Isolation and partial sequencing of Equid herpesvirus 5 from a horse in Iceland.

Horses are hosts to 2 types of gammaherpesviruses, Equid herpesvirus 2 and 5 (EHV-2 and EHV-5, respectively). Both EHV-2 and EHV-5 are common in horses in Iceland. An Icelandic EHV-5 isolate was recovered by sequential culture in primary fetal horse kidney and rabbit kidney cells. Glycoprotein B, glycoprotein H, and DNA terminase genes of the isolate were fully sequenced, and the DNA polymerase gene was partly sequenced. To date, the glycoprotein B gene of EHV-5 was the only gene that has been reported to be completely sequenced in addition to small parts of the glycoprotein H, DNA polymerase, and DNA terminase genes. The present report, therefore, is a significant addition to previously reported EHV-5 sequences.

The simian varicella virus uracil DNA glycosylase and dUTPase genes are expressed in vivo, but are non-essential for replication in cell culture.

Neurotropic herpesviruses express viral deoxyuridine triphosphate nucleotidohydrolase (dUTPase) and uracil DNA glycosylase (UDG) enzymes which may reduce uracil misincorporation into viral DNA, particularly in neurons of infected ganglia. The simian varicella virus (SVV) dUTPase (ORF 8) and UDG (ORF 59) share 37.7% and 53.9% amino acid identity, respectively, with varicella-zoster virus (VZV) homologs. Infectious SVV mutants defective in either dUTPase (SVV-dUTPase(-)) or UDG (SVV-UDG(-)) activity or both (SVV-dUTPase(-)/UDG(-)) were constructed using recA assisted restriction endonuclease cleavage (RARE) and a cosmid recombination system. Loss of viral dUTPase and UDG enzymatic activity was confirmed in CV-1 cells infected with the SVV mutants. The SVV-dUTPase(-), SVV-UDG(-), and SVV-dUTPase(-)/UDG(-) mutants replicated as efficiently as wild-type SVV in cell culture. SVV dUTPase and UDG expression was detected in tissues derived from acutely infected animals, but not in tissues derived from latently infected animals. Further studies will evaluate the pathogenesis of SVV dUTPase and UDG mutants and their potential as varicella vaccines.

Substrate specificity and molecular modelling of the feline herpesvirus-1 thymidine kinase.

Feline herpesvirus-1 (FHV-1) causes a severe upper respiratory and ocular disease in cats. An effective antiviral compound is required for treating FHV-1 infections. The virus-encoded thymidine kinase (TK) is the molecular basis for selective activation of commonly used antiviral nucleoside analogue drugs, e.g. acyclovir (ACV), penciclovir (PCV) and ganciclovir (GCV). The substrate specificity of a recombinant FHV-1 TK, expressed in Escherichia coli, was studied. FHV-1 TK efficiently phosphorylated its natural substrate deoxythymidine. However, it exhibited relatively lower affinity for the guanosine analogue substrates. PCV was most efficiently phosphorylated, followed by GCV, with approximately twofold reduction in the phosphorylation rate. The lowest phosphorylation rate was recorded for ACV. To correlate these biochemical data with structural features of the FHV-1 TK, a three-dimensional (3D) model of this enzyme was constructed based on sequence homology with two other herpesviral TKs, encoded by equine herpesvirus-4 (EHV-4) and herpes simplex-1 (HSV-1). Mutational analysis of the amino acids forming the FHV-1 TK active site identified two residues (Y29 and F144) as being critical for the differential ability of this enzyme to phosphorylate nucleoside analogues. A double substitution of Y29H/F144Y resulted in a threefold increase in the ACV phosphorylation rate.

Inactivity of the bicyclic pyrimidine nucleoside analogues against simian varicella virus (SVV) does not correlate with their substrate activity for SVV-encoded thymidine kinase.

