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H C Kaerner

Publications and source records attributed to H C Kaerner.

At least 19 recordsLinked to original sources
PubMed1994

Replacement of glycoprotein B gene in the herpes simplex virus type 1 strain ANGpath DNA by that originating from nonpathogenic strain KOS reduces the pathogenicity of recombinant virus.

Herpes simplex virus type-1 (HSV-1) strain ANGpath and its recombinants, in which the 8.1 kbp BamHI G restriction fragment (0.345-0.399) containing the glycoprotein B (gBpath) gene (UL27) or its subfragments-coding either for cytoplasmic or surface domains of gB-had been replaced with the corresponding fragments from nonpathogenic KOS virus DNA (gBKOS), were tested for their pathogenicity for DBA/2 mice and rabbits. The recombinant ANGpath/B6KOS prepared by transferring the 2.7 kbp SstI-SstI subfragment (0.351-0.368) of the BamHI GKOS fragment still had the original sequence of ANGpath DNA coding for the syn3 marker in the cytoplasmic domain of gB and was pathogenic for mice as well as for rabbits. Virological and immunohistological studies in DBA/2 mice infected with the latter pathogenic recombinant and with ANGpath showed the presence of infectious virus and viral antigen at inoculation site (epidermis, subcutaneous connective tissue and striated muscle in the area of right lip), in homolateral trigeminal nerve and ganglion, brain stem, midbrain, thalamic and hypothalamic nuclei. In contrast, nonpathogenic recombinants ANGpath/syn+B6KOS (prepared by transferring the whole BamHI GKOS fragment) and ANGpath/syn+KOS (prepared by transferring the 0.8 kbp BamHI-SstI subfragment of the BamHI GKOS fragment) showed limited haematogenous and neural spread, but no evidence of replication in CNS; thus, their behaviour resembled that of the wild type strain KOS. The recombinant ANGpath/syn+KOS, which was not pathogenic for mice, still remained pathogenic for rabbits, a phenomenon indicating the presence of an additional locus in the gB molecule participating on virulence. Sequencing the 1478 bp SstI-SstI subfragment of the BamHI G(path) fragment (nt 53,348-54,826 of UL segment) showed the presence of at least 3 mutations as compared to the KOS sequence, from which the change of cytosine to thymine at nt 54,251 altered the codon for arginine to that for histidine (amino acid 515) in the gB polypeptide chain.

Animals↗
PubMed1994

Regions US6 and US7 of herpes simplex virus type 1 DNA encoding glycoproteins D and I may influence neuroinvasivity.

Recombinants were prepared by replacing a 1931 bp region of the BamHI J fragment (0.906-0.920) of the pathogenic ANGpath DNA-coding for glycoprotein D (gD) and a part of glycoprotein I (gI)--by the corresponding sequence of nonpathogenic KOS DNA (Kaerner et al., 1991) and tested in DBA/2 mice. The strain ANGpath and the control recombinant ANGpath/gD-gIpath, prepared by back transfer of the given ANGpath DNA fragment into ANGpath/gD-gIdellacZ+ DNA, were pathogenic after intraperitoneal inoculation. In contrast, mice infected with the strain KOS and the low-pathogenic recombinant ANGpath/gD-gIKOS survived peripheral virus administration. Both the strain KOS and the low-pathogenic recombinant ANDpath/gD-gIKOS spread by bloodstream to spleen, liver and adrenal glands but did not multiply in spinal cord. Nevertheless, the antigen of low-pathogenic recombinant ANGpath/gD-gIKOS was found in retroperitoneal vegetative nerves and ganglia. On the other hand, the strain ANGpath and the pathogenic recombinant ANGpath/gD-gIpath multiplied in cerebrospinal nerves and spinal cord causing typical hind leg paralysis.

Animals↗
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↗
PubMed1990

Spread of herpes simplex virus (HSV) strains SC16, ANG, ANGpath and its glyC minus and GlyE minus mutants in DBA-2 mice.

