Comparing ultrastable lasers at 7 × 10-17 fractional frequency instability through a 2220 km optical fibre network.

Ultrastable lasers are essential tools in optical frequency metrology enabling unprecedented measurement precision that impacts on fields such as atomic timekeeping, tests of fundamental physics, and geodesy. To characterise an ultrastable laser it needs to be compared with a laser of similar performance, but a suitable system may not be available locally. Here, we report a comparison of two geographically separated lasers, over the longest ever reported metrological optical fibre link network, measuring 2220 km in length, at a state-of-the-art fractional-frequency instability of 7 × 10-17 for averaging times between 30 s and 200 s. The measurements also allow the short-term instability of the complete optical fibre link network to be directly observed without using a loop-back fibre. Based on the characterisation of the noise in the lasers and optical fibre link network over different timescales, we investigate the potential for disseminating ultrastable light to improve the performance of remote optical clocks.

First industrial-grade coherent fiber link for optical frequency standard dissemination.

We report on a fully bidirectional 680 km fiber link connecting two cities for which the equipment, the setup, and the characterization are managed for the first time by an industrial consortium. The link uses an active telecommunication fiber network with parallel data traffic and is equipped with three repeater laser stations and four remote double bidirectional erbium-doped fiber amplifiers. We report a short-term stability at 1 s integration time of 5.4×10-16 in 0.5 Hz bandwidth and a long-term stability of 1.7×10-20 at 65,000 s of integration time. The accuracy of the frequency transfer is evaluated as 3×10-20. No shift is observed within the statistical uncertainty. We show a continuous operation over five days with an uptime of 99.93%. This performance is comparable with the state-of-the-art coherent links established by National Metrology Institutes in Europe. It is a first step in the construction of an optical fiber network for metrology in France, which will give access to an ultrahigh performance frequency standard to a wide community of scientific users.

Gender influences herpes simplex virus type 1 infection in normal and gamma interferon-mutant mice.

Gender influences the incidence and severity of some bacterial and viral infections and autoimmune diseases in animal models and humans. To determine a gender-based difference, comparisons were made between male and female mice inoculated with herpes simplex virus type 1 (HSV-1) by the corneal route. Mortality was higher in the male mice of the three strains tested: 129/Sv//Ev wild type, gamma interferon (IFN-gamma) knockout (GKO), and IFN-gamma receptor knockout (RGKO). Similarly, in vivo HSV-1 reactivation occurred more commonly in male mice, but the male-female difference in reactivation was restricted to the two knockout strains and was not seen in the 129/Sv//Ev control. Comparison among male mice of the three strains showed a higher mortality of the RGKO mice and a higher reactivation rate of the GKO and RGKO mice than of the 129/Sv//Ev males. In contrast, female RGKO and GKO mice did not differ from female 129/Sv//Ev controls in either mortality or reactivation. HSV-1 periocular and eyelid disease was also more severe in male and dihydrotestosterone (DHT)-treated female mice than in control female mice. These results show a consistent gender difference in HSV-1 infection, with a worse outcome in male mice. In addition, the results comparing GKO and RGKO mice to controls show differences only in male mice, suggesting that some effects of IFN-gamma, a key immunoregulatory molecule, are gender specific.

Gamma interferon (IFN-gamma) receptor null-mutant mice are more susceptible to herpes simplex virus type 1 infection than IFN-gamma ligand null-mutant mice.

Mouse strains with null mutations in the gamma interferon gene (Ifng) or the gamma interferon receptor gene (Ifngr) have been engineered. The use of these strains as animal models of viral and bacterial infections has enhanced our understanding of the role of gamma interferon (IFN-gamma) in the host immune response. However, direct comparisons between Ifng-/- (GKO) and Ifngr-/- (RGKO) mice have been problematic because previously available strains of these mice have had different genetic backgrounds (i.e., C57BL/6 and BALB/c for GKO mice and 129/Sv//Ev for RGKO mice). To enable direct comparison of herpes simplex virus type 1 (HSV-1) infections in GKO and RGKO mice, we introduced the IFN-gamma null mutation into the 129/Sv//Ev background. We report that, after HSV-1 inoculation, mortality was significantly greater in RGKO mice than in GKO mice (38 versus 23%, P = 0.0001). Similarly, the mortality from vaccinia virus challenge was significantly greater in RGKO mice than in GKO mice. With differences in genetic background excluded as a confounding issue, these results are consistent with the existence of an alternative ligand(s) for the IFN-gamma receptor that is also capable of mediating protection against viral challenge.