Simian varicella virus (SVV) and human varicella-zoster virus (VZV) are closely related viruses that share many structural and functional properties. 5-Substituted 2'-deoxyuridine derivatives (e.g., BVDU, BVaraU) and acyclic guanine nucleoside derivatives (i.e., ACV and GCV) show comparable antiviral efficacy against VZV and SVV in cell culture. In contrast, the novel bicyclic nucleoside analogues (BCNAs) are exquisitely inhibitory to VZV (EC50 in the lower nanomolar range) but completely inactive against SVV. The VZV-encoded thymidine kinase (TK) appeared to be essential for BCNA activation (phosphorylation) and anti-VZV activity. Also SVV TK is able to recognize the BCNAs as substrate, although with a different structure-affinity relationship. Thus, viral TK-catalyzed phosphorylation is necessary but not sufficient for the BCNAs to display antiviral activity. Our data suggest that the eventual target of the BCNAs against VZV is either absent in SVV or, alternatively, is insensitive for the (phosphorylated) BCNAs.

Detection of bovine herpesvirus 4 glycoprotein B and thymidine kinase DNA by PCR assays in bovine milk.

A polymerase chain reaction (PCR) assay was developed to detect bovine herpesvirus 4 (BHV4) glycoprotein B (gB) DNA, and a nested-PCR assay was modified for the detection of BHV4 thymidine kinase (TK) DNA in bovine milk samples. To identify false-negative PCR results, internal control templates were constructed, added to milk samples, and co-amplified with viral DNA using the same primers for both templates. Specificity, sensitivity, and reproducibility of the two PCR assays were examined. In both PCR assays, all 31 BHV4 strains examined were scored positive, whereas 14 unrelated viruses scored negative. Sensitivity studies showed that two-ten copies of BHV4 DNA were detectable by the gB-PCR, while one-three copies could be detected by the TK-PCR. For the detection of BHV4 in milk samples, the gB-PCR amplification was found to be ten-times, and the TK-PCR was found to be 55-times more sensitive than virus isolation. BHV4 DNA was detected by gB-PCR and TK-PCR in 93 and 95%, respectively, of 61 milk samples collected from cows infected intramammarily with BHV4, while only 61% were positive by virus isolation. Four out of 48 cows with clinical mastitis were positive for BHV4-gB and BHV4-TK DNA, whereas no BHV4 DNA was detected in milk from control cows. Considerable agreement was seen between the results of the two PCR assays, and both methods were considered as rapid and reliable tests for the screening of BHV4 DNA in bovine milk. The less laborious gB-PCR might be the recommended test of choice for screening large amounts of milk samples for the presence of BHV4.

Characterization of heterosubunit complexes formed by the R1 and R2 subunits of herpes simplex virus 1 and equine herpes virus 4 ribonucleotide reductase.

We report on the separate PCR cloning and subsequent expression and purification of the large (R1) and small (R2) subunits from equine herpes virus type 4 (EHV-4) ribonucleotide reductase. The EHV-4 R1 and R2 subunits reconstituted an active enzyme and their abilities to complement the R1 and R2 subunits from the closely related herpes simplex virus 1 (HSV-1) ribonucleotide reductase, with the use of subunit interaction and enzyme activity assays, were analysed. Both EHV-4 R1/HSV-1 R2 and HSV-1 R1/EHV-4 R2 were able to assemble heterosubunit complexes but, surprisingly, neither of these complexes was fully active in enzyme activity assays; the EHV-4 R1/HSV-1 R2 and HSV-1 R1/EHV-4 R2 enzymes had 50% and 5% of their respective wild-type activities. Site-directed mutagenesis was used to alter two non-conserved residues located within the highly conserved and functionally important C-termini of the EHV-4 and HSV-1 R1 proteins. Mutation of Pro-737 to Lys and Lys-1084 to Pro in EHV-4 and HSV-1 R1 respectively had no effects on subunit assembly. Mutation of Pro-737 to Lys in EHV-4 R1 decreased enzyme activity by 50%; replacement of Lys-1084 by Pro in HSV-1 R1 had no effect on enzyme activity. Both alterations failed to restore full enzyme activities to the heterosubunit enzymes. Therefore probably neither of these amino acids has a direct role in catalysis. However, mutation of the highly conserved Tyr-1111 to Phe in HSV-1 R1 inactivated enzyme activity without affecting subunit interaction.