Herpes simplex virus type 1 (HSV-1) strains SC16, ANG, its pathogenic variant ANGpath and the mutants ANG-pathgC18 glycoprotein C (glyC) negative and ANGpathI2-4 (glyE negative) were compared for their ability to spread in DBA-2 mice after peripheral inoculation. Virus infectivity assay in 9 organs at days 2, 3, 4, 5, 6, and 10 post-infection (p.i.) and morphologic examinations (immunofluorescence, PAP staining) showed the following: SC16, ANG, and ANGpath spread first (days 2-3 p.i.) by haematogenic route to spleen, liver, and adrenal gland. Since day 4 the invasion of the vegetative and peripheral nervous system took place in SC16 and ANGpath-infected mice, followed by virus spread to the spinal cord and brain stem. In ANG-infected mice the invasion of peripheral nervous system was minimal although both ANG as well as ANGpath spread along the axons. In ANG pathC18-infected mice a relatively prolonged viraemic phase (days 2-4 p.i.) represented with foci of virus antigen-containing cells in spleen, liver, and mesenterial connective tissue was accompanied with a low grade invasion of the peripheral nervous system (days 3-4 p.i.). No spread by any route of ANGpathI2-4 was observed after intraperitoneal inoculation. When comparing ANGpath and SC16, the latter seemed slightly more lethal, since ANGpath killed 67.2% of DBA-2 mice which were given 2 X 10(6) PFU/0.1 ml by i.p. route as compared to the 100% lethality of SC16-infected animals.

Animals↗
PubMed1990

Latency competence of herpes simplex virus strains ANG, ANGpath and its gC and gE minus mutans.

The latency competence of herpes simplex virus type 1 (HSV-1) strains SC16, KOS, ANG, ANGpath and its mutants ANGpathgC18 (gC minus, spontaneous point mutation), KOSgC39 (gC minus deletion), ANGpathI2-4 (gE minus deletion), and ANGpathgCI-8 (gE and gC minus double mutan) was compared and DBA/2 mice. While the latent SC16 and KOS reactivated spontaneously in explanted homolateral trigeminal ganglion fragments coming from Velaz DBA/2 mice, methylation inhibitor 5-azacytidine (5-AzaC) was required to achieve reactivation of SC16 in the ganglion explants from Hannover DBA/2 mice. Reactivation of ANGpath in the cultured trigeminal ganglia from both lines of DBA/2 mice occurred only in the presence of the drug. The compound also enhanced the reactivation incidence in the ganglion explants from ANG-infected Hannover DBA/2 mice but not from Velaz DBA/2 mice: in the latter it remained low even in the presence of the inducer. Both gE- mutants failed to establish latency as judged by the failure of reactivation either in the presence or the absence of 5-AzaC. This seemed in accordance with the absence of neural (quick axonal) spread of these mutans in mice (Rajcáni et al., 1990). In contrast, both gC- mutans established latency: ANGpathgC18 at an unchanged rate and KOSgC39 at a lower frequency than the parent strain.

Animals↗
PubMed1989

A viable HSV-1 mutant deleted in two nonessential major glycoproteins.

A HSV-1 recombinant lacking the two major glycoproteins gC and gE was isolated from cells co-infected with mutants negative for only one of these glycoproteins. The deletions of the appropriate genes were shown to be the same as on the respective parental genomes. In cell culture, the gC/gE minus recombinant virus replicated to titers similar to those obtained with the gC minus parental strain.

Chromosome Deletion↗
PubMed1987

Replacement of glycoprotein B gene sequences in herpes simplex virus type 1 strain ANG by corresponding sequences of the strain KOS causes changes of plaque morphology and neuropathogenicity.

