Tk Gene Research Articles

Promiscuous patching of broken chromosomes in mammalian cells with extrachromosomal DNA.

To study double-strand break (DSB)-induced mutations in mammalian chromosomes, we stably transfected thymidine kinase (tk)-deficient mouse fibroblasts with a DNA substrate containing a recognition site for yeast endonuclease I-SceI embedded within a functional tk gene. Cells were then electroporated with a plasmid expressing endonuclease I-SceI to induce a DSB, and clones that had lost tk function were selected. In a previous study of DSB-induced tk-deficient clones, we found that approximately 8% of recovered tk mutations involved the capture of one or more DNA fragments at the DSB site. Almost half of the DNA capture events involved the I-SceI expression plasmid, and several events involved retrotransposable elements. To learn whether only certain DNA sequences or motifs are efficiently captured, in the current work we electroporated an I-SceI expression plasmid along with HaeIII fragments of φX174 genomic DNA. We report that 18 out of 132 tk-deficient clones recovered had captured DNA fragments, and 14 DNA capture events involved one or more fragments of φX174 DNA. Microhomology existed at most junctions between φX174 DNA and genomic sequences. Our work suggests that virtually any extrachromosomal DNA molecule may be recruited for the patching of DSBs in a mammalian genome.

Monitoring signal transduction in cancer: cDNA microarray for semiquantitative analysis.

This study targeted the development of a novel microarray tool to allow rapid determination of the expression levels of 58 different tyrosine kinase (tk) genes in small tumor samples. The goals were to define a reference probe for multi-sample comparison and to investigate the variability and reproducibility of the image acquisition and RT-PCR procedures. The small number of tk genes on our arrays enabled us to define a reference probe by artificially mixing all genes on the arrays. Such a probe provided contrast reference for comparative hybridization of control and sample DNA and enabled cross-comparison of more than two samples against one another. Comparison of signals generated from multiple scanning eliminated the concern of photo bleaching and scanner intrinsic noise. Tests performed with breast, thyroid, and prostate cancer samples yielded distinctive patterns and suggest the feasibility of our approach. Repeated experiments indicated reproducibility of such arrays. Up- or downregulated genes identified by this rapid screening are now being investigated with techniques such as in situ hybridization.

Capture of DNA sequences at double-strand breaks in mammalian chromosomes.

To study double-strand break (DSB)-induced mutations in mammalian chromosomes, we transfected thymidine kinase (tk)-deficient mouse fibroblasts with a DNA substrate containing a recognition site for yeast endonuclease I-SceI embedded within a functional tk gene. To introduce a genomic DSB, cells were electroporated with a plasmid expressing endonuclease I-SceI, and clones that had lost tk function were selected. Among 253 clones analyzed, 78% displayed small deletions or insertions of several nucleotides at the DSB site. Surprisingly, approximately 8% of recovered mutations involved the capture of one or more DNA fragments. Among 21 clones that had captured DNA, 10 harbored a specific segment of the I-SceI expression plasmid mapping between two replication origins on the plasmid. Four clones had captured a long terminal repeat sequence from an intracisternal A particle (an endogenous retrovirus-like sequence) and one had captured what appears to be a cDNA copy of a moderately repetitive B2 sequence. Additional clones displayed segments of the tk gene and/or microsatellite sequences copied into the DSB. This first systematic study of DNA capture at DSBs in a mammalian genome suggests that DSB repair may play a considerable role in the evolution of eukaryotic genomes.

Convection-enhanced delivery of AAV-2 combined with heparin increases TK gene transfer in the rat brain.

Adeno-associated virus type2 (AAV-2) binds to heparan-sulfate proteoglycans on the cell surface. In vivo, attachment of viral particles to cells adjacent to the injection tract limits the distribution of AAV-2 when infused into the CNS parenchyma and heparin co-infusion might decrease the binding of AAV-2 particles to cells in the vicinity of the infusion tract. We have previously shown that heparin co-infusion combined with convection enhanced delivery enhances distribution of the GDNF family trophic factors (heparin-binding proteins) in the rat brain. In this work we show that heparin co-infusion significantly increases the volume of distribution of AAV-2 as demonstrated by immunoreactivity to the transgene product 6 days after infusion into the rat striatum.

