Biomedical subjects

M Kúdelová

Publications and source records attributed to M Kúdelová.

At least 19 recordsLinked to original sources

PubMed2005

Murine herpesvirus pathogenesis: a model for the analysis of molecular mechanisms of human gamma herpesvirus infections.

Murine herpes virus (MHV), a natural pathogen originally isolated from free-living rodents, constitutes the most amenable animal model for human gamma herpesviruses. Based on DNA sequence homology, this virus was classified as Murid Herpesvirus 4 to subfamily Gammaherpesvirinae. Pilot studies in our laboratory, using mice inoculated by the intranasal route, showed that MHV infects macrophages, B lymphocytes, lung alveolar as well as endothelial cells. From the lungs the virus spreads via the bloodstream to spleen and bone marrow and via the lymphatics to the mediastinal lymph nodes. Similarly to other gamma herpesviruses, MHV established life-long latency maintained in host B lymphocytes and macrophages. An IM-like syndrome (per analogy to EBV) may develop during acute MHV infection, in which the atypical T/CD8+ lymphocytes eliminate viral DNA carrying B cells expressing the M2 latency associated protein. During latency, the MHV LANA (a KSHV LANA homologue) maintains the latent viral genome, assuring its copying and partition to new carrier cells in the course of division of the maternal cell. The nonproductive latency is turned onto virus replication by means of Rta protein. The chronic lymphoproliferative syndrome of unclear pathogenesis, which occurs in a certain part of latent MHV carriers, is related to the expression of gamma herpesvirus common latency-associated genes such as v-cyclin and/or to that of a virus-specific (M11/bcl-2) gene. This review attempts to summarize our knowledge concerning the function of MHV genes (either gamma herpesvirus common or MHV specific) related to immune evasion, latency and lymphoproliferation when highlighting the unsolved problems and/or controversial opinions.

Animals↗

PubMed2004

Expression of herpes simplex virus 1 glycoprotein D in prokaryotic and eukaryotic cells.

Recombinant plasmids encoding either the full-length glycoprotein D (FLgD) or truncated gDs were constructed. The recombinant plasmids were expressed in Escherichia coli and BHK-21 cells. The strongest expression was obtained with the recombinant plasmid encoding a truncated gD which corresponded to the gD ectodomain. The cells transformed with this plasmid showed good exponential growth ensuring satisfactory yields of the expressed polypeptide in the form of the fusion protein. The fusion protein was biotinylated and efficiently purified. The shortest truncated gD, which contained the main continuous antigenic locus VII binding neutralization antibody and additional continuous antibody binding epitopes, still reacted with specific antibody as proven by immunoblot analysis. In addition, a shuttle vector for expression of FLgD in mammalian cells was constructed. This vector-transfected BHK-21 cells expressed gD for 40 days during 9 consecutive passages. The expression of gD began on day 2 and culminated at day 9 post transfection (p.t.).

Animals↗

PubMed2003

Gamma herpesviruses: pathogenesis of infection and cell signaling.

Altered cell signaling is the molecular basis for cell proliferation occurring in association with several gamma herpesvirus infections. Three gamma herpesviruses, namely EBV/HHV-4, KSHV/HHV-8 and the MHV-68 (and/or MHV-72) and their unusual cell-pirated gene products are discussed in this respect. The EBV, KSHV as well as the MHV DNA may persist lifelong in an episomal form in the host carrier cells (mainly in lymphocytes but also in macrophages, in non-hornifying squamous epithelium and/or in blood vessel endothelial cells). Under conditions of extremely limited transcription, the EBV-infected cells express EBNA1 (EB nuclear antigen 1), the KSHV infected cells express LANA1 (latent nuclear antigen 1), while the MHV DNA carrier cells express the latency-associated protein M2. With the full set of latency-associated proteins expressed, EBV carrier cells synthesize additional EBNAs and at least one LMP (latent membrane protein 1). The latent KSHV carrier cells, in addition to LANA1, may express a viral cyclin, a viral Fas-DD-like ICE inhibitor protein (vFLIP) and a virus-specific transformation protein called kaposin (K12). In MHV latency with a wide expression of latency-associated proteins, the carrier cells express a LANA analogue (ORF73), the M3 protein, the K3/IE (immediate early) proteins and M11/bcl-2 homologue proteins. During the period of limited gene expression, the latency-associated proteins serve mainly for the maintenance of the latent episomal DNA (a typical example is EBNA1). In contrast, during latency with a broader spectrum gene expression, the virus-encoded products activate transcription of otherwise silenced cellular genes, which leads to the synthesis of enzymes capable of promoting not only viral but also cellular DNA replication. Thus, the latency-associated proteins block apoptosis and drive host cells towards division and immortalization. Proliferation of hemopoetic cells, which had become gamma herpesvirus DNA carriers, can be initiated and strongly enhanced in the presence of inflammatory cytokines and by virus-encoded analogues of interleukins, chemokines and IFN regulator proteins. At early stages of tumor formation, many proliferating hemopoetic and/or endothelium cells, which had became transcriptionally active under the influence of chemokines and cytokines, may not yet be infected. In contrast, at later stages of oncogenesis, the virus-encoded proteins, inducing false signaling and activating the proliferation pathways, bring the previously infected cells into full transformation burst.

