Biomedical subjects

J G Stevens

Publications and source records attributed to J G Stevens.

At least 55 records · Page 3Linked to original sources

PubMed1988

The herpes simplex virus latency-associated transcript is spliced during the latent phase of infection.

The herpes simplex virus type 1 latency-associated transcript (LAT) is expressed as a major species 2,100 to 2,200 bases in length and a less abundant one ca. 730 bases shorter in latently infected mouse and rabbit neurons. RNA blot hybridization experiments using 20- to 22-base synthetic oligonucleotides and mung bean nuclease protection assays have demonstrated that the smaller LAT species is colinear with the larger one, except for a 730-base intron. On the basis of Northern blot analysis, the spliced species which comprises as much as 50% of the total LAT in latent infections of mice with several strains of herpes simplex virus type 1 and latent infections of rabbits with either the McKrae or the KOS(M) strains of virus is not present in the acute phase of infection. Further and rather surprisingly, in mice latently infected with the KOS(M) strain of virus, the spliced LAT species is considerably less abundant. This suggests that both the strain of virus and the animal in which the latent infection occurs are important in either the processing or stability of spliced LAT. Finally, an exhaustive series of experiments failed to provide convincing evidence that a unique, poly(A)+ species of LAT exists in the latent phase of infection.

Acute Disease↗

PubMed1988

Physical characterization of the herpes simplex virus latency-associated transcript in neurons.

RNA transfer (Northern) blot analysis was used to perform the physical characterization of the transcript expressed in murine sensory nerve ganglia latently infected with herpes simplex virus type 1. Most of this latency-associated transcript (LAT) was isolated in the poly(A)- fraction from ganglia. A smaller RNA species was also detected at less than 10% the abundance of the major one. LAT was not detected with probes from DNA outside the limits of the larger species. In situ hybridization data correlated well with Northern blot analysis; however, low levels of hybridization were seen with probes immediately outside the region of viral DNA giving positive Northern blot signals. S1 nuclease and primer extension mapping were used to locate the 5' end of the LAT 510 bases to the left of a KpnI site at 0.783 map units. The 3' end of the major latency-associated species was mapped to just within a 310-base-pair SmaI fragment located 660 to 970 base pairs to the right of the SalI site at 0.790 map units. These data were correlated with an analysis of the sequence of the DNA encoding this transcript and its possible function in the latent phase of infection.

Amino Acid Sequence↗

PubMed1988

Localization of a herpes simplex virus neurovirulence gene dissociated from high-titer virus replication in the brain.

Previous studies with the herpes simplex virus type 1 X type 2 intertypic recombinant RS6 suggested that the genomic region from 0.11 to 0.14 map units is involved in neurovirulence (R. T. Javier, R. L. Thompson, and J. G. Stevens, J. Virol. 61:1978-1984, 1987). To study this further, we isolated an RS6-derived herpes simplex virus intertypic recombinant (R13-1) which has a genetic defect within this area. After inoculation into mouse brains, R13-1 was found to be approximately 10,000-fold less neurovirulent than either the wild-type type 1 or type 2 parental virus. However, R13-1 replicated in the mouse brain to titers resembling those of the wild-type parents. Further comparisons with wild-type counterparts indicated that R13-1 expressed equivalent levels of the enzyme thymidine kinase and replicated to intermediate levels in primary mouse embryo fibroblasts maintained at the normal body temperature for mice. Using marker rescue techniques combined with in vivo selection, we found that recombination between unit-length R13-1 DNA and a cloned type 1 DNA fragment spanning the region from 0.11 to 0.14 map units (EcoRI-d, 0.079 to 0.192 map units) generated viruses with a wild-type neurovirulence phenotype. To further refine the genomic region of interest, we performed marker rescue experiments using two EcoRI-d subclones, EcoRI/BamHI dc (0.079 to 0.143 map units) and BamHI/EcoRI and (0.143 to 0.192 map units), representing the left and right halves of the EcoRI d fragment, respectively. In these experiments the EcoRI/BamHI dc clone, but not the BamHI/EcoRI ad clone, yielded recombinant viruses exhibiting wild-type neurovirulence. These results show that at least one herpes simplex virus gene function associated with neurovirulence is located within a 9.1-kilobase region at 0.079 to 0.143 map units of the viral genome. Perhaps more significantly, the results indicate that this neurovirulence property functions independently of high-titer virus replication in the brain.

