated DNA fragment containing only ORF12, while no transactivation was associated with plasmids linearized by restriction enzyme digestion at a single site within ORF12. These data indicate that, despite its predicted lack of an acidic carboxy terminus (a region essential in HSV-1 alpha-TIF for trans-inducing activity), the EHV-1 ORF12 product is a functional alpha-TIF.     

Absence of human herpesvirus 8 and Epstein-Barr virus genome sequences in cutaneous epithelial neoplasms arising in immunosuppressed organ-transplant patients

Recent studies have implicated herpesvirus 8 and Epstein-Barr virus in the development of cutaneous malignancies in immunosuppressed patients. In order to examine the strength of this association, we examined 37 malignant, pre-malignant and benign cutaneous epithelial neoplasms removed from immunosuppressed organ recipients for the presence of human herpesvirus 8 and Epstein-Barr viral genome sequences using polymerase chain reaction (PCR) and in situ hybridization. We examined 2 actinic keratoses, 1 benign keratosis, 11 invasive squamous cell carcinomas, 17 squamous cell carcinomas in situ and 6 basal cell carcinomas. We also examined 4 basal cell carcinomas, 1 invasive squamous cell carcinoma and 3 squamous cell carcinomas in immunocompetent hosts. In contrast to findings reported by other investigators, we were unable to detect viral genome sequences in any of the biopsies examined. Our findings suggest that human herpesvirus 8 and Epstein-Barr virus likely do not play an etiologic role in cutaneous epithelial oncogenesis in immunocompromised patients.     

Cosmid library of the turkey herpesvirus genome constructed from nanogram quantities of viral DNA associated with an excess of cellular DNA

A protocol was designed for the rapid and efficient construction of cosmid libraries from cell-associated viral genomes available in very low quantities. Purification of viral DNA from cellular DNA was unnecessary. The vast excess of cellular DNA compensated for the limited amount of available viral DNA, enabling titration of the restriction endonuclease partial digest. A cosmid library of the turkey herpesvirus DNA genome was constructed from 1.5 micrograms of cellular DNA containing approximately 6 nanograms of viral DNA.     

The mitochondrial genome integrity gene, MGI1, of Kluyveromyces lactis encodes the beta-subunit of F1-ATPase

In a previous report, we found that mutations at the mitochondrial genome integrity locus, MGI1, can convert Kluyveromyces lactis into a petite-positive yeast. In this report, we describe the isolation of the MGI1 gene and show that it encodes the beta-subunit of the mitochondrial F1-ATPase. The site of mutation in four independently isolated mgi1 alleles is at Arg435, which has changed to Gly in three cases and Ile in the fourth isolate. Disruption of MGI1 does not lead to the production of mitochondrial genome deletion mutants, indicating that an assembled F1 complex is needed for the "gain-of-function" phenotype found in mgi1 point mutants. The location of Arg435 in the beta-subunit, as deduced from the three-dimensional structure of the bovine F1-ATPase, together with mutational sites in the previously identified mgi2 and mgi5 alleles, suggests that interaction of the beta- and alpha- (MGI2) subunits with the gamma-subunit (MGI5) is likely to be affected by the mutations.