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N W Fraser

Publications and source records attributed to N W Fraser.

At least 55 records · Page 3Linked to original sources
PubMed1995

In situ DNA PCR and RNA hybridization detection of herpes simplex virus sequences in trigeminal ganglia of latently infected mice.

The presence of herpes simplex virus (HSV-1) DNA in the trigeminal ganglia of latently infected mice was detected by an in situ DNA polymerase chain reaction (PCR), which includes a DNA:DNA hybridization step (indirect in situ PCR). These results were compared to the number of neurons possessing the HSV-1 latency associated transcript (LAT), as detected by in situ RNA hybridization with LAT probes. Sensitivity assays were shown to detect a single copy cellular gene in 48% of neuronal cell bodies. The results suggest that in situ PCR is an effective method to locate and detect HSV-1 within latently infected neurons. Moreover, the number of neurons found to be harboring HSV-1, by the method of in situ PCR, which does not depend upon virus gene expression, is within threefold of the number detected by in situ hybridization for LAT. Therefore, this report describes the first detection of HSV-1 DNA in latently infected murine trigeminal ganglia by the method of indirect in situ PCR, and compares the findings to the number of neurons expressing LAT, as assessed by conventional in situ hybridization.

Animals↗
PubMed1995

Replication, establishment of latent infection, expression of the latency-associated transcripts and explant reactivation of herpes simplex virus type 1 gamma 34.5 mutants in a mouse eye model.

The herpes simplex virus type 1 (HSV-1) gamma 34.5 gene is located within a region that is transcriptionally active during latent HSV-1 infection. To determine whether the gamma 34.5 gene deletion affects latency-associated transcript (LAT) gene expression or latent HSV-1 infection, a gamma 34.5 gene deletion mutant, 1716, and a stop codon insertion mutant, 1771, were studied in the mouse eye model. Although the gamma 34.5 gene is not essential, 1716 and 1771 replicated poorly in mouse eyes and trigeminal ganglia (TG). When mice were inoculated with 1716, infectious virus was detected in eyes only on the first day post-infection (p.i.), and was not detected at any time point in TG. Following inoculation with 1771, a small amount of virus was detected in the eyes on days 2 and 4 p.i., and in the TG of one animal on day 2 p.i. Reactivation of virus from mice latently infected with 1716 (0/30 TG) and 1771 (1/20 TG) was extremely low compared with the parental strain, 17+, and appropriate rescuants (80 to 100% reactivation), even though latent 1716 DNA was detected by PCR in 50% of TG. These results differ from those obtained following footpad inoculation; in the footpad there was limited 1716 replication and reactivatable latent infection was established in some dorsal root ganglia. The data support the hypothesis that the role of gamma 34.5 may be tissue and/or cell type specific. The synthesis, processing, and stability of the 2.0 kb LAT during 1716 and 1771 replication was not affected by these mutations in the gamma 34.5 gene. However, during latent infection of 1716 in mice the LATs were not detectable in TG by Northern blot, and were present in reduced amounts (approximately 10-fold less) during 1771 latency. The LATs from 1716 were barely detectable in a few neurons by in situ hybridization. Therefore, the gamma 34.5 gene might (i) affect replication in the eye, and reduce the amount of virus available to establish latent infection, be directly involved in (ii) establishment of latency, and/or (iii) the reactivation process.

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PubMed1995

The replicating intermediates of herpes simplex virus type 1 DNA are relatively short.

Herpes simplex virus type 1 (HSV-1) replication is thought to occur via a rolling circle type of mechanism, generating large DNA concatemers from which unit length genomes are subsequently cleaved. In this report, we have employed field inversion gel electrophoresis (FIGE), Southern blot hybridization, and endonuclease digestion, to identify and characterize these DNAs. Two species of HSV-1 DNA: (1) genome-length and (2) DNA that remained at the electrophoresis origin (referred to as well-associated DNA) were detected. To ascertain that the latter was large in size and not virion DNA trapped at the origin with high molecular weight cellular DNA, the infected cell DNA was digested with a restriction enzyme that does not cut the viral DNA. In order to do this HSV-1 strain 1702, lacking any XbaI sites in its genome, was utilized. After digestion of samples with XbaI, and FIGE, cellular DNA was seen to migrate into the gel; however, the viral DNA remained in the sample wells. Pulse labeling experiments showed that this large DNA was processed to 150 kb genome lengths. Endonuclease digestion of the well-associated DNA revealed that it contained a greater ratio of joint to terminal fragments than virion DNA-a characteristic of long concatemers. Quantitation of the terminal fragments revealed mainly L termini. Surprisingly, the ratio of joint to terminal fragments was 2.5 suggesting that the lengths of concatemers were short (in the order of 1-2 genome lengths) and that the well association was due to conformation rather than concatemeric length. Because one of these genome lengths is present as the replication intermediate, the growing tail must be less than genome length. Thus genome lengths must be processed from the replication intermediate soon after they are completed.