The equine herpes virus 4 thymidine kinase is a better suicide gene than the human herpes virus 1 thymidine kinase.

The herpes simplex virus type 1 thymidine kinase suicide gene (HSV1tk) together with ganciclovir (GCV) have been successfully used for in vivo treatment of various experimental tumors, and many clinical trials using this system have been launched. With the aim to improve this therapeutic system, we compared the potential efficacy of different herpes virus derived thymidine kinases (HSV1, varicella-zoster virus, equine herpes virus type-4 and Epstein-Barr virus) as suicide genes in association with the nucleoside analogs acyclovir, ganciclovir and bromovinyldeoxyur- idine. Using various murine and human cell lines expressing these viral tk, we show that HSV1- and EHV4tk are the more efficient suicide genes for the different nucleoside analogs tested. Moreover, EHV4tk expressing murine and human cells were three- to 12-fold more sensitive to GCV than HSV1tk expressing cells. This was correlated with the presence of five-fold higher amounts of the toxic triphosphated-GCV in EHV4- versus HSV1tk expressing cells. Altogether, these experiments underline the potential advantages of the EHV4tk as a suicide gene.

Identification and analysis of the simian varicella virus thymidine kinase gene.

The thymidine kinase (TK) of herpesviruses, in contrast to cellular TKs, phosphorylates a variety of substrates including antiherpetic nucleoside analogues. This study reports the identification and DNA sequence of the simian varicella virus (SVV) TK gene. A 32P-labeled varicella zoster virus (VZV) TK DNA probe hybridized to the HindIII B subclone of the SVV BamHI B restriction endonuclease (RE) fragment, indicating the presence of a SVV DNA sequence homologous to the VZV TK gene. DNA sequence analysis of the SVV HindIII B subclone revealed a 1014 base pair (bp) open reading frame (ORF) encoding a 337 amino acid polypeptide homologous to herpesvirus TKs. The predicted SVV and VZV TK polypeptides share 51.3% identity, and alignment of the putative protein sequence of several TK homologues suggests the position of a conserved nucleotide binding site and a nucleoside (substrate) binding site in the SVV TK. Identification of the 5' end of the SVV TK transcript by primer extension analysis allowed a comparison of the SVV and VZV TK promoter regions indicating extensive conservation of the DNA sequence and transcription factor binding sites. Plaque reduction assays demonstrate that the SVV TK is active based on the susceptibility of SVV to acyclovir treatment and that SVV is less sensitive to acyclovir than VZV and herpes simplex virus (HSV-1) in infected Vero cells. Identification of the SVV TK ORF will facilitate studies that examine the role of viral TKs in pathogenesis and antiviral sensitivity and provides a potential insertion site for the expression of foreign genes.

Sequences of the ribonucleotide reductase-encoding genes of equine herpesvirus 4.

The equine herpesvirus 4 (EHV-4) genes encoding the two subunits of the enzyme ribonucleotide reductase (RR) were cloned and their nucleotide (nt) sequences determined. The large subunit (RR1) is predicted to comprise 789 amino acids (aa), which compares with lengths of 790, 775 and 1137 aa for the RR1 proteins encoded by equine herpesvirus 1 (EHV-1) gene 21, varicella zoster virus (VZV) gene 19 and herpes simplex virus type 1 (HSV-1) UL39, respectively. In common with VZV RR1, the EHV-4 RR1 protein lacks the N-terminal domain of HSV-1 RR1 which possesses protein kinase activity. EHV-4 RR1 demonstrates identities of 88, 52 and 29% with the RR1 proteins of EHV-1, VZV and HSV-1, respectively. The small subunit (RR2) is predicted to be 320 aa in length, which compares with lengths of 321, 306 and 340 aa for the RR2 proteins encoded by EHV-1 gene 20, VZV gene 18 and HSV-1 UL40, respectively. The EHV-4 RR2 protein exhibits identities of 90, 60 and 55% with the RR2 proteins of EHV-1, VZV and HSV-1, respectively.