Role for gamma interferon in control of herpes simplex virus type 1 reactivation.

Observation of chronic inflammatory cells and associated high-level gamma interferon (IFN-gamma) production in ganglia during herpes simplex type 1 (HSV-1) latent infection in mice (E. M. Cantin, D. R. Hinton, J. Chen, and H. Openshaw, J. Virol. 69:4898-4905, 1995) prompted studies to determine a role of IFN-gamma in maintaining latency. Mice lacking IFN-gamma (GKO mice) or the IFN-gamma receptor (RGKO mice) were inoculated with HSV-1, and the course of the infection was compared with that in IFN-gamma-competent mice with the same genetic background (129/Sv//Ev mice). A time course study showed no significant difference in trigeminal ganglionic viral titers or the timing of establishment of latency. Spontaneous reactivation resulting in infectious virus in the ganglion did not occur during latency in any of the mice. However, 24 h after the application of hyperthermic stress to mice, HSV-1 antigens were detected in multiple neurons in the null mutant mice but in only a single neuron in the 129/Sv//Ev control mice. Mononuclear inflammatory cells clustered tightly around these reactivating neurons, and by 48 h, immunostaining was present in satellite cells as well. The incidence of hyperthermia-induced reactivation as determined by recovery of infectious virus from ganglia was significantly higher in the null mutant than in control mice: 11% in 129/Sv//Ev controls, 50% in GKO mice (P = 0.0002), and 33% in RGKO mice (P = 0.03). We concluded that IFN-gamma is not involved in the induction of reactivation but rather contributes to rapid suppression of HSV once it is reactivated.

Genetic and functional complementation of the HSV1 UL27 gene and gB glycoprotein by simian alpha-herpesvirus homologs.

Utilizing co-transfection of DNA from glycoprotein gB- strain of HSV1 and cloned fragments of several simian alpha-herpesviruses containing the UL26, UL27 (gB glycoprotein), and UL28 gene homologs, replication-competent recombinant viruses were produced. Genetic analysis of one HSV1/SA8 recombinant (HSV1/SgB) demonstrated the presence of SA8 DNA comprising the entire UL27 (gB) gene and parts of the UL28 and UL26 ORFs in an otherwise HSV1 genome. The recombinant was shown to express the SA8 gB and p40 proteins (UL27 & UL26.5 gene products, respectively); all other proteins were indistinguishable from those of HSV1. The recombinant behaved like SA8 in gB-specific virus neutralization and cell surface antibody binding assays, while plaque morphology and replication kinetics were very similar to HSV1. Despite its overwhelming HSV1 genetic constitution, the recombinant displayed a pathogenic phenotype in mice very different from the parental HSV1. While HSV1 produced corneal disease in ocularly infected mice and readily spread to the nervous system. HSV1/SgB was markedly impaired in both respects. These results demonstrate the functional equivalency of the cercopithecine monkey virus gB glycoproteins and genes (including transcriptional regulatory elements) in HSV1, the functional nature of HSV1/SA8 chimeric UL28 and UL26 genes/proteins, and that UL28, gB and/or p40 proteins may effect the pathogenicity of HSV1.

Characterization of an essential HSV-1 protein encoded by the UL25 gene reported to be involved in virus penetration and capsid assembly.