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PubMed1995

Therapy of experimental human brain tumors using a neuroattenuated herpes simplex virus mutant.

BACKGROUND: Engineered herpes simplex virus (HSV) strains previously have been shown to offer a potential therapeutic alternative to conventional treatment modalities for brain tumors. Because HSV Type 1 strain 1716 has a deletion in the gamma 34.5 neurovirulence gene that renders it avirulent in the mouse central nervous system, we have assessed its potential to induce selective lysis of tumor cells versus neurons in vitro and in vivo. EXPERIMENTAL DESIGN: To do this, we studied parental HSV-1 strain 17+ and strain 1716 using human embryonal carcinoma cells (NT2 cells). These cells resemble neuronal progenitor cells and can be induced to differentiate into neurons (NT2N) with retinoic acid. Intracerebral grafts of NT2 cells into the brains of nude mice resulted in lethal brain tumors, and grafts of NT2N cells resulted in the integration of NT2N cells. RESULTS: In vitro studies showed that strain 1716 replicates in and spreads on monolayers of NT2 cells but not in NT2N cells. In vivo, strain 1716 replicated preferentially in NT2 tumors as evidenced by immunohistochemical staining for viral antigens, by in situ hybridization for HSV-specific transcripts, and by titration of virus from brains with tumor after intracranial injection of the virus into these mice. The temporal regression of NT2 tumors in mice treated with strain 1716 was demonstrated in vivo by magnetic resonance imaging. Electron microscopy and studies of DNA fragmentation suggested that regression of NT2 brain tumors in strain 1716-treated mice was mainly caused by a nonapoptotic, lytic mode of cell death. Finally, strain 1716-treated NT2 tumor-bearing mice survived more than twice as long as mock-treated tumor-bearing mice, and these differences in survival (25 vs. 9 weeks) were statistically significant (p < 0.03). CONCLUSIONS: We conclude from these studies that strain 1716 induces regression of human neural tumors established in the brains of nude mice, resulting in their prolonged survival.

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PubMed1995

An HSV-1 containing the rat beta-glucuronidase cDNA inserted within the LAT gene is less efficient than the parental strain at establishing a transcriptionally active state during latency in neurons.

The herpes simplex virus vector 17/LAT-RGUSB has previously been shown to express beta-glucuronidase enzyme activity stably in the trigeminal ganglia and brain stems of beta-glucuronidase-deficient mutant mice. However, the number of beta-glucuronidase expressing cells in trigeminal ganglia latently infected with 17/LAT-RGUSB was smaller than expected. Using normal mice for further characterization of 17/LAT-RGUSB latent infection, no appreciable differences were found between the vector and wild-type virus in: (1) their abilities to replicate in acutely infected ganglia; (2) their abilities to reactivate from latently infected ganglia: or (3) the quantities of viral DNA in tissues during the acute or the latent phases of infection. Using a minor LAT (mLAT)-specific probe to detect transcription by in situ hybridization, it was found that the intensity of the signal from individual cells latently-infected with 17/LAT-RGUSB or wild-type virus was similar. However, the vector-infected ganglia had only 20% as many positive cells as in wild-type infection. These data suggest that 17/LAT-RGUSB virus established latency similarly to wild-type virus, but that the LAT-promoter driven gene expression was compromised.

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PubMed1995

Oral inoculation of SCID mice with an attenuated herpes simplex virus-1 strain causes persistent enteric nervous system infection and gastric ulcers without direct mucosal infection.