Spindle poisons induce allelic loss in mouse lymphoma cells through mitotic non-disjunction

Aneuploidy is an important contributor to reproductive failure and tumor development. It arises spontaneously or as a result of exposure to aneugenic agents through non-disjunction. Two spindle poisons, colchicine (COL) and vinblastine (VBL) are mutagenic in the mouse lymphoma assay (MLA), a gene mutation assay that targets the heterozygous thymidine kinase ( tk) gene on chromosome 11 in mouse lymphoma L5178Y tk+/− 3.7.2c cells. To investigate the mechanisms of spindle poison mutagenesis, we analyzed the COL- and VBL-induced TK mutants at the molecular and cytogenetic level. Loss of heterozygosity (LOH) analysis employing a microsatellite region within the tk locus revealed that almost all mutants had lost the functional tk allele. To determine the extent of the LOH, we further examined LOH mutants for heterozygosity at nine microsatellite loci spanning the entire chromosome 11. Interestingly, every microsatellite marker showed LOH in all COL- and VBL-induced LOH mutants, suggesting that these mutants were generated by loss of the whole chromosome 11 through mitotic non-disjunction. Chromosome painting analysis supported this hypothesis; there were no mutants showing structural changes such as deletions or translocations involving chromosome 11. In contrast, spontaneous TK mutants followed from point mutations, deletions and recombinational events as well as whole chromosome loss. Our present study indicates that spindle poisons induce mutations through mitotic non-disjunction without structural DNA changes and supports a possible mechanism in which a recessive mutation mediated by aneuploidy may develop tumors.

Insulinoma-induced hypoglycemic death in mice is prevented with beta cell-specific gene therapy.

Tumor-specific gene therapy can be achieved if a tumor-specific promoter can be identified. In this study the authors investigated the use of the rat insulin promoter (RIP) for insulinoma-specific expression of a reporter gene. Insulinoma-specific cytotoxicity using the suicide gene thymidine kinase (tk) was studied both in vitro and in vivo. RIPtk gene therapy, delivered by a nontoxic, noninflammatory liposomal delivery system, was used in an insulinoma ICR/SCID mouse model to prevent hypoglycemic death. Rat insulin promoter (0.502 kb) was ligated to the reporter gene lacZ and ligated to the tk gene. These two genes were transfected into a mouse insulinoma (NIT) cell line to ascertain insulinoma-specific expression and insulinoma-specific cytotoxicity in vitro. Reverse transcriptase-polymerase chain reaction and electrophoretic mobility-shift assays were performed on NIT-1 cell RNA and nuclear extract, respectively, to determine the transcription factors present and responsible for RIP activation in NIT-1 cells. A mouse insulinoma model was created with NIT-1 cells. These mice were treated with the RIPtk gene, and both blood sugars and animal viability were monitored. Only NIT-1 cells stained blue after X-gal staining or had detectable levels of beta-galactosidase protein. A significant decrease in cell survival was observed in NIT-1 cells transfected with RIPtk in vitro. Messenger RNA for both BETA2 and PDX-1 was found in NIT-1 cells, and a supershift was observed for both BETA2 and PDX-1. Experimental mice treated with the RIPtk gene, delivered by a liposomal gene delivery system, maintained their blood glucose levels, and the animals did not die of hypoglycemia. The data suggest that the RIP is an insulinoma-specific promoter. An ICR/SCID mouse insulinoma model was used to show that insulinoma-specific cytotoxicity can be accomplished by RIP coupled to a suicide gene in vivo, preventing hypoglycemic death.