Animals↗

PubMed2001

A simple procedure for expression and purification of selected non-structural (alpha and beta) herpes simplex virus 1 (HSV-1) proteins.

The expression and isolation of herpes simplex virus 1 (HSV-1) immediate early (alpha) IE63 (ICP27) and of the early (beta) thymidine kinase (Tk) polypeptides in Escherichia coli JM 109 cells transformed with the PinPoint Xa-1 (Promega) plasmid construct carrying either the HSV-1 UL54 or UL23 genes are described. The resulting biotinylated fusion protein(s) could be easily induced and were purified in appropriate amounts by means of a monomeric avidin-conjugated resin (SoftLink Soft Release Avidin Resin, Promega) provided that: (1) the exponential growth of the selected transformed cells was monitored carefully; (2) the post-induction harvest interval was properly chosen; and (3) the period for adsorption to the avidin resin suitably adjusted. The isolated protein(s), although partially digested in the case of the IE63 polypeptide, were suitable antigen(s) for immunization of various animal species. Co-purification of trace amounts of endogenous biotinylated protein(s) produced in E. coli was eliminated by shortening the duration of adsorption to the avidin resin.

Electrophoresis, Polyacrylamide Gel↗

PubMed2001

Early shutoff of host protein synthesis in cells infected with herpes simplex viruses.

Herpes simplex viruses 1 (HSV-1) and 2 (HSV-2) are capable of suppressing the host cell protein synthesis even without viral gene expression. This phenomenon is known as the early shutoff or as the virion-associated host shutoff (vhs) to emphasize that it is mediated by a component of infecting virions which is a product of the UL41 (vhs) gene. The UL41 encoded protein is a functional tegument protein also present in light (L) particles and is not essential for virus replication. The major product of UL41 gene is a 58 K phosphoprotein. At least two forms of UL41 protein differing in the extent of phosphorylation are present in HSV-1-infected cells. HSV-2 compared to HSV-1 strains display a stronger vhs phenotype. However, in superinfection experiments the less strong vhs phenotype is dominant. UL41 protein triggers disruption of polysomes and rapid degradation of all host and viral mRNAs and blocks a reporter gene expression without other HSVs proteins. The available evidence suggests that UL41 protein is either itself a ribonuclease (RNase) or a subunit of RNase that contains also one or more cellular subunits. UL41 protein is capable of interacting with a transactivator of an alpha-gene, the alpha-transinducing factor (alpha-TIF). Interaction of UL41 protein with alpha-TIF down regulates the UL41 (vhs) gene activity during lytic infection. The possible role of other viral proteins in the shutoff is discussed.

Cell Line↗

PubMed2001

The UL9 ori-binding protein of herpes simplex virus 1: its expression and localization in vero cells.

The ori-binding protein (OBP), an early protein which is encoded by the herpes simples virus 1 (HSV-1) UL9 gene and initiates the replication of viral DNA, was expressed in Escherichia coli, purified on an avidin resin and used for preparation of a mouse antiserum to OBP (OBP antiserum). Expression and localization of OBP in HSV-1-infected Vero cells was assessed by reverse transcription-polymerase chain reaction (RT-PCR) and indirect immunofluorescence test. RT-PCR revealed the presence of abundant UL9 transcripts from 3 to 12 hrs post infection (p.i). Traces of UL9 mRNA were detected already at 1.5 hr p.i. The OBP antiserum detected clumps of irregularly shaped structures in the nuclei of infected Vero cells first at 4 hrs p.i. These nuclear structures peaked at 5-6 hrs p.i. and later on (at 8-12 hrs p.i.) they changed into fine granules filling the whole nucleus.

