A mouse model for varicella-zoster virus latency.

Following primary infection with varicella-zoster virus (VZV), the virus establishes a latent infection in humans. The molecular pathogenesis of VZV latency is not well understood, mainly due to the lack of an adequate animal model. We report here that we have developed a mouse model for VZV infection that involves corneal inoculation of mice. Although infected animals showed no signs of disease, most of the animals could not eliminate the virus early after infection. By PCR, we demonstrated that at 33 days post-infection (p.i.), viral DNA was still present in more than 60% of the animals (14/21). VZV DNA was most frequently detected in the trigeminal ganglia (7/14) followed by the brain stem (10/21), kidneys (4/21), spleen (3/20), liver (2/21) and brain (1/21). By in situ hybridization, a few cells positive for VZV mRNA were detected in the trigeminal ganglia, brain stem, cerebellum and spleen of a small number of the infected animals as late as 33 days p.i. No viral proteins were detected at the site of inoculation or in any other tissue by immunostaining. Our results suggest that VZV spreads in mice by both viraemia and axonal transport and establishes a non-productive (latent) infection.

The HSV-1 latency associated transcript (LAT) variants 1704 and 1705 are glycoprotein C negative.

The latency associated transcripts (LAT) of herpes simplex virus type 1 (HSV-1) are encoded by diploid genes that are expressed during latent infections. Two LAT variant viruses, 1704 and 1705, were compared with the parental strain 17+. Variant 1705 has a deletion affecting one copy of the LAT genes, expresses LATs during latent infection and reactivates normally. Variant 1704 has deletions affecting both LAT gene copies, does not express LATs during latent infection, and its reactivation is impaired (Steiner et al., 1989). Comparison of infected cell proteins by immunoprecipitation and Western blot analysis revealed one significant difference between HSV-1 strain 17+, 1704 and 1705; glycoprotein C (gC) was not synthesized by 1704 or 1705. Since both 1704 and 1705 are gC minus, the reactivation defect of 1704 is most likely related to the absence of the LATs, and not to the absence of gC.

Retroviral sequences related to human T-lymphotropic virus type II in patients with chronic fatigue immune dysfunction syndrome.

Chronic fatigue immune dysfunction syndrome (CFIDS) is a recently recognized illness characterized by debilitating fatigue as well as immunological and neurological abnormalities [Straus, S.E. (1988) J. Inf. Dis. 157, 405-412]. Once thought to be caused by Epstein-Barr virus, it is now thought to have a different but unknown etiology. We evaluated 30 adult and pediatric CFIDS patients from six eastern states for the presence of human T-lymphotropic virus (HTLV) types I and II by Western immunoblotting, polymearse chain reaction, and in situ hybridization of blood samples. The majority of patients were positive for HTLV antibodies by Western blotting and for HTLV-II gag sequences by polymerase chain reaction and in situ hybridization. Twenty nonexposure healthy controls were negative in all assays. These data support an association between an HTLV-II-like virus and CFIDS.

A review of the molecular mechanism of HSV-1 latency.

The neurotropic herpes viruses, as typified by herpes simplex virus type 1, are noted for their ability to form latent infections. The latent infection differs from the acute infection both in gene expression and the physical state of the viral genome. Latency can be divided into several stages--establishment, maintenance of reactivation--each of which are active areas of research. This review describes the molecular biology of HSV-1 latency and presents the current level of understanding of the molecular mechanism of HSV-1 latency.

Value of bile acid determination for the diagnosis of neonatal jaundice.

Serum concentrations of bile acids and bilirubin, and activity of alanine transferase and alkaline phosphatase as well as bile acid and bilirubin levels in duodenal contents were determined in 90 infants aged 1-44 weeks (including 49 under 10 weeks of age) admitted to hospital for prolonged jaundice. Infants with extrahepatic cholestasis were found to have statistically higher serum bile acid and bilirubin concentrations. Oral administration of cholestyramine produced a statistically significant decrease in serum bile acids and bilirubin in infants with intrahepatic cholestasis under 10 weeks of age. In 24 out of the 30 infants with biliary tract obstruction total absence of bile acids in the duodenal contents was demonstrated while in the others the concentration did not exceed 0.2 mmol/l. The mean bile acid concentration in infants with intrahepatic cholestasis was 2.81 mmol/l while in 8 infants out of the 60 bile acids were either absent or present in trace amounts. The method had an 84.4% sensitivity.

Antigenic cross-reaction between a varicella-zoster virus nucleocapsid protein encoded by gene 40 and a herpes simplex virus nucleocapsid protein.