The 2.3-kb BamHI-U DNA fragment (map units 0.319-0.335) of herpes simplex virus type 1 (HSV-1) genome contains the complete UL25 open reading frame (ORF). It specifies an essential viral protein reported previously to be involved in virus penetration and capsid assembly (C. Addison, F. J. Rixon, J. W. Palfreyman, M. O'Hara, and V. G. Preston, Virology 138, 246-259, 1984). To identify the protein encoded by the UL25 gene, the UL25 ORF was cloned in a eukaryotic expression vector (p91023) downstream of the adenovirus major late promoter to generate the expression plasmid p9-UL25. Synthesis of a 60-kDa protein was observed in COS-7 cells transfected with p9-UL25 plasmid DNA, but not in cells transfected with p91023 control plasmid DNA. To identify and characterize the UL25 protein from HSV-1-infected cells, we prepared a rabbit antiserum by using UL25-GST fusion protein expressed in Escherichia coli as immunogen. This rabbit antiserum readily immunoprecipitated the 60-kDa UL25 protein from HSV-1-infected cells. In HSV-1-infected cells, UL25 protein was expressed as a late (gamma) or a leaky late (gamma 1) viral protein. The rabbit antiserum raised against HSV-1 UL25 protein immunoprecipitated a UL25-homologue of identical size from HSV-2-infected cells. However, the reactivity of the antiserum with HSV-2 UL25-homologue was weaker than compared to the corresponding HSV-1 protein. Consistent with its classification as a virion component, the UL25 protein was found to be associated with purified HSV-1 virions.

Deletion of the carboxy-terminus of herpes simplex virus type 1 (HSV-1) glycoprotein B does not affect oligomerization, heparin-binding activity, or its ability to protect against HSV challenge.

A recombinant vaccinia virus designated VgBt which expresses a truncated secreted herpes simplex virus gB (gBt) was constructed and compared to V11gB, a vaccinia recombinant previously studied which expresses gB exclusively on the surface of infected cells. Indirect immunofluorescence assay (IFA) revealed that gBt was strongly associated with the surface of infected cells despite being released slowly into the cell culture medium. Both gB and gBt existed as oligomers, and both membrane bound and secreted forms of gBt exhibited heparin-binding activity. In protection studies VgBt and V11gB conferred equivalent protection against both homologous (HSV-1) and heterologous (HSV-2) challenge with HSV.

Gamma interferon expression during acute and latent nervous system infection by herpes simplex virus type 1.

This study was initiated to evaluate a role for gamma interferon (IFN-gamma) in herpes simplex virus type 1 (HSV-1) infection. At the acute stage of infection in mice, HSV-1 replication in trigeminal ganglia and brain stem tissue was modestly but consistently enhanced in mice from which IFN-gamma was by ablated monoclonal antibody treatment and in mice genetically lacking the IFN-gamma receptor (Rgko mice). As determined by reverse transcriptase PCR, IFN-gamma and tumor necrosis factor alpha transcripts were present in trigeminal ganglia during both acute and latent HSV-1 infection. CD4+ and CD8+ T cells were detected initially in trigeminal ganglia at day 5 after HSV-1 inoculation, and these cells persisted for 6 months into latency. The T cells were focused around morphologically normal neurons that showed no signs of active infection, but many of which expressed HSV-1 latency-associated transcripts. Secreted IFN-gamma was present up to 6 months into latency in areas of the T-cell infiltration. By 9 months into latency, both the T-cell infiltrate and IFN-gamma expression had cleared, although there remained a slight increase in macrophage levels in trigeminal ganglia. In HSV-1-infected brain stem tissue, T cells and IFN-gamma expression were present at 1 month but were gone by 6 months after infection. Our hypothesis is that the persistence of T cells and the sustained IFN-gamma expression occur in response to an HSV-1 antigen(s) in the nervous system. This hypothesis is consistent with a new model of HSV-1 latency which suggests that limited HSV-1 antigen expression occurs during latency (M. Kosz-Vnenchak, J. Jacobson, D.M. Coen, and D.M. Knipe, J. Virol. 67:5383-5393, 1993). We speculate that prolonged secretion of IFN-gamma during latency may modulate a reactivated HSV-1 infection.

Herpes simplex virus DNA in normal corneas: persistence without viral shedding from ganglia.