DNA sequences encoding glycoprotein B (gB) derived from herpes simplex virus type 1 (HSV-1) strain KOS321 were transferred to HSV-1 ANG. In cotransfection experiments the cloned HSV-1 KOS BamHI G fragment served as donor, and genomic DNA of two ANG variants as recipients. One of these variants, HSV-1 ANG path, expresses gC and the other, C18, was a spontaneous gC-negative mutant. Both ANG strains are of the syncytial (syn) phenotype whereas HSV-1 KOS321 is non-syncytial (syn+). Recombinants were identified by means of a monoclonal antibody which selectively recognizes gBKOS. Among the HSV-1 ANG path/gBKOS recombinants, the majority displayed an altered plaque morphology, i.e. they were of the syn+ phenotype. In contrast all of the C18/gBKOS recombinants were of the syn phenotype. The possibility that the mutant C18 carries a syn mutation not present in the parental strain could be excluded. Marker transfer experiments involving subfragments of the gB gene mapped the syn mutation of HSV-1 ANG path to a locus within the gene that has been previously termed syn 3. Subclones of HSV-1 ANG path were established either directly or after intermittent transfection or cotransfection with the KOS BamHI G fragment. The pathogenicity in mice of these clones was compared. The data obtained indicated that at high frequency, the BamHI G fragment confers apathogenicity.

Animals↗
PubMed1987

Herpes simplex virus type 1 glycoprotein E is not indispensable for viral infectivity.

A mutant of the herpes simplex virus type 1 Angelotti was isolated in which 87% of the coding region of glycoprotein E (gE) was deleted and replaced by a functional neomycin resistance gene of the Tn5 transposon. The mutant was characterized by restriction enzyme analyses and Southern blotting. Western blotting of proteins and immunofluorescence assays revealed that gE was completely absent and that the Fc receptor was not expressed in cells infected with the mutant. The fact that this mutant was viable and that it replicated to a slightly lower titer than did the wild-type virus suggests that the presence of gE is not a prerequisite of viral infectivity in tissue culture.

Cell Line↗
PubMed1986

The DNA replication origins of herpes simplex virus type 1 strain Angelotti.

The nucleotide sequences of the origins of DNA replication (ori) of the S- and L-component (oriS, oriL) of the herpes simplex virus type 1 (HSV-1) standard genome were determined from HSV-1 strain Angelotti (ANG). In contrast to other HSV-1 strains, the ANG oriS sequence revealed an insertion of an TA-dinucleotide in an otherwise very conserved but imperfect palindromic sequence of 47 bp. The oriL sequence of the standard ANG genome was found to be identical to that of an ANG class II defective genome which exhibits a duplication of a 144 bp palindrome. A model is presented to explain the origination of the amplified ANG oriL sequences from the parental genome with a single copy of oriL via illegitimate recombination. Alignment of the ori sequences of HSV, adeno- and papovaviruses unveiled that the HSV ori region can be subdivided into two distinct sites of homology to the DNA initiation signals of papova- and adenoviruses, suggesting that the HSV origins of replication comprise elements for DNA replication by both, cellular and virus-encoded DNA polymerases.

Animals↗
PubMed1986

Neurovirulence and latency in inbred mice of two HSV-1 intrastrain variants of divergent pathogenicity.

The pathogenicity pattern of the HSV-1 strain ANG which is nonencephalitogenic in mice is compared with that of a selected neurovirulent variant of this strain in DBA-2 mice. After i.p. inoculation both variants replicate to high titers in the mouse peritoneum and build up a virus reservoir in the spleen. Both viruses have no effect on visceral mouse organs other than the spleen; both viruses lead to an inefficient and masked viraemia and both replicate efficiently in CNS tissue after direct intracranial injection. Only the pathogenic variant, however, spreads to the CNS and leads to lethal encephalitis upon intraperitoneal infection. The assumption that infection of the CNS would be mediated by hematogenous transport is not supported by the data obtained from transfer and cocultivation experiments with lymphocytes or experiments involving artificial viraemia. In a model to analyse the capacity of the viruses to invade nerve axons and to induce a latent infection both viruses were found to be latency positive in dorsal root ganglia. It is clear that non-neurovirulent HSV-1 strains are subjected to a postganglionic block of virus spread from the periphery to the CNS. The experiments led to the hypothesis that axonal transport even beyond the dorsal root ganglia to the CNS proceeds unrestricted, whereas lethal CNS invasion is prevented by a restriction of viral replication of HSV-1 ANG in the CNS by a virus-induced host defence mechanism.