Animals↗

PubMed2000

Herpes simplex virus 1 (HSV-1) strain HSZP glycoprotein B gene: comparison of mutations among strains differing in virulence.

The nonpathogenic HSZP strain of HSV-1 induces large polykaryocytes due to a syn3 mutation (His for Arg at residue 858) in the C-terminal endodomain of glycoprotein B (gB) (40). We determined the nucleotide (nt) sequence of the UL27 gene specifying the gB polypeptide of HSZP (gBHSZP) and found 3 mutations in its ectodomain at aminoacids (aa) 59, 79 and 108. The ANGpath virus, which also has a syn3 mutation in the C-terminal endodomain of gB (Val for Ala at residue 855) is pathogenic for adult mice (39), but can be made nonpathogenic by replacing the gBANGpath gene by the corresponding gBKOS sequence (21). The gBANGpath had three ectodomain mutations (at aa 62, 77 and 285), while gBKOS had at least four ectomain mutations (aa 59, 79, 313, and 553). Two mutations (aa 59 and 79) in the latter, located in the variable antigenic site IV/D1 were common for gBKOS and gBHSZP. These together with the gBANGpath mutations at aa 62 and 77 create a cluster of 4 mutations in diverse region of the N-terminal part of gB (between aa 59-79), in which the gBs of pathogenic ANGpath and 17 viruses differ from the gBs of nonpathogenic HSZP and KOS viruses. The lower pathogenicity of KOS as related to gBKOS, is furthermore associated with the change of Ser to Thr at aa 313 (locus III/D2). The possibility is discussed that mutations in both above mentioned antigenic loci could result in higher immunogenicity of the corresponding antigenic epitopes, which, in turn, would contribute to the decreased virulence of HSZP and KOS viruses.

Amino Acid Substitution↗

PubMed2000

Characterization of glycoprotein C of HSZP strain of herpes simplex virus 1.

Sequences of UL44 genes of strains HSZP, KOS and 17 of herpes simplex virus 1 (HSV-1) were determined and the amino acid sequences of corresponding glycoproteins (gC) were deduced. In comparison with the 17 strain, the HSZP strain showed specific changes in 3 nucleotides and in 2 amino acids (aa 139 and 147, both from Arg to Trp) in the antigenic locus LII. The change at aa 147 was situated within the GAG-binding epitope. In a similar comparison, KOS strain had changes in 3 nucleotides and 3 amino acids (aa 3, 14, and 300). The UL44 genes of HSZP and KOS strains were expressed in insect Sf-21 cells by means of the baculovirus (Bac-to-Bac) expression system. As shown by immunoblot analysis, both the recombinant baculoviruses (B1-HSZP and B6-KOS) expressed a glycosylated gC, the M(r) of which (116 K) was lower than that of gC synthesized in Vero cells (129 K) infected with strains HSZP or KOS. In addition, smaller gC-specific proteins (of apparent M(r) of 50-58 K and 98 K) corresponding to a non-glycosylated precursor polypeptide and/or incomplete forms of the partially glycosylated gC were found. When Balb/c mice were immunized with Sf-21 cells expressing gC, the recombinant gC-HSZP represented a more efficient immunogen possibly due to its stronger expression in these cells. The corresponding gC-HSZP antiserum reacted in enzyme-linked immunosorbent assay (ELISA) equally well with HSZP and KOS virion antigens and neutralized HSZP strain at a low titer. Both gC-HSZP and gC-KOS antisera detected the homologous as well as the heterologous gC antigens in Vero cells regardless whether infected with strains HSZP, KOS or 17, revealing the presence of gC from 6 to 16 hrs post infection (p.i.) in the cytoplasm, on the nuclear membrane and at the cell surface.

Animals↗

PubMed1999

Characterization of strain HSZP of herpes simplex virus type 1 (HSV1).

