Comparison of bacillary indexes in slit-skin smears, skin and nerve biopsies; a study from Malawi.

Data analyzed in this paper were collected within the framework of the Lepra Evaluation Project, an epidemiological study of leprosy in Karonga District, northern Malawi. For 212 patients information on the number of skin lesions, slit-skin smear and skin biopsy results were available. Among 61 patients with a single lesion none were slit-skin-smear positive and two had bacilli detected in skin biopsies. In contrast, among 119 patients with four or more lesions 34 (28.6%) versus 59 (49.6%) had bacilli detectable in slit-skin smears or skin biopsies, respectively. In a further 47 patients skin biopsy results could be compared with split-nerve biopsy results. In 20 of 47 patients the bacterial indexes (BIs) were identical in skin and nerve biopsies, while in 26 of 47 patients the BIs were higher in nerve than in skin biopsies. This difference, which is consistent with several other studies in the literature, provides an insight into the pathogenesis of leprosy.

Multibacillary nerve histology in clinically diagnosed borderline tuberculoid leprosy patients.

The classification of leprosy into multibacillary (MB) and paucibacillary (PB) patients in almost all clinics is entirely dependent on clinical examination. In a study of 21 patients clinically classified as borderline tuberculoid (BT) and, therefore, belonging to the PB group, skin smears and skin and nerve biopsies were examined. Four patients did not have any histopathological evidence of leprosy. Skin smears showed that 1 patient was positive for acid-fast bacilli (AFB), 2 skin biopsies belonged to the borderline lepromatous (BL) category and showed AFB in their lesions, and AFB were present in 10 nerve biopsies classified as BL. It is possible that reported relapses among PB patients may be in those patients with demonstrable AFB in the lesions, including nerves. A careful follow-up study of this particular group of patients after PB multidrug therapy is suggested to resolve this question.

Interaction of cellular proteins with the poliovirus 5' noncoding region.

The 5' noncoding region (NCR) of poliovirus RNA is folded into a complex structure comprised of multiple, critically spaced, stem-loop domains. Mutations in at least one of these domains markedly affects the neurovirulence of the virus. Two proteins have been identified recently which bind and apparently mediate functions of the 5' NCR in translation. We have demonstrated specific binding of three additional proteins in a Hela cell ribosomal salt wash that can be crosslinked to specific stem-loop segments of the 5' NCR. These same RNA segments inhibit translation of polio RNA in vitro, presumably by competing for protein binding. The Sabin vaccine strain of polio RNA exhibits a reduced affinity of binding for specific proteins. The determinant for this reduction appears to be a single nucleotide difference at position 480 between the neurovirulent and attenuated viral strains.

Failure to transmit bovine spongiform encephalopathy to mice by feeding them with extraneural tissues of affected cattle.

Single tissue pools of brain and five extraneural tissues were prepared from four clinical cases of bovine spongiform encephalopathy (BSE) and fed to C57B1 and/or CRH mice. The disease was transmitted only to C57B1 mice fed brain. Intracerebral passages at 12 and 18 months of spleen and spinal cord homogenates from this group of mice to C57B1 mice resulted in reduced incubation periods. Similar blind passages from all other groups failed to produce evidence of infection in a cumulative incubation period of 42 months. The results suggest that the probability of human infection occurring as a result of the consumption of non-neural bovine tissues is remote.

Herpes simplex virus type 1 dUTPase mutants are attenuated for neurovirulence, neuroinvasiveness, and reactivation from latency.