Abnormal angiogenesis but intact hematopoietic potential in TGF-beta type I receptor-deficient mice

Deletion of the transforming growth factor beta1 (TGF-beta1) gene in mice has previously suggested that it regulates both hematopoiesis and angiogenesis. To define the function of TGF-beta more precisely, we inactivated the TGF-beta type I receptor (TbetaRI) gene by gene targeting. Mice lacking TbetaRI die at midgestation, exhibiting severe defects in vascular development of the yolk sac and placenta, and an absence of circulating red blood cells. However, despite obvious anemia in the TbetaRI(-/-) yolk sacs, clonogenic assays on yolk sac-derived hematopoietic precursors in vitro revealed that TbetaRI(-/-) mice exhibit normal hematopoietic potential compared with wild-type and heterozygous siblings. Endothelial cells derived from TbetaRI-deficient embryos show enhanced cell proliferation, improper migratory behavior and impaired fibronectin production in vitro, defects that are associated with the vascular defects seen in vivo. We thus demonstrate here that, while TbetaRI is crucial for the function of TGF-beta during vascular development and can not be compensated for by the activin receptor-like kinase-1 (ALK-1), functional hematopoiesis and development of hematopoietic progenitors is not dependent on TGF-beta signaling via TbetaRI.

Microcarriers enhance the efficacy of PA317/HSV-1 tk gene therapy in 9L rat glioma model

Microcarriers enhance the efficacy of PA317/HSV-1 tk gene therapy in 9L rat glioma model

ZIDOVUDINE (AZT) RESISTANCE IN H9 CELLS DUE TO DECREASED TK EXPRESSION IS ASSOCIATED WITH HYPERMETHYLATION OF TK GENE

AZT resistant human T-lymphoid H9 cells, deficient in TK gene expression, re-expressed TK mRNA and regained the ability to metabolize AZT by exposure to the demethylation agent azacytidine (AzaCd). Cytotoxic and anti-HIV-1 effects of AZT were increased in H9 AZT resistant cells treated with AzaCd when compared to untreated cells. This leads to the assumption that drug induced DNA hypermethylation was involved in the TK gene-silencing mechanism. Our results suggest approaches using modulation of gene methylation for increasing antiviral efficiency of drugs.

Experimental study on human pulmonary adenocarcinoma cell A549 transfected with HSV₁-TK gene in vitro and in vivo

To observe killing effect of HSV₁-TK/GCV system on human pulmonary adenocarcinoma cell A549 in vitro and in vivo. A retroviral vector containing TK gene was constructed and transduced into pulmonary adenocarcinoma cell A549 by electroporation. The sensitivity of transfected cell to GCV in vitro and bystander effect and cellular apoptosis were observed. The recombination and expression of TK gene were examined by DNA PCR and in situ hybridization individually. In addition, the therapeutic effect of GCV on subcutaneous tumors inoculated with transfected and parental cells respectively was observed. The transfected cells were irregular in shape, polyangular and easy of vacuolization. The double time of A549,A549-PLXSN and A549-TK was 36.15±3.27,40.82±3.75 and 42.06±4.12 hours respectively (P>0.05). The sensitivity of transfected cells to GCV was 46 times higher than that of parental cells and bystander effect was more apparent in high density inoculation cells than in low density. Apoptotic bodies and semimoon feature in nuclear were observed in transfected cells, but not in parental cell. Apoptotic cells were found significantly more in transfected cells than in parental cells by FCM and TUNEL (P<0.001). The recombination and expression of TK gene were positive in the transfected cells. In vivo, growth of tumors which formed by transfected cells was significantly inhibited by GCV, however, there was no similar inhibitive effect found in control group. The transfected cells have obtained sensitivity to GCV. The killing effect of TK/GCV system on tumor cells is probably related to apoptosis. GCV could inhibit growth of tumors inoculated by transfected cells.

Gene therapy for hepatocellular carcinoma based on tumour-selective suicide gene expression using the alpha-fetoprotein ( AFP) enhancer and a housekeeping gene promoter

The aim of this study was to examine whether the human α-fetoprotein ( AFP) enhancer could be used to induce hepatocellular carcinoma (HCC)-selective expression of the herpes simplex virus thymidine kinase (HSV- tk) gene which is under the control of the phosphoglycerate kinase ( pgk) promotor. The human AFP enhancer was linked with the non-tissue-specific, human housekeeping pgk promoter in a retroviral vector. AFP-producing HCC cells infected with retroviruses carrying the HSV- tk gene under the control of the AFP enhancer/ pgk promoter were much more susceptible to the prodrug, ganciclovir (GCV), than those infected with the same retroviruses without the AFP enhancer. Non-HCC cells infected with retroviruses carrying the HSV- tk gene under the control of the AFP enhancer/ pgk promoter exhibited profoundly increased resistance to GCV compared with those infected with the same retroviruses without the AFP enhancer. Northern blot analysis revealed that the AFP enhancer caused enhanced HSV- tk expression in AFP-producing HCC cells and suppressed HSV- tk expression in non-HCC cells. Our results indicate that the AFP enhancer could give HCC selectivity to the pgk promoter, and that this novel strategy may be useful for HCC-selective cancer gene therapy.