The genetic background of HSZP virus, an HSV1 strain with extensive passage history, was analyzed by parallel comparative sequencing of four relevant genes (UL27/gB, UL41/vhs, UL44/gC and UL53/gK) of HSZP and additional three selected viruses [strains ANGpath, strains KOS(a) and KOS(b) and the prototype strain 17]. Mutation at position 858 (His for Arg) in gB of HSZP was found to be responsible for giant cell formation (syn3gB mutation) similarly as the 855 mutation (Val for Ala) in the gB of ANGpath. No syn1gK mutations were detected in the UL53 gene either of HSZP or of ANGpath viruses. The reduced virulence of HSZP for adult mice after peripheral inoculation, similarly as that of KOS virus, seems to be related (at least in part) to numerous mutations in the gB ectodomain. Of these, two mutations located in the antigenic domain IV were the same in gBHSZP as well as in gBKOS (at amino acids 59 and 79), at least two (amino acids 313 and 553) were specific for gBKOS, while one mutation (Ser for Ala at position 108) was specific for gBHSZP. The abolished shutoff function of the HSZP virus was related to at least four out of six specific mutations seen in the vhs polypeptide (vhsHSZP) encoded by the UL41 gene, of which three (amino acids 374, 386, 392) were clustered in the semiconservative box A of vhsHSZP (the truncation of which abrogates the inhibition provided by this protein) and one mutation (at amino acid 18) was situated in the highly conservative locus I of vhsHSZP. In addition, the two vhsKOS specific mutations (amino acids 19 and 317) not found in vhsHSZP, enhanced the early host shutoff function of the vhsKOS protein. Finally, gCHSZP had two specific mutations (amino acids 137 and 147) located in the antigenic domain II of gC, which is responsible for binding of HSV1 virions to the glycosoaminoglycan (GAG) receptor. When expressed in Sf21 cells using the recombinant baculovirus system (Bac-to-Bac), gCHSZP and gCKOS showed no essential antigenic differences.

Amino Acid Sequence↗

PubMed1999

Interference of the low-pH inactivated herpes simplex virus type 1 (HSV-1) strain HSZP with the early shutoff function of superinfecting HSV-1 strain KOS.

In former studies, we described that the HSZP strain of herpes simplex virus type 1 (HSV-1) was defective with respect to the early shutoff of host protein synthesis but was effective at interfering with the early shutoff function of the HSV-1 strain KOS, even when heat-inactivated or neutralized by antibody. However, the HSZP strain failed to interfere when inactivated with zinc ions or purified from cells treated with 2-deoxy-D-glucose. In this study, we provide evidence that the ability of the purified low-pH inactivated (citrate buffer, pH 3.0) and gel-filtered (Sephadex G-25) HSZP virions to adsorb host cells was not significantly affected. However, their ability to induce interference with the early shutoff function of the superinfecting HSV-1 strain KOS was restricted. In comparison with native virus, up to eight times more low-pH inactivated HSZP virions were needed to interfere with the shutoff by strain KOS. The interference was not due to exclusion of strain KOS by HSZP at the level of adsorption and/or penetration. The restriction was partially overcome by treatment of the cells with polyethylene glycol after adsorption of the low-pH inactivated HSZP virions. This observation indicates that the direct fusion of the virion envelope of low-pH inactivated HSZP with the plasma cell membrane was predominantly hampered.

Animals↗

PubMed1999

Glycoprotein K of herpes simplex virus: a transmembrane protein encoded by the UL53 gene which regulates membrane fusion.

Glycoprotein K (gK) encoded by the UL53 gene is the ninth out of eleven HSV glycoproteins (gps). The precursor gK (pgK) is a transmembrane protein with four hydrophobic domains, which consists of 338 amino acids. The UL53 gene has two initiation codons: the upper overlaps with the UL52 ORF, while the lower is located 55 codons downstream and specifies a truncated precursor of the gK polypeptide. The UL53 gene and the upstream located UL52 gene have a common polyadenylation signal downstream from the UL53 stop codon so that the UL53 mRNA is completely nested within the UL52 transcript. The syn1 mutations in several KOSsyn mutants and in the MPsyn virus, which had been fine mapped to DNA coordinates 0.735-0.740, were later on located to the UL53 gene, especially to its portion which specifies the first 120 amino acids (aa) from the N-terminus (most frequently residue 40) and to a less precisely defined locus between aa 301-310 (close to the C-terminus). Point mutations in the N-terminal ectodomain of gK, which are related to syn formation, impair the putative ability of this region to down-regulate membrane fusion. The two N-glycosylated mannose core oligosaccharides are attached to the Asn residues of the gK polypeptide at positions 48 and 58, respectively. In infected cells, gK is localized mainly in the nuclear and endoplasmic reticulum (ER) membranes. It is not clear, whether gK becomes incorporated into the envelope of mature HSV particles. Studies with the insertion/deletion gK mutants showed the importance of gK for capsid envelopment, for the transportation and egress or virions from infected cells. It seems that gK has an essential role in virion egress, even though this glycoprotein acts in accord with gH and with another membrane protein encoded by the UL20 gene.