BACKGROUND: After placement of herpes simplex virus type 1 (HSV-1) into the esophageal lumen of BALB/c mice, the virus replicated in enteric neurons within the esophagus and stomach and was transported to the sensory ganglia of the vagus nerve (nodose ganglia), where viral replication also occurs and where ultimately a long term latent infection is established. This described infection of immunocompetent mice primarily involved neuronal cells and associated satellite cells. EXPERIMENTAL DESIGN: Severe combined immunodeficient (SCID) mice were orally infected with an attenuated strain of HSV-1 to better identify sites of viral involvement in the gastrointestinal tract, particularly the mucosa. RESULTS: Three to five weeks after oral inoculation of SCID mice with HSV-1 strain in1814, a persistent viral infection of the gastrointestinal tract was established in most of the mice. Extensive viral replication was detected by immunohistochemistry throughout pathways of the vagus nerve and within the intrinsic enteric nervous system. Despite this ultimately fatal infection, viral replication in the gut occurred almost exclusively in enteric neurons and their processes; viral proteins were occasionally seen in smooth muscle cells immediately adjacent to heavily infected enteric ganglia. More than 50% of these persistently infected mice, when killed 18 to 31 days postinoculation, had gastric ulcers that were identified grossly and histologically. Only one of the 40 gastric ulcers was found to contain viral Ag. The remaining ulcers, although devoid of viral proteins, were found adjacent to virus-infected ganglia. CONCLUSIONS: HSV-1 can enter enteric neurons with minimal initial mucosal involvement, and once inside the nervous system, the virus is contained there despite the absence of a specific host immune response. Furthermore, chronically infected enteric neurons may provide an indirect mechanism for the pathogenesis of gastric ulcers in these immune-deficient mice.

Administration, Oral↗
PubMed1994

The HSV-1 2-kb latency-associated transcript is found in the cytoplasm comigrating with ribosomal subunits during productive infection.

We have examined the nuclear and cytoplasmic distribution of the latency-associated transcripts (LATs) of HSV-1. During latency these transcripts accumulate in the nuclei of neurons in the peripheral and central nervous system of infected animals. However, our Northern blot analyses demonstrate that the 2-kb LAT is found in the cytoplasm of HSV-1-infected CV-1 cells, and brainstems of HSV-1 productively infected mice. Like the nuclear LAT from latently infected tissue, most of the cytoplasmic 2-kb LAT from lytically infected CV-1 cells is unpolyadenylated. In order to determine if cytoplasmic LAT could be translated, we compared its distribution with that of glycoprotein C mRNA in polysome profiles from HSV-1-infected tissue culture cells. Specific association of RNAs to polysomes was verified by disruption of polysomes with EDTA or puromycin. Analyses of numerous experiments indicate that most of the cytoplasmic 2-kb LAT migrates at the position of ribosomal subunits in polysome profiles. Thus, the 2-kb LAT may not be efficiently translated during productive infection. This suggests that if the 2-kb LAT is indeed translated, its translation may be tightly regulated during HSV-1 infection, possibly in a cell type- or cell cycle-specific manner. Another possibility is that the 2-kb LAT is not a translated RNA but may have another function, possibly related to translation as indicated by its apparent association to ribosomal complexes.

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PubMed1994

N-linked oligosaccharides on herpes simplex virus glycoprotein gD are not essential for establishment of viral latency or reactivation in the mouse eye model.

Glycoprotein D (gD) is an essential component of the herpes simplex virus (HSV) envelope. It is essential for viral penetration and for cell to cell spread of virus in vitro, and is also important for neuroinvasiveness. We investigated the contribution of N-linked oligosaccharides (N-CHO) on gD to viral pathogenesis. We used F-gD(QAA), a mutant virus derived from strain F of HSV-1. This virus contains three mutations in the gD gene which eliminate all signals for addition of N-CHO. These mutations affect the antigenic structure of gD and also lead to a small plaque phenotype. Otherwise the virus appears normal in in vitro assays. We used the mouse eye model of HSV latency to examine whether the mutations alter the phenotype of the virus in vivo. At 4 days postinfection similar amounts of F-gD(QAA) and F-gD(WT), its wild-type parent, were found in either eyes or trigeminal ganglia (TG) of infected mice. Moreover, both mutant and wild-type viruses exhibited the same ability to establish, maintain, and be reactivated from latency. We conclude that N-CHO on gD are not essential for HSV-1 pathogenesis in this model.

Animals↗
PubMed1994

Restricted herpes simplex virus type 1 gene expression within sensory neurons in the absence of functional B and T lymphocytes.

Herpes simplex virus type I (HSV-1) establishes a latent infection in sensory ganglion neurons. During latency no viral-specific proteins are detected and virus gene expression is restricted to the latency-associated transcripts. We report here that trigeminal ganglia of mice with severe combined immunodeficiency contain individual sensory neurons exhibiting restricted viral gene expression characteristic of latency; this occurred during acute (4-6 days) infection with the wild-type HSV-1 strain 17+ and after prolonged (4 weeks) infection with the replication impaired HSV-1 mutant in 1814. These results indicate that T and B lymphocytes, while important for the recovery from viral infections, are not required for the establishment or maintenance of latency in neurons.