Human sera from varicella-zoster virus (VZV) and herpes simplex virus type 1 (HSV-1) seropositive individuals contain antibody to a 155-kilodalton (155 kDa) viral protein. In this study, we show that monoclonal antibodies (mAb10.1 and mAb1A1.4) prepared against VZV and HSV-1 proteins, respectively, reacted with nuclear antigens and recognized a 155 kDa protein in the infected cells. Immunoprecipitation of whole virions and viral nucleocapsids with these mAbs showed that the 155 kDa protein is located in VZV and HSV-1 nucleocapsids. In addition, immunofluorescence and cross-reaction experiments revealed the antigenic cross-reactivity between the VZV and HSV-1 155 kDa nucleocapsid proteins. To map the coding region of the VZV 155 kDa protein, a truncated DNA fragment from the predicted open reading frame 40 was cloned into an in vitro transcription vector (pGEM). The RNA transcribed from the inserted DNA was translated in vitro and immunoprecipitated with mAb10.1. The reactivity of the in vitro translation products with mAb10.1 indicated that the 155 kDa nucleocapsid protein is encoded by VZV gene 40. These findings demonstrated that the VZV 155 kDa nucleocapsid protein encoded by gene 40 induces humoral response which cross-reacts with both VZV and HSV.

Detection of HSV-1 proteins prior to the appearance of infectious virus in mouse trigeminal ganglia during reactivation of latent infection.

Following corneal inoculation of mice HSV-1 produces an acute infection and establishes a latent infection in trigeminal ganglia. The latent virus can be reactivated in vitro by explantation of ganglionic tissue. Viral protein expression was studied in trigeminal ganglia during acute infection of mice and explant reactivation of latent infection. HSV-1 proteins were detectable by immunoprecipitation and immunostaining, in mouse ganglia only from 3-5 days post infection. Although during explant reactivation it has been demonstrated that at 24 h post-explant the trigeminal ganglia are all infectious virus negative (Spivack, O'Boyle II and Fraser (1987) J. Virol. 61, 3288-3291), we have found that three HSV-1 proteins, of 175 kDa, 110 kDa and 90 kDa, are present in latently infected trigeminal ganglia as early as 6-21 h post explantation. Initially, only neuronal cells were positive by immunostaining with anti HSV-1 polyclonal serum for HSV-1 antigens, but at later times HSV-1 antigens were seen in non neuronal cells as well. These proteins may play a role in the initial stages of the reactivation process.

Human choroid plexus cells can be latently infected with human immunodeficiency virus.

The human immunodeficiency virus (HIV) penetrates the central nervous system, particularly the cerebrospinal fluid, early in the course of HIV infection, and may cause a progressive encephalopathy in patients prior to the development of the acquired immunodeficiency syndrome. Neither the specific mechanism for penetration of the virus into the central nervous system nor the pathophysiological basis for these abnormalities is well understood. We cultured cells from the choroid plexus of 3 individuals who died of causes unrelated to the acquired immunodeficiency syndrome and demonstrated that these cells can be infected with type 1 HIV. Infection of cells of the choroid plexus may provide an initial route of entry of HIV into the cerebrospinal fluid and, together with the macrophage, a route of entry into the brain.

herpes simplex virus infection accelerates the age-related reactivity of mouse trigeminal ganglia neurons with anti-mouse IgG antibody in immunostaining.

The detection of viral proteins is a major goal of research on herpes simplex virus type 1 (HSV-1) latency. We used immunostaining to detect viral proteins in neuronal cells of trigeminal ganglia of Balb/c mice after corneal inoculation with HSV-1 virus. Viral proteins were detected in the neurons during the acute stage of infection, i.e. within one week after inoculation. However, the detection of viral antigens at the latent stage of HSV-1 infection has proven difficult. We have detected age-dependent non-specific reactivity with anti-mouse IgG antibody in the neurons of 10-week-old or older uninfected mice. This reactivity is accelerated in HSV-1 infected mice, being seen at 6 weeks of age (2 weeks post infection). The accelerated reaction and impact of this effect is discussed in relation to detection of viral proteins during latency.

Recognition of similar epitopes on varicella-zoster virus gpI and gpIV by monoclonal antibodies.