Herpes simplex virus type 1 (HSV-1) encodes a dUTPase which has been shown to be dispensable for normal viral replication in cultured cells (S. J. Caradonna and Y. Cheng, J. Biol. Chem. 256:9834-9837, 1981; F. B. Fisher and V. G. Preston, Virology 148:190-197, 1986). However, the importance of this enzyme in vivo has not been determined. In this report, HSV-1 strain 17 syn+ and two isogenic engineered dUTPase-negative mutants were characterized in the mouse model. Both mutants replicated with wild-type kinetics and achieved wild-type titers in cultured cells. The mutants were 10-fold less neurovirulent than 17 syn+ following intracranial inoculation and more than 1,000-fold less virulent following footpad inoculation. The dUTPase- mutants replicated with wild-type kinetics in the footpad and entered and replicated efficiently in the peripheral nervous system of the mouse. However, their replication in the central nervous system was significantly reduced. The dUTPase- strains established latent infections but displayed a greatly reduced reactivation frequency in vivo. Neurovirulence, neuroinvasiveness, and reactivation frequency were all restored by recombination with wild-type dUTPase sequences. These results have important implications with regard to anti-herpesvirus therapeutic strategies.

Infectious Japanese encephalitis virus RNA can be synthesized from in vitro-ligated cDNA templates.

Japanese encephalitis virus (JEV) is a positive-stranded enveloped RNA virus that belongs to the family Flaviviridae. Genomic JEV RNA is approximately 11 kb long and encodes 10 proteins, 3 structural and 7 nonstructural. A full-length cDNA copy of the JEV genome was constructed by in vitro ligation of two cDNA fragments which encode the 5' (nucleotide positions 1 to 5576) and 3' (nucleotide positions 5577 to 10976) halves of the genome. T7 RNA polymerase transcripts of the ligated full-length cDNA template were infectious when transfected into BHK-21 cells. To identify the recombinant virus, a silent mutation was introduced into the clone encoding the 3' half of the genome, which abolished an XbaI site at nucleotide position 9131. Virus recovered by transfection with the transcripts contained this silent mutation, confirming its identity. Recombinant and parent viruses were identical with respect to growth and plaque production in BHK-21 cells, envelope protein expression in C6/36 cells, and neurovirulence and immunogenicity in mice. Repeated attempts to obtain infectious RNA by transcription from full-length JEV genome cDNA templates cloned into plasmid vectors were unsuccessful. Synthesis of infectious JEV RNA from in vitro-ligated JEV cDNA templates will be useful for molecular and genetic studies of flavivirus replication and virulence.

Synchronous appearance of antigen-positive and latently infected neurons in spinal ganglia of mice infected with a virulent strain of herpes simplex virus.

Studies with replication-defective mutants of herpes simplex virus (HSV) have defined the minimum requirements for establishment of latency, but their behaviour may not reflect the course of events following infection by wild-type HSV, in which ability to express viral genes has not been precluded by a genetic lesion. To address this issue we devised a strategy for studying establishment of latency by a virulent strain of HSV, based on the distinctive molecular characteristics of latently infected neurons. By combining in situ hybridization for detection of latency-associated transcripts with immunohistochemical analysis of viral proteins we demonstrate here that antigen-positive and latently infected neurons appear synchronously in spinal ganglia during the earliest stages of acute ganglionic infection. This is consistent with early divergence of the molecular pathways leading to productive and latent infection, supporting and extending the results obtained with viral mutants.

Two patterns of persistence of herpes simplex virus DNA sequences in the nervous systems of latently infected mice.

The number of herpes simplex virus (HSV) genome equivalents recovered from latently infected mouse spinal ganglia was compared with the proportion of neurons containing latency-associated transcripts (LATs). Two distinct patterns of HSV persistence were observed, depending on the anatomical location of ganglia with respect to the site of cutaneous inoculation. The location of the bulk of latent viral DNA did not correspond with the highest prevalence of LAT+ neurons. Viral DNA was most abundant in spinal ganglia directly innervating the inoculation site and the amount recovered, which was similar to that found previously in human trigeminal ganglia, suggested that LAT+ neurons each contain hundreds of copies of HSV DNA. In stark contrast, although LAT+ neurons were most abundant in neighbouring ganglia, viral DNA was scarce (approx. 20 copies/LAT+ cell). These data indicate that amplification of HSV DNA sequences is greatest in ganglia previously shown to be associated with viral antigen expression during the productive phase of primary infection.