Stimulation of intercellular communication of poor-communicating cells by gap-junction-competent cells enhances the HSV-TK/GCV bystander effect in vitro.

We have previously shown that gap-junctional intercellular communication (GJIC) appears to play a role in the bystander effect that is observed in anticancer suicide gene therapy mediated by herpes simplex virus (HSV) thymidine kinase (tk) and ganciclovir (GCV). We now report that when connexin-expressing (Cx+) cells are present within a noncommunicating population of cells (Cx-), there is GJIC between the Cx+ and Cx- cells and that due to this stimulation of GJIC, the bystander effect also occurs when the 2 cell types are mixed. We transfected HeLa cells, which do not express any detectable level of connexin, with Cx43. The Cx+ and Cx- HeLa cells were further transfected with the tk gene, giving 4 phenotypes: Cx+tk-, Cx+tk+, Cx-tk+ and Cx-tk-. We observed GJIC between Cx+ and Cx- cells, but not between Cx- and Cx- cells, regardless of the tk genotype. Similarly, we observed the HSV-tk/GCV bystander effect in Cx+tk-/Cx-tk+ and Cx+tk+/Cx-tk- cocultures. The extent of the bystander effect in cocultures of Cx+tk- and Cx-tk+ cells was stronger than in cocultures of Cx+tk+ and Cx-tk- cells when each mixture had the same ratio of Cx+ and tk+ cells. These results suggest that Cx-expressing HeLa cells stimulate GJIC capacity between them and non-Cx-expressing HeLa cells, which mediates the bystander effect in mixtures of Cx+ cells and Cx- cells in vitro. Thus, Cx expression even in only a limited fraction of tumor cells may enhance the efficacy of the HSV-tk/GCV strategy by inducing a bystander effect.

Gene therapy utilizing the Cre/loxP system selectively suppresses tumor growth of disseminated carcinoembryonic antigen-producing cancer cells.

Recent clinical trials of cancer gene therapy have shown encouraging results for controlling localized tumors. However, to control metastatic or disseminated tumor cells, further modification of vectors is required to enhance specificity and infectivity against targets. We investigated whether utilization of the Cre recombinase(Cre)/loxP system contributes to enhanced antitumor effects together with minimal adverse reactions in specific gene therapy against disseminated carcinoembryonic antigen (CEA)-producing cancer cells in the peritoneal cavity of mice. CEA-producing cancer would be a good therapeutic target because it is found in lung, stomach and colon sites, which account for most cancers. We constructed a pair of recombinant adenoviral vectors (Ads), one of which expresses the Cre gene under the control of the CEA promoter (Ad.CEA-Cre); the other expresses the herpes simplex virus thymidine kinase (HSV-TK) gene (Ad.lox-TK), or the beta-galactosidase gene (beta-gal) by Cre (Ad.lox-beta-gal). Intraperitoneal coinjection of Ad.CEA-Cre and Ad.lox-beta-gal into mice with peritonitis carcinomatosa by CEA-producing tumor cells showed selective expression of the beta-gal gene in tumor foci. Coinfection of Ad.CEA-Cre and Ad.lox-TK followed by ganciclovir (GCV) administration significantly suppressed the total tumor weight in the peritoneal cavity of the mice to 13% of that of the untreated mice and 22% of that of the mice treated with Ad.CEA-TK/GCV, an Ad that expressed the HSV-TK gene driven by the CEA promoter alone. Moreover, treatment with Ad.CEA-Cre and Ad.lox-TK/GCV completely suppressed tumors in 4 of 10 (40%) mice without significant weight loss, although 2 of 10 mice treated with Ad.CAG-TK/GCV, an adenovirus vector that strongly but nonspecifically expressed the TK gene, died due to severe side effects including diarrhea, weight loss and liver dysfunction. These findings suggest that cell type-specific gene therapy using the Cre/loxP system is effective against disseminated cancer cells without significant side effects.