Two monoclonal antibodies, MAb43.2 and MAb79.0, prepared against varicella-zoster virus (VZV) proteins were selected to analyze VZV gpIV and gpI, respectively. MAb43.2 reacted only with cytoplasmic antigens, whereas MAb79.0 recognized both cytoplasmic and membrane antigens in VZV-infected cells. Immunoprecipitation of in vitro translation products with MAb43.2 revealed only proteins encoded by the gpIV gene, whereas MAb79.0 precipitated proteins encoded by the gpIV and gpI genes. Pulse-chase analysis followed by immunoprecipitation of VZV-infected cells indicated reactivity of MAb43.2 with three phosphorylated precursor species of gpIV and reactivity of MAb79.0 with the precursor and mature forms of gpI and gpIV. These results indicated that (i) MAb43.2 and MAb79.0 recognize different epitopes on VZV gpIV, (ii) glycosylation of gpIV ablates recognition by MAb43.2, and (iii) gpIV is phosphorylated. To map the binding site of MAb79.0 on gpI, the pGEM transcription vector, containing the coding region of the gpI gene, was linearized, and three truncated gpI DNA fragments were generated. RNA was transcribed from each truncated fragment by using SP6 RNA polymerase, translated in vitro in a rabbit reticulocyte lysate, and immunoprecipitated with MAb79.0 and human sera. The results revealed the existence of an antibody-binding site within 14 amino acid residues located between residues 109 to 123 on the predicted amino acid sequences of gpI. From the predicted amino acid sequences, 14 residues on gpI (residues 107 to 121) displayed a degree of similarity (36%) to two regions (residues 55 to 69 and 245 to 259) of gp IV. Such similarities may account for the binding of MAb79.0 to both VZV gpI and gpIV.

Molecular cloning of human T-cell lymphotrophic virus type I-like proviral genome from the peripheral lymphocyte DNA of a patient with chronic neurologic disorders.

Human T-cell lymphotropic virus type 1 (HTLV-I), the etiologic agent of human T-cell leukemia, has recently been shown to be associated with neurologic disorders such as tropical spastic paraparesis, HTLV-associated myelopathy, and possibly with multiple sclerosis. In this communication, we have examined one specific case of neurologic disorder that can be classified as multiple sclerosis or tropical spastic paraparesis. The patient suffering from chronic neurologic disorder was found to contain antibodies to HTLV-I envelope and gag proteins in his serum and cerebrospinal fluid. Lymphocytes from peripheral blood and cerebrospinal fluid of the patient were shown to express viral RNA sequences by in situ hybridization. Southern blot analysis of the patient lymphocyte DNA revealed the presence of HTLV-I-related sequences. Blot-hybridization analysis of the RNA from fresh peripheral lymphocytes stimulated with interleukin 2 revealed the presence of abundant amounts of genomic viral RNA with little or no subgenomic RNA. We have cloned the proviral genome from the DNA of the peripheral lymphocytes and determined its restriction map. This analysis shows that this proviral genome is very similar if not identical to that of the prototype HTLV-I genome.

Induction of antibody against in vitro translation products encoded by varicella-zoster virus glycoprotein genes.

Antibodies were raised in rabbit against the in vitro translation products encoded by the varicella-zoster virus (VZV) glycoprotein genes gpI and gpIV. The antisera neutralized VZV infectivity and specifically identified two late VZV glycoproteins, gpI and gpIV, in VZV-infected cells and in the envelope of VZ virions. Pulse-chase experiments revealed a 55K precursor protein to gpIV (60K) and a 82K precursor protein to gpI (95K). Immunoprecipitation of 32P-labeled VZV-infected cells showed that the precursor-products of gpI are phosphorylated. These results demonstrate that translation products synthesized in vitro can be used to produce antibodies that recognize native viral proteins and therefore facilitate the identification and analysis of viral gene products in the infected cells.

HTLV-I infection of cerebrospinal fluid T cells from patients with chronic neurologic disease.

Antibodies reacting with HTLV-I, the etiologic agent of acute T cell leukemia/lymphoma and a transforming agent for T4-positive lymphocytes in vitro, have recently been described in sera of patients with chronic neurologic disease in the absence of lymphoproliferative disorders. The largest number of such cases was described in Japan and in the Caribbean and parts of South America. We report here two cases of patients with chronic neurologic disease whose cerebrospinal fluid (CSF)-derived T cells contain HTLV-I specific RNA sequences and antigens and are expressing retroviral particles. Only one of these patients has demonstrable antibody to HTLV-I in serum or CSF.

New common nomenclature for glycoprotein genes of varicella-zoster virus and their glycosylated products.

The accumulation of recent data concerning the reactivity of monoclonal antibodies with particular varicella-zoster virus (VZV) glycoproteins and the mapping of several of their respective genes on the VZV genome has led to a unified nomenclature for the glycoprotein genes of VZV and their mature glycosylated products. Homologs to herpes simplex virus glycoprotein genes are noted.

Possible relationship between antigenic properties and isolation history of HSV-1 strains.

29 monoclonal anti-herpes simplex virus (HSV)-1 antibodies were produced and characterized with regard to virus neutralizing activity, intracellular or cell-surface location of viral antigens and, where possible, molecular weight of the viral protein recognized. 13 antibodies recognized viral antigens expressed on the surface of infected cells and 16 were directed to intracellular viral components. Only two antibodies exhibited virus neutralizing activity. Application of these antibodies to an antigenic comparison of standard laboratory HSV-1 strains F, HFEM, mP, Glasgow-17 and MAC revealed unique antigenic differences among these strains. The antibodies were further used in an antigenic comparison of 45 human HSV-1 isolates with defined isolation history. Except for two paired isolates from left and right trigeminal ganglia of two human cadavers, the antibody panel revealed antigenic differences among all isolates, including paired isolates from three additional cadavers. Overall, isolates from different human donors showed greater antigenic dissimilarity from each other in cell-surface associated than in intracellular antigens. The data suggest the possibility of a correlation between antigenic and biologic properties of HSV-1.