Peripheral replication and latency reactivation kinetics of the non-neurovirulent herpes simplex virus type 1 variant 1716.

The terminal portion of the herpes simplex virus (HSV) genome long repeat region has been shown to contain a neurovirulence gene. Both HSV-1 and HSV-2 mutants deleted in this gene fail to cause central nervous system (CNS) disease in mice. The HSV-1 strain 17 variant 1716, which has a 759 bp deletion encompassing the gene, grows normally in tissue culture but fails to grow following intracerebral inoculation of mice. This paper demonstrates that 1716 is capable of peripheral replication in the footpads of mice. However, no acute replication of virus is detectable in dorsal root ganglia up to 10 days after footpad inoculation. These results imply that the replication defect in 1716 is not host-specific, but is tissue- and/or cell type-specific. Latency reactivation kinetics demonstrate that 1716 is capable of establishing a latent infection, but the kinetics of reactivation are significantly impaired compared to wild-type virus and are dose-dependent. Lack of acute ganglionic replication combined with impaired reactivation kinetics support the conclusion that a proportion of 1716 genomes initiate a lytic infection which then aborts, and a proportion enter the latent state. The results with 1716 imply that its inability to replicate in CNS and peripheral nervous system neurons is specific, and that the block in replication is beyond the stage of adsorption and entry. A prerequisite for any live attenuated HSV vaccine is an inability to initiate CNS involvement following peripheral inoculation. In this respect, 1716 has prototype vaccine potential with the proviso that a direct extrapolation is being made from mouse to man.

Presence, distribution and spread of productive varicella zoster virus infection in nervous tissues.

Nervous tissue lesions were retrospectively studied for detection of productive varicella zoster virus (VZV) infection in 33 autopsied cases, including 19 herpes zoster (HZ) (10 trigeminal, nine spinal) and 14 cases of nodular brainstem encephalitis without HZ. Immunocytochemistry for VZV antigens and in situ hybridization with a biotinylated VZV DNA probe were used on formol-fixed paraffin sections. Peripheral and central nervous system, skin and striated muscle were investigated in serial sections; available tissue blocks, however, varied between cases. Varicella zoster virus production (both antigen and DNA) in nervous tissue was found in HZ cases but only of short survival after a rash of up to 7 wks (eight out of 12 patients). Varicella zoster virus was visualized in nerve cells, glial cells, Schwann cells and blood vessels. In the central nervous system (CNS), VZV was detected in trigeminal nuclei (one out of 10 brains) or disseminated nodular brainstem lesions (one out of 10 brains), in subependymal microvessels (one out of 10 brains) or vasculitic arteries (two out of 19 brains or spinal cords). In the peripheral nervous system (PNS), VZV (DNA and antigen) was found in neurons and satellite cells of sensory ganglia (four out of seven cases with sampling of ganglia), and in damaged nerve fibres including a muscle nerve in one case; myositis with VZV in affected muscle fibres was found in the latter case. In nodular brainstem encephalitis, one case contained VZV within nodular lesions. We conclude that (i) VZV neural spread is suggested by detectable virus in ganglia, nerve fibres and CNS target nuclei; (ii) haematogenous spread of VZV is suggested by detection of virus in CNS microvessels and in disseminated brainstem encephalitis; (iii) VZV myositis may occur in zosteric myotomes; and (iv) VZV is a possible agent in nodular brainstem encephalitis.

Infectivity of human T-lymphotropic virus type I to human nervous tissue cells in vitro.