Herpesvirus Saimiri Pathogenicity Enhanced by Thymidine Kinase of Herpes Simplex Virus

Herpesvirus saimiri can be used as an efficient gene expression vector for human T lymphocytes and thus may allow applications in experimental leukemia therapy. We constructed recombinant viruses for the functional expression of the thymidine kinase (TK) of herpes simplex virus type 1 (HSV) as a suicide gene. These viruses reliably allowed the targeted elimination of transduced nonpermissive human T cells in vitro after the administration of ganciclovir. To test the reliability of this function under the most stringent permissive conditions, in this study we analyzed the influence of the prodrugs ganciclovir and acyclovir in common marmosets on the acute leukemogenesis induced by either wild-type herpesvirus saimiri C488 or by a recombinant derivative expressing TK of HSV. Antiviral drug treatment did not influence the rapid development of acute disease. In contrast, the presence of the HSV tk gene resulted in a faster disease progression. In addition, HSV TK-expressing viruses showed faster replication than wild-type virus in culture at low serum concentrations. Thus, HSV TK accelerates the replication of herpesvirus saimiri and enhances its pathogenicity. This should be generally considered when HSV TK is applied as a transgene in replication-competent DNA virus vectors for gene therapy.

Experimental study on pancreatic cancer gene therapy using the HSV-TK gene mediated by a retroviral vector

OBJECTIVE: To study the value of the herpes simplex virus type I thymidine kinase (HSV-TK) gene mediated by a retroviral vector in the treatment of human pancreatic cancer cell line 8988. METHODS: The HSV-TK gene was directionally cloned into a site following the SV40 promoter that was adjacent to the 5′ LTR (long terminal repeats) and the neomycin phosphotransferase gene in the retroviral vector pMNSM. This enabled the TK gene to integrate into the chromatin of the host cell. The recombinant plasmid pMNS-TK-M was then transfected into the retrovirus-packaging cell Pa317. Finally, the HSV-TK gene was transfected into pancreatic cancer cell line 8988 by the recombinant retrovirus after selection with G418. RESULTS: The HSV-TK gene was stably integrated into the host cell and its expression confirmed by Southern blotting and drug-sensitivity tests. In vitro studies showed that the acyclovir (ACV) sensitivity level in the 8988/TK+ cells was higher than that of the parent cells. The cells that were transfected with the TK gene were significantly susceptible to ACV. In vivo studies in nude mice showed that intraperitoneal injection of ACV might postpone the formation of implanted tumors and produce a treatment effect on the tumors. CONCLUSIONS: This study demonstrated that the HSV-TK/ACV retroviral system could be used in vivo to treat pancreatic cancer.

Acyclovir-Resistant Bilateral Keratitis Associated with Mutations in the HSV-1 Thymidine Kinase Gene

Purpose: To evaluate the contribution of molecular methods for the diagnosis of an acyclovir-resistant HSV-1 bilateral keratitis in an AIDS patient and to report a new point mutation in the nucleotide sequence of the thymidine kinase (tk) gene involved.Methods: A 31 year old HIV-positive female presented with severe, active, bilateral and sight-threatening keratitis of 6 months duration, which was treated unsuccessfully with acyclovir. After corneal biopsy, samples were analysed by standard virological procedures, in situ hybridization, and PCR. The tk gene was cloned and subsequently sequenced.Results: Conventional virological methods remained inconclusive. However, in situ hybridization and PCR rapidly confirmed the diagnosis of HSV-1 keratitis. The tk gene sequence revealed the presence of five variations previously described in two reference strains, but also a new point mutation at nucleotide position 431 which leads to an amino-acid change at position 144 that supported the hypothesis of a putatively altered functional form of the enzyme. Intravenous foscarnet treatment in an induction regimen was effective and cicatrization occurred within 3 weeks.Conclusions: PCR and in situ hybridization are effective and powerful techniques when other virological procedures are non-contributive, particularly in immunocompromised patients previously treated with antiviral drugs. The new point mutation identified in the tk gene may be associated with resistance to acyclovir.