Animals↗

PubMed1988

Pathogenesis of a lethal mixed infection in mice with two nonneuroinvasive herpes simplex virus strains.

We previously reported that simultaneous inoculation of mice on abraded rear footpads with two nonneuroinvasive viruses (herpes simplex virus type 1 ANG and KOS) resulted in the deaths of 62% of the animals (R. T. Javier, F. Sedarati, and J. G. Stevens, Science 234:746-748, 1986). In the current study, to better understand the events responsible for the pathogenesis of this virus mixture, we investigated replicative capacity and spread of the virus mixture within specific tissues. We found that, compared with neuroinvasiveness of ANG or KOS alone, neuroinvasiveness of the virus mixture related to significantly increased amounts of the virus within spinal cords and brains of the mice. This finding indicates that ANG and KOS have defects in their capacities to spread and replicate within spinal cords. We also examined whether the increased neuroinvasiveness of the virus mixture related to complementation between viruses in tissues of the nervous system, generation and selection of neuroinvasive recombinants, or both. It was found that, although neuroinvasive recombinant viruses could be detected in the spinal cords of the infected animals, most of the viruses (both recombinants and nonrecombinants) isolated from all tissues tested were nonneuroinvasive (i.e., no mice died as a result of footpad infection with high doses of such plaque-purified isolates). As a result of these findings, we propose that the virulence of the virus mixture is a consequence of the complementation as well as the generation and selection of neuroinvasive recombinants in spinal cords of these mice.

Animals↗

PubMed1987

RNA complementary to a herpesvirus alpha gene mRNA is prominent in latently infected neurons.

In initial attempts to define the molecular events responsible for the latent state of herpes simplex virus, in situ hybridization was utilized to search for virally encoded RNA transcripts in latently infected sensory neurons. The use of cloned probes representing the entire viral genome indicated that transcripts encoded within terminal repeats were present. When the alpha genes encoding ICP-0, ICP-4, and ICP-27 and the gamma 1 gene encoding VP-5 were employed, only RNA transcripts hybridizing to the ICP-0 probe were detected. In latently infected cells, the ICP-0--related transcripts were localized principally in the nucleus; this was not the case in acutely (productively) infected neurons or in neurons probed for RNA transcripts coding for actin. In Northern blotting experiments, an RNA of 2.6 kilobases was detected with the ICP-0 probe. When single-stranded DNAs from the ICP-0 region were used as probes, RNA from the strand complementary to that encoding ICP-0 messenger RNA (mRNA) was the major species detected. This RNA species may play a significant role in maintaining the latent infection.

Animals↗

PubMed1987

Biological basis for virulence of three strains of herpes simplex virus type 1.

Herpes simplex virus type 1 (HSV-1) strains F, HF and HFEM were studied with respect to pathogenicity in mice and growth characteristics in vivo and in vitro compared to the neurovirulent HSV-1 strains 17 syn+ and KOS. All three viruses demonstrated reduced virulence in mouse brains and were completely avirulent after footpad inoculation. They were shown to express high levels of thymidine kinase activity. Investigations concerning the virulence phenotype indicated that the defect(s) in strains F, HF and HFEM related to general replication deficiency in mouse cells. It was also shown that although the replication restriction observed for strains F and HF was specific for murine cells, strain HFEM did not replicate well in any cell type tested. Additional studies indicated that the avirulence phenotype which followed peripheral inoculation was related to different genotypes, since strain F complemented HF and HFEM and, as expected, the latter two agents did not complement each other. All three agents were found to complement the non-neuroinvasive strain KOS. Finally, the data also show that two herpesviruses which are highly restricted in murine cells (e.g. strains F and HF) can still interact in the animal and produce a lethal infection.

Animals↗

PubMed1987

Genetic and biological analyses of a herpes simplex virus intertypic recombinant reduced specifically for neurovirulence.