Herpes simplex virus type 1 (HSV-1) DNA has been shown to persist in the cornea not only after inoculation of experimental animals but also in surgical samples from patients with herpes keratitis. The further observation of corneal HSV-1 DNA in subjects without known HSV eye disease prompted the present study of the presence and distribution of HSV-1 in eye bank corneas. Prior to DNA extraction, the corneas were trephined, separating the central and peripheral cornea. With polymerase chain reaction (PCR) for HSV-1 thymidine kinase (TK) and glycoprotein D (gD) gene sequences, we found HSV-1 in 10 of 24 eye bank corneas, from the 4 mm wide corneal rim in 8 eyes and from the 8 mm diameter central cornea in 2 eyes. In 9 subjects, both eyes were assayed, and HSV-1 was detected in 6 subjects. In only one subject was HSV-1 detected in both eyes and in only one subject was HSV-1 detected in the central and peripheral cornea of the same eye. The biological role of HSV-1 DNA corneal sequences is unknown. To investigate this, a rabbit animal model was established by transplantation of corneas containing viral DNA sequences in HSV-1 naive recipients. Followed for 5 months, there was no evidence of sheeding of HSV-1 in the tear film or seroconversion of the recipient rabbits. At the end of this time, HSV-1 DNA was detected in the corneal graft at a similar intensity to the PCR signal from the donor rims.(ABSTRACT TRUNCATED AT 250 WORDS)

Detection of herpes simplex virus DNA sequences in human blood and bone marrow cells.

Herpes simplex virus type 1 (HSV1) establishes latent infections in neural tissues of humans and experimental animals. Utilizing a sensitive polymerase chain reaction (PCR) assay we detected HSV DNA sequences in blood cells of healthy prospective bone marrow transplant (BMT) donors and patients. In three healthy individuals studied, HSV DNA sequences were found in all blood cell types and also in bone marrow cells as well as in stem cell progenitor colonies isolated from in vitro cultures. Studies of BMT donor-recipient pairs suggested that HSV reactivation may occur in hematopoietic cells after transplantation, as the PCR signal intensity increased over time simultaneous with an increased antibody titer to HSV. In a mouse model for HSV infection, HSV DNA sequences were found in blood and bone marrow cells at the latent stage of infection, after intravenous (IV) inoculation, but not after ocular inoculation. These studies suggest that bone marrow cells may be an additional site of HSV latency capable of reactivation after BMT. These studies have broad implications for understanding pathogenesis of HSV disease and are of particular significance in situations where allogeneic bone marrow cells are given therapeutically.

In vitro and in vivo effects of synthetic ribozymes targeted against BCR/ABL mRNA.

Chronic myelogenous leukemia (CML) is associated with a translocation of the BCR and the ABL genes, t(9;22). Results of this event are transcription and translation products that are unique to malignant cells. We therefore designed synthetic ribozymes which are capable of exclusively cleaving the BCR/ABL B3A2-type mRNA without altering normal cellular transcripts. Synthetic B3A2-type transcripts could only be cleaved by B3A2-type mRNA targeted ribozymes and not by any of the controls. The B3A2-type mRNA directed ribozyme, on the other hand, did not cleave any of the control transcripts. The effective delivery of ribonucleic acids by lipofection into K562 cells could be demonstrated by fluorescent microscopy, slot blot analysis, and RNA polyacrylamide gel electrophoresis. In vivo, we were able to induce a significant inhibition of the proliferation of K562 cells with ribozymes directed against the B3A2-type mRNA. Quantitative PCR analyses showed an up to fivefold reduction of the relative number of BCR/ABL mRNA molecules per single cell after exposure to ribozymes compared to controls. We conclude that ribozymes targeted against the B3A2-type BCR/ABL mRNA function in vitro and in vivo.

Antisense oligonucleotides as antiviral agents: prospects and problems.

One possible strategy for the development of antiviral drugs is to synthesize short antisense oligonucleotides that interfere specifically with RNA transcription and processing to prevent expression of protein. This can be readily achieved, but there are formidable technical problems to solve before routine clinical applications can be envisaged.

Persistence of herpes simplex virus DNA in rabbit corneal cells.