Alphaherpesvirinae↗

PubMed1998

Syn strains HSZP and ANGpath [correction of ANG] of herpes simplex virus type 1 do not contain mutations in the regions of UL53 gene relevant to syncytium formation.

Parallel sequencing of UL53 gene of four strains of herpes simplex virus type 1 (HSV-1), two of which (HSZP and ANGpath) were of the syn phenotype while another two (KOS and 17) were of the non-syn phenotype, showed in three strains amino acid mutations unrelated to the already described syn1 glycoprotein K (gK) mutations (Dolter et al., 1994). The only mutations which altered encoded amino acids were found in strains HSZP (Gln to Arg at position 198) and ANGpath (Val to Ile at position 137). Both mutations were localised outside of the two mutation clusters suspected for affecting syncytium formation. In addition, a CG/GC variation was found at positions 245-246 and 669-670. These compressions affected three codons altering amino acids (aa) 82 (Cys or Ser), 223 (Me or Ile) and 224 (Leu or Val), respectively.

Animals↗

PubMed1998

Strain variability of plum pox virus isolates from western Slovakia.

Leaf tissues of stone fruit trees (plum, apricot, peach and myrobalan) carrying symptoms of plum pox virus (PPV) infection and of peach GF 305 seedlings and Nicotiana benthamiana infected experimentally with PPV were assayed for PPV by polymerase chain reaction (PCR). The expected 243 bp PCR products were subjected to restriction fragment length polymorphism (RFLP) analysis with restriction endonucleases AluI and RsaI. All of the PCR products contained the AluI site. The RsaI restriction profiles of the PCR products demonstrated the prevalence of PPV-M subgroup in the tested samples from western Slovakia.

Plum Pox Virus↗

PubMed1997

Herpes simplex virus type 1 (HSV-1) strain HSZP host shutoff gene: nucleotide sequence and comparison with HSV-1 strains differing in early shutoff of host protein synthesis.

The UL41 gene of the HSZP strain of herpes simplex virus type 1 (HSV-1) defective with respect to the early shutoff of host protein synthesis was sequenced and compared with the corresponding HSV-1 strain KOS and 17 gene sequences. In comparison with strain 17, nine mutations (base changes) were HSZP specific, five KOS specific and four were common for both strains. Nine mutations caused codon changes. Three of these mapped to the nonconserved regions and the others to the conserved regions of the functional map of UL41 gene. One KOS specific mutation mapped to the region responsible for the binding of the virion host shutoff (vhs) protein to the alpha-transinducing factor (VP16). The possible relationship between mutations and host shutoff function is discussed. The nucleotide sequence data of the UL41 gene of HSZP and KOS have been submitted to the Genbank nucleotide database and have been assigned the accession numbers Z72337 and Z72338.

Amino Acid Sequence↗

PubMed1996

The syn3 strain HSZP of herpes simplex virus type 1 (HSV-1) is not pathogenic for mice and shows limited neural spread.

Strain HSZP of the herpes simplex virus type 1 (HSV-1) forms large giant cells in vitro. This property was found associated with a mutation that alters the codon CGC (in the strain KOS or 17 sequence) to CAC (in the HSZP sequence), changing the amino acid 857 from arginine to histidine in the cytoplasmic domain of the glycoprotein B (gB) polypeptide chain. Giant cell formation by ANGpath was attributed to a mutation that alters the codon GCC (in KOS and strain 17 sequences) to GTC (in ANGpath sequence) changing the amino acid 854 in the same (syn3) region of the gB molecule. In contrast to the ANGpath virus, which is pathogenic (1 LD50 < 1 x 10(4) PFU) for adult DBA/2 mice after peripheral inoculation, strain HSZP was never found to be lethal for adult mice. Whereas ANGpath-infected mice which survived acute infection frequently (79%) developed latency in the regional sensory ganglion (as proved by virus reactivation during explantation), latent HSZP reactivated in ganglion culture at a considerably reduced rate (21%). Only 10-day-old DBA/2 mice were sensitive to HSZP infection. In these, HSZP spread from the site of peripheral administration mainly by hematogenous route. The neural spread of HSZP in suckling DBA/2 mice was manifested by the involvement of vegetative neurons in the wall of the small intestine and in the retroperitoneal vegetative ganglia. We conclude that HSZP, a polykaryocyte-forming strain with a mutation in the syn3 region II, shows limited neuroinvasity for mice after peripheral administration.