Animals↗
PubMed1985

A herpes simplex virus type 1 mutant with a deletion in the polypeptide-coding sequences of the ICP4 gene.

A deletion mutant derived from herpes simplex virus type 1 (HSV-1) strain ANG was analysed. The deletion mapped within the polypeptide-coding region of the immediate-early ICP4 gene. Based on DNA sequence data the deletion was shown to comprise 84 base pairs. In the wild-type genome of strain ANG these sequences were almost completely homologous to the known sequences of HSV-1 strain 17. The ICP4 polypeptide induced by the mutant was similar in size to the wild-type ICP4 protein and was recognized by a monoclonal antibody against ICP4. The data presented suggest that the deletion corresponds to a region on the ICP4 polypeptide that is nonessential for the replication of the virus in vitro.

Base Sequence↗
PubMed1985

Passive immune protection by herpes simplex virus-specific monoclonal antibodies and monoclonal antibody-resistant mutants altered in pathogenicity.

Virus-neutralizing monoclonal antibodies specific for 13 different genetically defined epitopes of glycoproteins gC, gB, and gD of herpes simplex virus type 1, strain KOS-321, were compared for their ability to provide passive immunity to DBA-2 mice challenged intracranially. Protection was highly specific, since individual monoclonal antibodies failed to protect against infection with monoclonal antibody-resistant (mar) mutants altered in the single epitope recognized by the injected antibody. The dose-response kinetics of passive immunity paralleled the in vitro neutralization titers for each antibody. No correlation was observed between immune protection and antibody isotype or complement-dependent in vitro neutralization titers. This suggests that virus neutralization was not the protective mechanism. In general, antibodies reactive with epitopes of gC were protective at the lowest antibody doses, antibodies specific for gB were less efficient in providing immunity, and antibodies against gD were the least effective. mar mutants with single epitope changes in gC and multiple epitope changes in gB showed highly reduced pathogenicity, requiring up to 5 X 10(6) PFU to kill 50% of infected animals. These findings indicated that antigenic variation affects virus growth and spread in the central nervous system. Thus, mutations which affect antigenic structure also can alter virus pathogenicity. The alteration of these epitopes does not, however, appreciably reduce the development of resistance to infection. Infection of mice with these mutants or inoculation of mice with UV-inactivated, mutant-infected cells before challenge rendered the animals resistant to infection with wild-type herpes simplex virus type 1.

Animals↗
PubMed1984

Sequence of the putative origin of replication in the UL region of herpes simplex virus type 1 ANG DNA.

Interest has been stimulated concerning the region mapping between 0.38 and 0.42 on the prototype configuration of the herpes simplex virus type 1 (HSV-1) genome due to the high probability of the presence there of a second origin of DNA replication. A 960 bp restriction fragment (HinfI E) of a class II defective HSV-1 ANG DNA has been sequenced using the viral DNA rather than molecularly cloned DNA. This fragment includes the BamHI U/R cleavage site, mapping at approximately 0.4. Part of the sequence derived in this study displays homology with the origins of DNA replication contained in TRS/IRS of HSV-1 and HSV-2 DNA. The homologous region comprising 76 bp occurs as two copies, each of which contains two palindromically arranged copies of an 8 bp sequence identical to the 'consensus' sequence reported to be part of the origin of DNA replication at the terminus of the mammalian adenoviruses. It can be deduced from a comparison of this structure to the TRS/IRS origin of HSV-1 and HSV-2 that there are two origins of replication in the UL region of HSV-1 ANG DNA. Assuming that the orientation of the consensus sequence is relevant to the direction of DNA replication, one can conclude that the UL origin(s) of HSV-1 ANG is (are) bidirectional. It has not yet been possible to clone DNA fragments molecularly which include the region spanning the UL origin(s) of HSV-1 DNA.

Base Sequence↗
PubMed1983

Herpes simplex virus defective genomes: structure of HSV-1 ANG defective DNA of class II and encoded polypeptides.