Animals↗
PubMed1994

A thymidine kinase-negative HSV-1 strain establishes a persistent infection in SCID mice that features uncontrolled peripheral replication but only marginal nervous system involvement.

A detailed knowledge of the pathogenesis of infections caused by thymidine-kinase (TK)-deficient herpes simplex virus type 1 (HSV-1) strains is important because such mutants can arise during treatment of HSV infections with acyclovir--especially in immunocompromised patients--and also because TK-negative mutants may become useful for the therapy of intracranial tumors. In this work, we studied the pathogenesis of a genetically engineered TK-negative HSV-1 strain dlsptk, in SCID mice (mice with severe combined immunodeficiency) after corneal infection. We found that dlsptk established a persistent infection that kills SCID mice within 80.2 +/- 21.3 days. The cause of death seemed to be related to uncontrolled viral replication in the superficial and deep facial tissues of the animals. Viremia probably did not occur, as judged by the inability to detect infectious virus and viral gene expression in various internal organs. However, the virus did reach the nervous system, most probably by axonal transport from the primary site of the infection. Virus-specific DNA reached low but detectable levels in the trigeminal ganglia and the brainstems by 7 days p.i. and remained at low levels for up to 50 days p.i. as determined by spot blot analysis. By in situ hybridization and immunostaining we determined that, in some of the neurons of the trigeminal ganglia infected by the virus, viral latency was established. However, our results suggested that in other infected neurons viral replication occurred and virus spread to surrounding nonneuronal cells and to the central nervous system. This work provides a new model in which the pathogenesis of infections caused by TK-deficient HSV strains in immunocompromised hosts can be effectively studied and which may also help to identify the potential side effects of the therapy of intracranial tumors with TK-negative HSV strains.

Animals↗
PubMed1994

The herpes simplex virus type 1 strain 17+ gamma 34.5 deletion mutant 1716 is avirulent in SCID mice.

Laboratory animal models are important tools for the identification of avirulent herpes simplex virus type 1 (HSV-1) strains which have potential for use in humans as vaccine strains or gene therapy vectors. We have studied an HSV-1 17+ variant, 1716, that has a deletion in the gamma 34.5 gene and which replicates poorly in the footpads of mice and is unable to grow in the mouse central nervous system or dorsal root ganglia (DRG) of the peripheral nervous system following peripheral inoculation. However, 1716 is known to be capable of establishing latent infections in the DRG of mice. Here we show that 1716 is avirulent after ocular infection and has low virulence after intracranial inoculation in SCID mice. Since SCID mice are much more sensitive to HSV-1 infection than immunocompetent mice, our results clearly demonstrate the drastically reduced virulence of the variant 1716 and provide additional support for the hypothesis that this variant would be avirulent in humans.

Animals↗
PubMed1994

Oral-oesophageal inoculation of mice with herpes simplex virus type 1 causes latent infection of the vagal sensory ganglia (nodose ganglia).

Herpes simplex virus type 1 (HSV-1) gingivostomatitis during childhood is known to result in a latent infection of the trigeminal ganglion neurons, which innervate the oral mucosa. During latency the viral genome is maintained in a non-infectious state. However, stimuli such as stress, fever or localized trauma can cause HSV-1 to reactivate in neurons and produce recrudescent disease in the peripheral tissues. Recently, HSV-1 proteins and nucleic acids have been detected in biopsies from human duodenal and gastric ulcers, raising the possibility that HSV-1 latency within the enteric nervous system is involved in this chronic recurrent gastrointestinal disorder. The studies in mice described here were done to determine whether HSV-1 latency could be established in neurons that innervate the murine gut. We found that after either intraperitoneal or oral-oesophageal inoculation of mice, HSV-1 establishes a latent infection in nodose ganglia of the vagus nerve, whose sensory neurons project to the gastrointestinal tract. This animal model of HSV-1 latency in the vagal sensory ganglia will be useful to examine the possible relationship between HSV-1 and recurrent gastrointestinal disease.

Animals↗
PubMed1994

Long term herpes simplex virus type 1 infection of nerve growth factor-treated PC12 cells.

The behaviour of herpes simplex virus type 1 (HSV-1) strain 17 in tissue cultures of PC12 cells treated with nerve growth factor (NGF) was studied. PC12 cells respond to NGF by ceasing to proliferate and extending long neurites. After differentiation with NGF, cultures were infected with HSV-1 and maintained in the presence of the hormone for several weeks. These long-term infected cultures were tested for HSV DNA, transcripts and the ability to produce virus, before and after NGF removal. Before NGF removal, the cultures were characterized by little or no virus production and the presence of HSV-1 DNA in a predominantly endless form. In situ analysis of long-term infected cultures revealed latency-associated transcript expression in only a portion of the cells. However, as shown by an infectious centre assay, virus was present in almost all cells in the population. Moreover, removal of NGF from long-term cultures resulted in the appearance of significantly increased amounts of virus in the media. The degree to which this system resembles HSV latency in vivo is discussed.