Animals↗

PubMed1996

Determination of the DNA target sequence of poorly reactivable strain HSZP of herpes simplex virus type 1 by polymerase chain reaction.

HSZP strain of herpes simplex virus type 1 (HSV-1)-unlike strains KOS, SC16 and ANGpath-established latency in the homolateral trigeminal ganglion of mice at a limited rate (21%) when tested by reactivation of latent virus in culture. If a nested polymerase chain reaction (PCR) for virus DNA detection was used, the positivity rate was 63-100%. The detection rate of HSZP DNA in acutely infected gangla did not differ from that of SC16 DNA provided that the sensitivity of PCR was below 20 pg of HSZP DNA per 0.5 microgram of total ganglionic DNA. The nested PCR assessed at least 200 fg of HSZP DNA per 0.5 microgram of ganglionic DNA.

Animals↗

PubMed1995

Detection of herpes simplex virus DNA by polymerase chain reaction in the cerebrospinal fluid of patients with viral meningoencephalitis using primers for the glycoprotein D gene.

A novel set of primers for polymerase chain reaction (PCR) which amplified the portion of US6 sequence coding for the main type-common neutralizing epitope of glycoprotein D (gD) was used for detection of herpes simplex virus (HSV) DNA in 44 cerebrospinal fluid (CSF) samples from 29 patients with clinical symptoms of viral meningitis or meningoencephalitis. The primers in question amplified the DNA of 9 out of 10 low-passage HSV-1 isolates and of 5 out of 10 HSV-2 low-passage isolates as well as the DNA of all laboratory strains examined when tested in the supernatant fluid of infected cells cultures. The PCR was positive in 5 CSF samples (taken on days 2, 4, 8, 10 and 56 after the onset of symptoms, but not later than day 8 after starting acyclovir (ACV) therapy) obtained from 4 patients with intrathecal antibody response. The PCR was repeatedly negative in CSF of 15 patients who had antibodies to HSV in serum and CSF, but did not show intrathecal antibody production. It was also negative in 10 patients who had no HSV antibodies in CSF. Our results confirmed that positive PCR for HSV DNA in the CSF is an indication for starting and/or continuing ACV therapy even in the absence of classical symptoms of HSV encephalitis.

Acyclovir↗

PubMed1991

Pathogenicity and latency competence for rabbits of the herpes simplex virus type 1 ANGpath gC and gE defective mutants.

A total of 139 rabbits was infected to the right scarified cornea with HSV type 1 strains Kupka, ANG, ANGpath and their gE defective (ANGpathI2-4), gC defective (ANGpathgC18), gC/gE negative (ANGpathCI-8) and gC/ICP4 deletion (ANGpathY1) mutants. Strains ANG, ANGpath, ANGpathgC18 and ANGpathY1 were, in contrast to the two gE negative mutants, highly lethal, but 79% of rabbits infected with the non-encephalitogenic Kupka strain survived. Strain Kupka and strain ANGpath gE-negative mutants I2-4 and gCI-8 were tested for their latency competence. While Kupka established latency in the homolateral trigeminal ganglia from 80% of infected rabbits, I2-4 did so in one of 10 animals only, and the gC/gE mutant gCI-8 was not harboured in any of infected animals in an inducible form. Significant correlation was found between shedding into the culture fluid of reactivated virus from the explanted ganglion and brain stem fragments at one hand and the presence of the viral DNA in these organs on the other hand as judged by spot blot hybridization with the HSV-1 strain 17 Kpn I fragments h and i to DNA extracts prepared from these organs. Hybridizations were predominantly negative with the DNA from the corresponding non-cultured organs, except in a few cases of non-cultured ganglion and brain stem from rabbits previously infected with the gE deletion mutants which displayed positive hybridization, although no virus reactivation could be observed in corresponding explants.

Animals↗