Sequence organization and origin of HSV-1 strain Angelotti (ANG) class II defective DNA (HSV-1 ANG dDNA1) were examined in detail by establishing physical maps and by molecular cloning. dDNA1 consists of concatemers of tandem repeat units in which sequences from the UL region spanning map coordinates 0.37 to 0.415 of standard HSV ANG DNA are covalently linked to TRS/IRS sequences. The size of the repeat unit was determined to be about 8.9 kilobase pairs (kb), comprising sequences of 7.3 kb from UL and 1.6 kb from TRS/IRS regions. UL sequences were delineated by restriction enzyme sites KpnI N-P and EcoRI F-M, and were colinear with the corresponding sequences of the standard (wild-type) virus genome. Expression of dDNA1 was studied in African green monkey kidney cells and in Xenopus laevis oocytes. A major polypeptide of approx. mol. wt. 135 000 (135K) was overproduced, suggesting that this protein was encoded by dDNA1. By several parameters, e.g. size, immune cross-reactivity, and affinity for native and denatured DNA, the 135K polypeptide was identified as the major HSV DNA-binding protein. It was further shown that the repeat unit contains part of the DNA polymerase gene as demonstrated by its ability to rescue some mutations in this gene.

Base Sequence↗
PubMed1983

Neuropathogenicity of herpes simplex virus in mice: protection against lethal encephalitis by co-infection with a non-encephalitogenic strain.

Intraperitoneal infection of susceptible mice with an apathogenic herpes simplex virus type 1 (HSV-1) strain prevented the lethal outcome of a challenge infection with a pathogenic strain, even if the challenge preceded the protective infection. It was found that the protective inoculation blocks the initial replication of the challenge virus. In addition, intraperitoneal infection with the protective HSV-1 strain led to the induction of a refractory state in the central nervous system, resulting in resistance to direct intracranial infection with HSV-1. This state is also inducible locally by intracerebral inoculation of a non-replicating mutant virus. The results indicate that HSV-1 strains differing in neurovirulence may differ in the induction or the sensitivity to this protective effect. Experiments with non-replicating HSV-1 temperature-sensitive strains demonstrated that protection against lethal infection does not depend on replication or expression of late genes of the protective strain. Inoculation of animals with detergent-soluble extracts of infected cells or infected and u.v.-irradiated syngeneic cells protected the animals against co-infection with encephalitogenic challenge virus. The experiments define this protective effect as an antigen-induced-immediate host defence mechanism active within 24 h post-infection.

Animals↗
PubMed1983

Genetic variability of herpes simplex virus: development of a pathogenic variant during passaging of a nonpathogenic herpes simplex virus type 1 virus strain in mouse brain.

Herpes simplex virus type 1 ANG (HSV-1 ANG) is originally nonpathogenic for inbred mice upon intraperitoneal intravenous, or intravaginal inoculation. In contrast, mice died of encephalitis within 4 to 5 days after intracerebral inoculation with this strain. HSV-1 ANG was serially passaged in mouse brains. In two independent series, peripherally pathogenic virus variants had developed and accumulated in the virus progeny after 12 to 15 intracerebral passages. In mixed infections both nonpathogenic and pathogenic viruses replicated at the primary site of infection and spread to various organs. However, only the pathogenic phenotype could be recovered from the spinal cord and the brain. Comparison of the restriction enzyme cleavage patterns of pathogenic ANG and nonpathogenic ANG virus DNAs revealed distinct alterations in the S-segment (US) sequences bounded by coordinates 0.953 and 0.958 in the prototype orientation and by coordinates 0.862 to 0.867 in the IS orientation of the viral genome. However, it is not known whether these alterations are physiologically relevant to the observed changes in pathogenicity. When coinjected intraperitoneally at 50 to 100-fold excess, the nonpathogenic HSV-1 ANG protected mice against its own pathogenic variant as well as against other pathogenic HSV-1 strains. Pathogenic HSV-1 ANG proved to be genetically and phenotypically stable for at least 25 serial passages in tissue culture at either high or low multiplicity of infection.

Animals↗