Affinity-purified varicella-zoster virus glycoprotein gp1/gp3 stimulates the production of neutralizing antibody.

Varicella-zoster virus glycoprotein gp1/gp3 was purified by affinity chromatography using anti-gp1/gp3 monoclonal antibody 19.1 linked to CNBr-activated Sepharose CL-4B. Rabbits immunized with purified glycoprotein gp1/gp3 developed mono-specific neutralizing antibody.

Analysis of three late varicella-zoster virus proteins, a 125,000-molecular-weight protein and gp1 and gp3.

Two monoclonal antibodies were prepared against varicella-zoster virus proteins. One of the monoclonal antibodies (10.2) reacted only with the nuclei of infected cells and immunoprecipitated one nonglycosylated late viral protein (125,000 molecular weight). The other monoclonal antibody (19.1) with neutralizing activity, reacted with membrane antigens of infected cells and with the varicella-zoster virus envelope and immunoprecipitated two late major viral glycoproteins (gp1 and gp3). Synthesis of the 125,000-molecular-weight protein, gp1, and gp3 began at 20 to 22 h postinfection, 2 h after the peak of viral DNA synthesis, and continued until 29 h postinfection, when the first progeny virus appeared in infected cells. Pulse-chase experiments showed that during pulse-labeling, only gp1 was detected, whereas during the chase period, gp1 as well as gp3 was detected in infected cells. Under nonreducing conditions, gp3 migrated in sodium dodecyl sulfate-polyacrylamide gel electrophoresis as a 130,000-molecular-weight protein as compared with the 62,000-molecular-weight species obtained when gels were resolved under reducing conditions. This finding indicates that gp3 is a dimer that is disulfide linked.

Experimental demyelination produced by the A59 strain of mouse hepatitis virus.

Intracerebral inoculation of 4- to 6-week-old C57BL/6 mice with the A59 strain of mouse hepatitis virus (MHV), a murine coronavirus, produced biphasic disease. Acute hepatitis and mild meningoencephalitis were followed by subacute spastic paralysis with demyelinating lesions in the brain and spinal cord as determined by Epon-embedded toluidine-blue-stained sections and by electronmicroscopy. MHV-A59 was cultured by plaque assay from the blood, brain, spinal cord, and liver of infected mice during the acute phase, but not in the chronic stage. MHV-A59 antigen was detected by immunofluorescence (IF) until 3 months postinfection (PI). Serum anti-MHV-A59 antibodies were detected from 7 days to 5 months PI. The induction of demyelination by MHV-A59 provides a suitable system to study virus-induced demyelination further.

CSF antibodies to myelin basic protein and oligodendrocytes in multiple sclerosis and other neurological diseases.

Cerebrospinal fluid (CSF) from 18 multiple sclerosis (MS) patients, 13 subacute sclerosing panencephalitis (SSPE) patients, 22 other neurological disease (OND) patients, and 7 neurotic patients as controls were tested in an 125I-labeled anti-human F(ab')2 binding assay for the presence of antibodies to normal human brain cells from tissue culture, human fibroblasts, plasma membranes of MS and normal human brain, myelin basic protein (MBP) and bovine oligodendrocytes. Antibodies to MBP and to oligodendrocytes were found in the CSF of MS, SSPE and OND patients. Absorption of CSF with bovine CNS myelin significantly diminished binding activity to oligodendrocytes. Antibodies in the CSF against MBP and oligodendrocytes, on which some myelin determinants are expressed, seem to be a common feature of diseases in which demyelination is a component.

Molecular mimicry in virus infection: crossreaction of measles virus phosphoprotein or of herpes simplex virus protein with human intermediate filaments.

Using monoclonal antibodies, we demonstrate that the phosphoprotein of measles virus and a protein of herpes simplex virus type 1 crossreact with an intermediate filament protein of human cells. This intermediate filament protein, probably vimentin, has a molecular weight of 52,000, whereas the molecular weights of the measles viral phosphoprotein and the herpes virus protein are 70,000 and 146,000, respectively. Crossreactivity was shown by immunofluorescent staining of infected and uninfected cells and by immunoblotting. The monoclonal antibody against measles virus phosphoprotein did not react with herpes simplex virus protein and vice versa, indicating that these monoclonal antibodies recognize different antigenic determinants on the intermediate filament molecule. The significance of these results in explaining the appearance of autoantibodies during virus infections in humans is discussed.