Animals↗
PubMed1994

Herpes simplex virus type 1 gene expression and reactivation of latent infection in the central nervous system.

Restricted gene expression takes place during latent infection of herpes simplex virus type 1 (HSV-1) in the human peripheral nervous system and has been linked with viral reactivation. The state of HSV-1 gene expression in the central nervous system (CNS) during latency is unclear and we, therefore, examined gene expression in the brainstem of experimental mice and normal humans. Only part of the transcription pattern present during latent infection in peripheral sensory ganglia (PSG) was identified in the human brainstem by in situ hybridization and Northern blot analysis for HSV-1-specific transcripts. Instead of three HSV-1 latency-associated transcripts (LATs) present in PSG and demonstrated by Northern blot analysis, only one was identified in mouse brainstem and none was detected in human brainstem. These findings might be attributed to the relatively low amounts of HSV-1-specific latency-associated RNAs in brainstem tissue. Combined with our inability to reactivate HSV-1 from explanted mouse brainstem, these findings suggest that tissue levels of latency-associated gene expression play a role in HSV-1 reactivation and have relevance to the very low incidence of HSV-1-induced CNS disease compared with peripheral mucocutaneous disease.

Animals↗
PubMed1994

Analysis of a herpes simplex virus type 1 LAT mutant with a deletion between the putative promoter and the 5' end of the 2.0-kilobase transcript.

A herpes simplex virus type 1 strain 17 mutant with a deletion between genomic nucleotides 118880 and 119250 was constructed and called 17 delta Sty. The deletion removes most of a putative secondary LAT promoter (called LAPII) as well as 370 of the first 449 nucleotides of the proposed 8.5-kb transcript believed to be the precursor of 2.0-kb LAT. 17 delta Sty was shown to produce major 2.0-kb LATs in tissue culture. Moreover, trigeminal nerves from latently infected mice contained an intact 1.45- to 2.0-kb LAT as well as the minor LATs which are recognized by probes specific for regions downstream of the 2.0-kb LAT. Finally, 17 delta Sty reactivated with normal kinetics from the trigeminal ganglia of latently infected mice in the explant cocultivation assay and egressed from tissue culture cells as efficiently as wild-type virus. These results clearly show that the region deleted in 17 delta Sty is dispensable for intact 2-kb LAT production, viral egress in tissue culture, and normal reactivation from latently infected neurons in mice.

Animals↗
PubMed1994

An in vitro ligation and transfection system for inserting DNA sequences into the latency-associated transcripts (LATs) gene of herpes simplex virus type 1.

This report describes a simple, rapid and highly efficient method for introducing specific DNA sequences into a defined locus of the herpes simplex virus type 1 (HSV-1) genome by restriction enzyme cleavage and ligation. The genome of the HSV-1 strain HFEM contains a 4.1 kb deletion in one copy of the RL region, deleting one copy of the latency-associated transcript (LAT) gene. It does not contain any site for restriction enzyme PacI. Two unique PacI restriction enzyme sites flanking an HSV-1 ICP6 promoter-LacZ reporter gene cassette were engineered into the LAT region to generate a recombinant virus HFEM/ICP6-LacZ which produced blue plaques in the presence of X-gal. This viral vector allowed the insertion of foreign genes directly into the HSV-1 genome by restriction enzyme digestion and ligation. The system was tested by digesting the HFEM/ICP6-LacZ DNA with PacI and with SwaI (an endogenous unique restriction enzyme site upstream of the LAT promoter locus and inserting by in vitro ligation a LAT promoter-LacZ gene cassette into the HFEM/ICP6-LacZ genome. The new recombinant virus HFEM/LAT-LacZ was detected as white plaques in the presence of X-gal, since beta-galactosidase expression, when driven by the LAT promoter, is not detectable during viral replication in tissue culture. The high yield (approximately 100%) of the recombinant virus obtainable from this in vitro ligation and transfection procedure coupled with a blue-white or reversible white-blue plaque detection scheme makes this a powerful method for constructing HSV-1 vectors around the LAT promoter locus.(ABSTRACT TRUNCATED AT 250 WORDS)

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