Production of a Heterozygous Mutant Cell Line by Homologous Recombination (Single Knockout)

AbstractFormerly UNIT 9.16, this unit takes a more appropriate place in Chapter 23, and has been updated and revised for this publication. Gene targeting by homologous recombination allows the introduction of specific mutations into any cloned gene. In this unit, the gene of interest is inactivated by interrupting its coding sequence with a positive selectable marker (e.g., neo). Expression of neo is obtained by including the phosphoglycerate kinase (PGK) promoter in the construct. To enrich for clones in which the target gene has undergone homologous recombination over those in which random integration of the construct has occurred, a negative selectable marker, herpes simplex virus thymidine kinase (HSV‐TK), is included in the construct outside the region of homology to the target gene. Depending upon the target gene, it may be easier to assemble the construct by adding the neo and TK genes to the cloned target gene or by adding two fragments of the target gene to a plasmid containing the neo and TK genes.

Reactivation of acyclovir‐resistant thymidine kinase‐deficient herpes simplex virus harbouring single base insertion within a 7 Gs homopolymer repeat of the thymidine kinase gene

HSV infections are treated efficiently and prevented by acyclovir, although resistant strains have been reported. Resistance to acyclovir involves mainly mutations in the viral gene encoding thymidine kinase; mutations may lead to an altered or, more frequently, deficient TK. These acyclovir-resistant TK deficient strains are not able to reactivate from a latent infection in an experimental model, compared to TK positive strains. A case is reported of a bone marrow transplant child who developed HSV infection at 11 days post-transplantation. Acyclovir-resistant HSV 1 was isolated on day 19 post-transplantation. The patient was cured of his infection. A resistant virus was detected 20 months later that harboured the same TK gene mutation as the first resistant virus. This mutation is an insertion of one guanine in a homopolymer repeat of seven guanines located at codon 146 of TK. It has previously been reported and associated with the expression of a deficient TK activity and the ability to reactivate in mice. These results corroborate the clinical relevance of this mutation, which is associated with acyclovir-resistant recurrent infections in humans. J. Med. Virol. 62:247–250, 2000. © 2000 Wiley-Liss, Inc.

Targeted killing of carcinoembryonic antigen (CEA)-producing cholangiocarcinoma cells by polyamidoamine dendrimer-mediated transfer of an Epstein-Barr virus (EBV)-based plasmid vector carrying the CEA promoter.

The present study reports a novel nonviral method to efficiently and specifically target carcinoembryonic antigen (CEA)-producing cholangiocarcinoma (CC) cells in vitro. Epstein-Barr virus (EBV)-based and conventional plasmid vectors were constructed that possess the beta-galactosidase (beta-gal) or herpes simplex virus-1 (HSV-1) thymidine kinase (Tk) genes as well as tandem repeats of the human genomic sequence -82 to -42 bp from the transcriptional start site of the CEA gene. The plasmids were transfected by means of polyamidoamine dendrimer into CEA-positive (HuCC-T1) or -negative cell lines. Transfection of the conventional plasmid vector with the CEA promoter and beta-gal gene resulted in a very low or undetectable level of marker gene expression even in the CEA-positive cell line. Transferring the HSV-1 Tk gene by conventional plasmid did not affect the susceptibility of HuCC-T1 cells to ganciclovir. In marked contrast, strong beta-gal expression was specifically obtained in HuCC-T1 cells by transfecting the EBV-based plasmid in which the CEA promoter and a ubiquitous promoter (SRalpha) are employed to drive the EBV-encoded nuclear antigen 1 (EBNA1) and beta-gal genes, respectively (pTES.beta). Furthermore, CEA-positive but not -negative tumor cells were rendered highly susceptible to ganciclovir when transfected with the EBV-based vector that carries the CEA promoter-EBNA1 and SRalpha-HSV-1 Tk genes (pTES.Tk). These results strongly suggest that the EBV-based plasmid vector/cationic polymer system (EBV/polyplex) equipped with the CEA promoter provides an efficient nonviral method for the targeted gene therapy of CEA-producing malignancies.

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

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.