Corneal cell cultures were established from the corneas of rabbits killed during a period of latency 118 d after ocular infection with the RE strain of herpes simplex virus (HSV). DNA was isolated from frozen cell pellets of 42 cell cultures that did not develop viral cytopathic effects during 44 d in culture. Using the polymerase chain reaction (PCR) to amplify HSV thymidine kinase (TK) gene sequences, HSV-specific DNA was detected in 15 of 42 culture-negative cell cultures. Subsequent reamplification, using nested primers that were complementary to HSV TK sequences internal to the orginal primers, resulted in eight additional culture-negative samples showing positive hybridization for HSV TK DNA. Twenty three of the 42 virus culture-negative corneal cell cultures tested by PCR were found to contain HSV genetic material. Detailed examination of the clinical histories of the eyes from which the corneal cultures were obtained showed no correlation between increased frequency or severity of epithelial disease, stromal disease, or virus shedding and more frequent isolation of virus or detection of HSV-specific DNA. These studies document that HSV DNA residues in the corneas of HSV-infected rabbits up to 118 d post-infection. About 10% of the eyes contained virus that could be reactivated in culture, whereas an additional 55% of the eyes contained DNA sequences homologous to a portion of the HSV TK gene.

Antiviral effects of herpes simplex virus specific anti-sense nucleic acids.

We have targeted mRNA sequences encompassing the translation initiation codon of the essential herpes simplex virus type 1 (HSV-1) IE3 gene with three kinds of anti-sense molecule. Addition of a 15mer oligodeoxyribonucleoside methylphosphonate to tissue culture cells resulted in suppression of viral replication. HSV-1 replication was also inhibited in cultured cells containing anti-sense vectors expressing transcripts complementary to the IE3 mRNA. We have also constructed a ribozyme which upon base pairing with the target IE3 mRNA induces cleavage at the predicted GUC site. A major obstacle to anti-sense studies in animals is drug delivery of preformed antisense molecules to ganglionic neurons, the site of HSV latency and reactivation. We speculate as to how this may be accomplished through carrier compounds which are taken up by nerve terminals and transported by retrograde axoplasmic flow. By the same route, HSV itself may be used as an anti-sense vector.

Application of polymerase chain reaction assays to studies of herpes simplex virus latency.

We have amplified herpes simplex virus type 1 (HSV-1) DNA sequences from individual latently infected mouse trigeminal ganglia by polymerase chain reaction (PCR) assays. This report presents two useful modifications in the PCR technique. The first involves the use of two sets of closely spaced, oppositely oriented oligonucleotide primers and two rounds of 20-40 PCR cycles, first with the more widely spaced outer primers and then with the internal nested primers. This method enhanced the sensitivity of PCR detection as shown by assays of HSV-1 sequences in human brain. The second modification was designed to detect selectively HSV-1 sense or anti-sense RNA transcripts when both are present by adding a single primer during an initial reverse-transcriptase-mediated cDNA synthesis reaction. After destruction of the RNA template, standard PCR is initiated by the addition of the second primer and thermus aquaticus DNA polymerase (Taq). We show here applications of both of these modifications to amplify HSV-1 sequences from nervous system tissue.

The potential use of catalytic RNAs in therapy of HIV infection and other diseases.

This article describes the applications (both real and potential) of a new antiviral strategy, based on the use of antisense, catalytic RNAs (ribozymes) as therapeutic agents. An understanding of both antisense inhibition of gene expression and RNA autocleavage reactions are essential to the use of this technology. In addition, for the successful application of this technology in clinical settings, an interdisciplinary approach involving clinicians, molecular and cellular biologists, will be necessary. The following treatise will highlight several salient features of ribozyme technology, emphasizing its application as an antiviral as well as discuss some problems and potential solutions pertinent to the clinical application of this technology.

Detection of herpes simplex virus DNA sequences in corneal transplant recipients by polymerase chain reaction assays.

Polymerase chain reaction (PCR) assays were used to amplify herpes simplex virus type 1 (HSV-1) thymidine kinase (TK) sequences in DNA extracted from formalin-fixed, paraffin embedded corneas of patients undergoing corneal transplantation. PCR reamplification with an internal (nested) set of primers was required for detection in 10 of the 12 positive corneas indicating very low abundance of viral sequences. Three of the positive corneal samples were from failed corneal grafts. Overall, TK sequences were detected in 8 of 11 corneas from subjects with a past history of herpes keratitis and in 4 of 11 corneas from subjects with no past history of herpetic eye disease.