RS6 is a herpes simplex virus intertypic recombinant derived from type 1 strain 17 syn+ and type 2 strain HG52. With a 50% lethal dose of about 10(5) PFU after intracerebral inoculation of mice, RS6 was approximately 100,000 times less neurovirulent than either of its wild-type parental viruses were. When compared with strains 17 syn+ and HG52, RS6 replicated intermediately in primary mouse embryo fibroblasts in vitro at 38.5 degrees C (mouse temperature) and to wild-type peak titers in mouse feet in vivo. In contrast, following intracranial inoculation of mice, RS6 replicated significantly less well than did either of its parental viruses in brains. The genetic defect(s) responsible for the reduced neurovirulence of RS6 was stable after in vitro and in vivo serial passage, was not manifested as temperature-sensitive plaquing in vitro, and did not affect thymidine kinase expression. These data indicate that RS6 has a genetic defect(s) specifically affecting its ability to replicate in the mouse brain. Using marker rescue technologies, we increased the neurovirulence of RS6 and localized one genetic determinant(s) involved with the reduced neurovirulence of this agent to 0.72 to 0.87 map units (and, tentatively, to 0.79 to 0.83 map units) of the herpes simplex virus genome. When coupled with the work suggesting that thymidine kinase expression is essential for efficient replication in nerve tissues and earlier reports from this laboratory and others, the results presented in this study indicate that more than one herpes simplex virus gene is involved with neurovirulence.

Animals↗

PubMed1987

Defining herpes simplex genes involved in neurovirulence and neuroinvasiveness.

In this discussion, strategies for physically mapping HSV gene functions associated with significant biologic effects are presented and discussed. They represent an application of molecular biological technologies to complex biologic systems, and are a beginning step in understanding the fundamental basis of herpetic disease. Future studies will involve precise identification of relevant genes, and studies of the important properties of products encoded by these genes.

Animals↗

PubMed1986

Two avirulent herpes simplex viruses generate lethal recombinants in vivo.

While it is widely appreciated that infection with a virulent virus can produce disease in an animal, the ability of a mixture of avirulent viruses to produce disease by means of complementation or recombination in vivo has not been established. In this study, two weakly neuroinvasive herpes simplex virus type 1 (HSV-1) strains were simultaneously inoculated onto the footpads of mice. Many (62%) of the animals that received a 1:1 mixture of the viruses died, whereas the animals that received a similar or 100-fold higher dose of each agent alone survived. Of fourteen viruses isolated from the brains of ten mice that died after receiving the mixture of the two weakly neuroinvasive viruses, eleven were recombinants; three of these recombinants were lethal when reapplied to the footpads of mice. These results show that two avirulent HSV-1 variants may interact in vivo to produce virulent recombinants and a lethal infection. They also suggest that different genetic lesions account for the weakly neuroinvasive character of the HSV-1 strains ANG and KOS after footpad inoculation.

DNA Restriction Enzymes↗

PubMed1986

Herpes simplex virus immediate-early protein ICP4 in murine models of latency.

Herpes simplex virus-1 (HSV) infection of trigeminal and dorsal root ganglia was established in mice via corneal scarification and footpad injection, respectively. Trigeminal and dorsal root ganglia were removed during the acute and latent stages of infection and processed for the immediate-early HSV polypeptide ICP4 (VP175) using both a monoclonal reagent and polyclonal antiserum and the avidin biotin complex immunoperoxidase method. ICP4 (VP175) antigen was readily detected during the acute infection of both dorsal and trigeminal ganglia, but not during latency. This antigen could again be detected by reactivation of the latent virus by explanation and organ culture. The detection of ICP4 (VP175) during latency in a rabbit model but not in a murine model may correlate with biologic differences in each system (e.g., rabbits spontaneously reactivate). Alternatively, the discrepancy could reflect viral strain characteristics or may imply that ICP4 (VP175) need not be constitutively expressed (at detectable levels) in all models of latent infection.

Animals↗

PubMed1986

A herpes simplex virus mutant is temperature sensitive for reactivation from the latent state: evidence for selective restriction in neuronal cells.