Infectivity of human T-lymphotropic virus type I (HTLV-I) to human nervous tissue cells was explored using co-cultivation with X-irradiated, HTLV-I-producing MT2 cells. Examined cells included normal cerebellar cells, brain tumor cells (astrocytoma, medulloblastoma, meningioma, hemangioblastoma, and schwannoma), and various cell lines (astrocytoma, ependymoma, oligodendroglioma, medulloblastoma, and neuroblastoma). Successful HTLV-I infection was confirmed immunohistochemically using monoclonal antibodies to HTLV-I p19, p24, and pX product. All cell lines and primary cultures from normal cerebellar tissues and brain tumors could be infected with HTLV-I. Double immunostaining showed that glial fibrillary acidic protein-, S-100 protein- or vimentin-positive cells were susceptible to infection. Neurofilament- or neuron-specific enolase-positive cells in medulloblastoma could also be infected. Reverse-transcriptase assay revealed the productive infection in U251-MG (astrocytoma) and KG-IC (oligodendroglioma) lines. Co-cultivated U251-MG cells formed syncytial polykaryons after serial passages, and polymerase chain reaction assay detected HTLV-I genome in U251-MG syncytial polykaryons and p19+ mononuclear cells. HTLV-I viral RNA was also detected in infected U251-MG cells by in situ hybridization. These data show that HTLV-I may have a wide spectrum of infectivity in human nervous tissues.

The polymerase chain reaction: a new tool for the understanding and diagnosis of HIV-1 infection at the molecular level.

The polymerase chain reaction (PCR) is at present the most powerful analytical tool for detection of specific nucleic acid sequences. The method is based on the in vitro amplification of DNA segments before detection with conventional hybridization techniques or visualization following electrophoresis and staining. The current diagnostic methods for HIV-1 do not allow easy identification of subgroups of infected patients including infants born to seropositive mothers, individuals with delayed serological responses to the virus, infected patients with indeterminate serology results, and patients with dual retroviral infections. Furthermore, response to antiviral therapy cannot be evaluated with serological assays. The rationale for applying PCR in those situations is elaborated here. The applications of this technique for HIV-1 as a diagnostic test and for the understanding of the pathogenesis of this retrovirus are described. Potential limitations of this technique for diagnostic purposes include mainly the possibility of false-positive results due to contamination and false-negative reactions caused by Taq polymerase inhibition. Non-isotopic means for detection of amplified products have been described and should allow for a wider application of this technology. Modifications of PCR which make use of internal standards seem promising for quantitative analysis of nucleic acids. PCR has great potential for viral diagnosis but still requires further studies and better characterization.

[The detection of hepatitis B virus DNA in nerve tissue: the procedural aspects]. / Detección del DNA del virus de la hepatitis B en tejido nervioso: consideraciones mecanicistas.

A patient with HBsAg-positive chronic aggressive hepatitis and HBeAg and hepatitis B virus DNA (HBV DNA) developed fatal Creutzfeldt-Jakob disease. The investigation of cerebrospinal fluid and a temporal lobe brain tissue post mortem sample with radioactive probes of previously cloned HBV DNA showed the unquestionable presence of viral nucleotide sequences in the nervous tissue (about 9 viral genomes per cell). Although a pathogenetic role in the underlying neurologic disease cannot be attributed to HBV, our observation widens the spectrum of tissues where HBV has been detected, and supports the contention that there are replicative extrahepatic foci where the immunologic system of the host is permissive for the virus.

Observations on nerve tissue infected with M. leprae.

Nerve tissue from leprosy patients showed (i) small linear pinkish translucent crystalloid bodies, (ii) small round structures in relation to filamentous strands, (iii) short pieces of filaments with round spaces within them and (iv) miscellaneous structures like pink granules, brown bodies and dark masses. These structures are being studied for their relationship to leprosy.

Detection of viral DNA and activation of latent herpes simplex virus in the rabbit neural tissue.