When the replicative defect in the HSV-1 temperature sensitive mutant tsI was repaired, the agent derived (RI-1) was found to possess an additional temperature sensitive lesion limiting its reactivation from the latent state. Thus, when spinal ganglia from latently infected mice were scored for reactivation by cocultivating them with indicator cells in vitro, significantly more were found to be positive at 31 degrees than at 38.5 degrees. To assess a possible relationship between reactivation and replication in neurons, the replication of RI-1 in murine C1300 neuroblastoma cells was studied. In these cells, RI-1 was severely restricted, and viral replication was delayed at 38.5 degrees. Serial passage of RI-1 in neuroblastoma cells at the restrictive temperature resulted in selection of an agent which gained both the capacity to replicate efficiently in neuroblastoma cells and reactivate from the latent state at 38.5 degrees. However, the replication pattern of this neuron adapted virus in mouse embryo fibroblasts remained unchanged from the parental RI-1. Taken together, these results indicate that RI-1 possesses a neuron specific temperature sensitive replicative lesion which is also manifest during reactivation from the latent state.

Animals↗

PubMed1986

Passage of herpes simplex virus type 1 on chick embryo fibroblasts confers virulence for chick embryos.

The pathogenesis of chorioallantoic membrane (CAM) infection with herpes simplex virus 1 and 2 (HSV-1 and HSV-2) as well as chick embryo fibroblast (CEF) passaged HSV-1 was studied. It was found that HSV-2 is at least a million-fold more virulent than HSV-1 as measured by pfu/LD50 ratios for the embryo. Serial passage of HSV-1 in vitro on CEF cells selected for a virus (CEFP10) which, unlike its parental HSV-1 strain, is able to kill the embryo. The restriction endonuclease maps of CEFP10 and its parental strain are indistinguishable and the mechanism of the increased virulence of CEFP10 was demonstrated to be its enhanced replication in chick embryo cells both in vivo and in vitro. In contrast, despite its inferior replicative ability, HSV-2 was found to have a biologically important specific invasiveness function that is not simply related to overall viral replication. Finally, the ability to isolate HSV-1 (CEFP10) virulent for the chick embryo after passage in vitro illustrates that tissue culture passage of HSV in appropriate cells may actually increase virulence for the animal host.

Animals↗

PubMed1986

Vaccine potential of a live avirulent herpes simplex virus.

We previously described a herpes simplex intertypic recombinant that is completely and specifically non-neurovirulent following intracranial inoculation. In this report the vaccine potential of this mutant (RE6) is analyzed. It is shown that RE6 replicates to wild type levels at the peripheral site of inoculation (the mouse footpad) but, unlike its virulent parental strains, does not replicate to detectable levels in lumbosacral ganglia. A single vaccination with RE6 is sufficient to completely protect mice against subsequent challenge with either HSV-1 or HSV-2. However, this agent establishes latent infections efficiently, and is fully neurovirulent for newborn mice.

Animals↗

PubMed1986

Functional and molecular analyses of the avirulent wild-type herpes simplex virus type 1 strain KOS.

It has been documented that KOS, a laboratory strain of herpes simplex virus type 1, is several orders of magnitude less neurovirulent than most other wild-type strains. Studies initiated to determine the functional nature of the block to neuroinvasiveness and to establish the genes involved have determined that, after footpad inoculation of mice, strain 17 syn+ induced neuropathologic signs (paralysis) at titers of 10(2) and yielded a PFU/50% lethal dose ratio of 10(4). In contrast, KOS was not lethal and did not induce paralysis at inoculation doses of 10(8) PFU. This reduced neurovirulence of KOS could not be explained by the lack of thymidine kinase activity, its inability to replicate in mouse cells maintained in culture at 38.5 degrees C, or its inefficient replication in nonneural tissues in vivo. Kinetic experiments tracing the virus through the nervous system after footpad inoculation showed that KOS was blocked at the level of the spinal ganglia. A cosmid library of strain 17 syn+ was utilized in recombination and in vivo selection experiments with strain KOS to establish the genomic region involved in 17 syn+ neuroinvasiveness. A cosmid clone containing the HindIII A fragment (0.25 to 0.53 map units) of strain 17 syn+ in mixed transfections with full-length KOS DNA yielded recombinants with enhanced neuroinvasiveness.

Animals↗

PubMed1985

Rescue of a herpes simplex virus type 1 neurovirulence function with a cloned DNA fragment.

A herpes simplex virus type 1 (HSV-1) genetic function that is required for viral replication in the murine central nervous system was unambiguously localized. Thus, cosmid clones of either HSV-1 HindIII fragment C (0.64 to 0.87 map units) or fragment B (0.64 to 0.83 plus 0.91 to 1.0 map units) were employed to restore neurovirulence to an intertypic recombinant (RE6) that is specifically deficient in this property. The neurovirulent recombinants were generated in cell culture by cotransfecting the clone fragments and unit-length RE6 DNA and then selected in mouse brains. Either fragment efficiently conferred neurovirulence to RE6, demonstrating that no short region unique sequences are required. Analyses of the genomic structures of the neurovirulent recombinants showed that, in every case, HSV-1 information from 0.71 to 0.83 map units was incorporated into the RE6 genome. Cleavage of HindIII fragment C with EcoRI eliminated its capacity to rescue RE6. Virulence could be restored by the addition of HSV-1 BamHI fragment L (0.71 to 0.74 map units) that spans an EcoRI site at 0.72 map units. The precise location of this HSV-1 neurovirulence function is discussed.

Animals↗

PubMed1984

Latent herpesviruses of humans.

The herpesviruses that infect humans characteristically establish a latent infection that may be reactivated later. The consequences of reactivation range from asymptomatic shedding to severe disseminated infection. Varicella-zoster and herpes simplex viruses are both highly neurotropic, establishing nonreplicating infections in sensory ganglia. Latent herpes simplex virus is known to reside in neurons, and the virus-cell interactions involved have been defined to an extent. Cytomegalovirus and Epstein-Barr virus interact with peripheral blood leukocytes. Latent cytomegalovirus infection of human leukocytes has not been proved, although studies in a murine model have implicated B lymphocytes as a repository of latent virus. Epstein-Barr virus is known to persist in a non-replicating state as extrachromosomal DNA in B lymphocytes and to cause "immortalization" of the infected cell; persistence of the viral genome in epithelial cells may also result in malignant transformation, such as nasopharyngeal carcinoma.

Animals↗

PubMed1983

Shope papilloma virus DNA is extensively methylated in non-virus-producing neoplasms.

The extent of viral DNA methylation in non-virus-producing benign and malignant tumors induced by the Shope (rabbit) papilloma virus was investigated with the two isoschizomeric restriction endonucleases MspI and HpaII. The overall extent of methylation is variable in papillomas and uniformly higher in carcinomas. In a transplantable Shope virus-induced carcinoma and in two transplantable carcinoma derived cell lines, the viral DNA is even more highly methylated than in carcinomas induced by the virus directly. However, even in these cells at least two MspI sites remain essentially unmethylated and these sites are also unmethylated in carcinomas induced by the virus directly. At least one additional MspI site is unmethylated in papillomas but is partly methylated in carcinomas. Viral DNA from papillomas, which consists mostly of unit-sized form I and form II DNA and from carcinomas in which large oligomeric forms predominate was separated into fractions containing viral plasmids of different size and conformation. The analysis of the different fractions showed that methylation patterns do not correlate with the size of viral DNA, but they rather correlate with the degree of neoplasia.

Animals↗

PubMed1983

Biological characterization of a herpes simplex virus intertypic recombinant which is completely and specifically non-neurovirulent.

In this study, a pre-existing "library" of Herpes Simplex Virus (HSV) intertypic recombinants was found to be not useful for mapping HSV genes controlling viral neurovirulence in mice. Most of these agents were significantly less virulent than either parental type following intracranial inoculation, and in the general case this lessened virulence could be attributed to inefficient replication in any cell type at 38.5 degrees (the normal temperature of the mouse). One agent tested (recombinant RE6) was completely non-neurovirulent following intracranial inoculation of as much as 3.2 X 10(7) PFU. Since about 10 PFU of either 17 Syn+ or HG52 (the "parental" strains of this recombinant) were lethal for mice, RE6 is at least 10 million-fold less neurovirulent than the wild-type strains from which it was produced. The function of the defective gene(s) in RE6 is not yet known, but it is not required for the expression of viral thymidine kinase, efficient replication in cultured cells at 38.5 degrees, or replication in non-neural mouse tissue in vivo. Therefore, the defect in RE6 is in an HSV gene function(s) which is absolutely required for neurovirulence but not for general viral replication. Several possibilities for the molecular nature of the defect in RE6 are discussed.

Animals↗