Both trigeminal ganglia, brain stem, and cornea from rabbits with established latent herpes simplex virus type 1 (HSV-1) infection were examined by explantation and by spot blot hybridization using strain 17 Kpn I fragments i, d, and h and the DNA extracted from above mentioned tissues. Correlation between positive hybridization and reactivation of infectious virus in the cultured explants was documented by enhanced hybridization with the DNA extracts from explanted ganglion samples. In addition, we found positive hybridization in some noncultured ganglion and brain stem samples which did not yield infectious virus by explantation. Keeping in mind the pitfalls of false positive hybridization, the results may indicate during latency the presence in neural tissues of HSV DNA sequences which did not spontaneously reactivate in culture.

Human cells of neural origin are permissive for bovine leukemia virus.

Bovine leukemia virus (BLV) propagated in a cell clone of fetal lamb kidney origin was transmitted by cell contact to different mammalian cells including human cells. The transmission of the BLV genome was effectively achieved by cocultivation of mitomycin-C-killed, virus-producing cells of the cell clone with recipient cells. In particular, human cells of neural origin were highly susceptible to BLV infection, while some other cells were resistant. The transmission of the BVL genome from virus-nonproducing cells failed which suggests the existence of virus specific receptors on the cells. The donor cells contained three integrated BLV proviruses. In recipient cells only one provirus was found. The majority of cells contained both unintegrated and integrated BLV provirus. In the cells containing the transmitted BLV, the viral genome was expressed to its protein products. The results indirectly suggest that retroviruses with similar properties could cause various neural diseases in man.

Type 1 human poliovirus binds to human synaptosomes.

Poliovirus is a neurotropic virus that selectively infects human motoneurons in vivo. The basis for the specificity of this infection is not fully understood. It has been suggested that this tropism occurs because motoneurons are the only neurons to express poliovirus receptors. We have examined this hypothesis by measuring the binding of 125I-labeled poliovirus type 1 to neural tissues. With this assay we have detected highly specific binding sites in human but not rodent neural tissue. Regional assays of binding in human central nervous system homogenates demonstrate that binding sites are not confined to motoneurons. Rather, they are widely distributed throughout the human neuraxis. Particularly in the forebrain, binding is more abundant in gray than white matter. For this reason, we performed tissue fractionation studies which indicate that poliovirus binding sites are enriched in synaptosomes, the subfraction of central nervous system gray matter tissue rich in synaptic endings. The preferential expression of poliovirus binding sites in synaptic endings may be an important factor in the motor tropism of this virus, inasmuch as the major category of neurons with synaptic endings outside the central nervous system are motoneurons; thus, particles of virus may preferentially bind to this cell type during poliovirus viremia.

Spread of HSV and establishment of latency after corneal infection in inbred mice.

The spread of herpes simplex virus (HSV) through neural tissues was studied in three inbred mouse strains that differ in susceptibility to HSV stromal keratitis. The left eyes of BALB/c, C57BL/6, and DBA/2 mice were inoculated topically with HSV type 1. The optic and trigeminal nerves, trigeminal ganglia, and eyes were assayed for infectious virus on days 1, 2, 3, 4, 7, 9, 11 and 14 after inoculation. At 2-4 months post-inoculation, eyes and trigeminal ganglia were assayed for latent virus. Up to 7 days post-inoculation, infectious virus was present at a similar frequency in the inoculated eyes of mice from all three strains. The quantity of virus recovered, however, was mouse strain-dependent: DBA mice yielded the most virus; C57BL/6, the least. The frequency of virus recovery and the quantity of virus recovered from trigeminal nerves and ganglia also varied according to mouse strain. Infectious virus was recovered from the uninoculated right eye of some DBA and C57BL/6 mice 1 wk after inoculation. The overall incidence of latency differed among inbred mouse strains. However, in mice that developed ocular disease (blepharitis, dendritic keratitis, or stromal keratitis), there was no host strain-related difference in the incidence of latency. These results support the hypothesis that host genetic factors play a role in controlling HSV replication and the spread of virus to neural tissues after ocular HSV inoculation. This control may influence the development and severity of disease. However, once infection occurs, latency is established in both susceptible and resistant mouse strains.