Human papovavirus (JC): induction of brain tumors in hamsters.

Eighty-three percent of hamsters inoculated at birth with JC virus, a human papovavirus isolated from brain tissue of a case of progressive multifocal leukoencephalopathy, developed malignant gliomas within 6 months. Three brain tumors have been serially transplanted as subcutaneous tumors. JC virus was isolated from five of seven tumors tested. Cells from four tumors were cultivated in vitro. These cells contained an intranuclear antigen with the characteristics of a T antigen, and this antigen was antigenically related to SV40 T antigen. Although virus was not recovered from extracts of serially cultured tumor cells, JC virus was rescued when one tumor cell line was fused with permissive cells.

Brain tumors in owl monkeys inoculated with a human polyomavirus (JC virus).

Owl monkeys were inoculated intracerebrally, subcutaneously, and intravenously with JC, BK, or SV40 virus. Two of four adult owl monkeys inoculated with JC virus, a human polyomavirus, developed brain tumors at 16 and 25 months after inoculation, respectively. A grade 3 to grade 4 astrocytoma (resembling a human glioblastoma multiforme) was found in the left cerebral hemisphere and brainstem of one monkey. The second monkey developed a malignant tumor in the left cerebral hemisphere containing both glial and neuronal cell types. Impression smears prepared from unfixed tissue of this tumor showed cells that contained polyomavirus T antigen. Virion antigens were not detected. Tumor cells cultured in vitro also contained T antigen but were negative for virion antigen. Infectious virus was not isolated from extracts of this tumor.

Modification of hydroxyindole-O-methyltransferase activity in experimental pineocytomas induced in hamsters by a human papovavirus (JC).

Hydroxyindole-O-methyltransferase (HIOMT) (HIOMT) activity was studied and compared in 7 pineal tumors (pineocytomas) induced in vivo in Syrian hamsters after postnatal inoculation with strains of human papovavirus (JC). Levels of tumor HIOMT activity were correlated with degree of cytologic differentiation of the pineocytes of each neoplasm, as judged from electrom micrographs. The relative capacities of the HIOMT in the tumors to methylate three related substrates gave similar ratios in the individual tumors and were not different from those of HIOMT in normal hamster pineal glands.

Induction of peripheral neuroblastomas in Syrian hamsters after injection as neonates with JC virus, a human polyoma virus.

Neuroblastomas developed in 10 of 31 Syrian hamsters inoculated intraocularly with JC virus, a human polyoma virus. The latent period was 6 to 11 months. Primary tumors occurred in the abdominal cavity, pelvis, mediastinum, and neck region. The origin of one tumor from the adrenal gland was demonstrated. Metastases were seen in the liver, bone marrow, and lymph nodes. Two neuroblastomas arising in this experiment were transplanted serially in weanling hamsters, and a tissue culture cell line was established from one of the transplanted tumors. T-antigen was detected in three of five primary tumors tested and in the transplanted tumors. Antibody against T-antigen was demonstrated in sera from five of six animals with neuroblastomas. Neuroblastomas also developed after combined s.c. and i.p. injection of JC virus.

Differential neurooncogenicity of strains of JC virus, a human polyoma virus, in newborn Syrian hamsters.

The neurooncogenicity recently isolated strains of the human polyoma virus, JC virus, was determined by intracerebral inoculation of newborn Syrian golden hamsters. All three strains produced malignant brain tumors in a majority of inoculated animals during a 6.5-month observation period. The results obtained with the MAD-2 strain, 19 of 20 animals with cerebellar medulloblastomas and 0 of 20 animals with pineal gland tumors, were quite similar to those observed previously with the prototypic strain of JC virus, MAD-1. Inoculation of the MAD-4 strain, however, resulted in 10 of 22 animals with pineal gland tumors and only 10 of 22 animals with tumors in the cerebellum. The MAD-3 strain was neurooncogenic, but too few animals lived to be weaned to provide significant additional information. The basis for the apparent predilection of the MAD-4 strain for the pineal gland is unknown. Two hamsters in the experiment developed extracranial neuroblastomas.

Vertical Distribution of Rotylenchulus reniformis in Cotton Fields.

The possible impact of Rotylenchulus reniformis below plow depth was evaluated by measuring the vertical distribution of R. reniformis and soil texture in 20 symptomatic fields on 17 farms across six states. The mean nematode population density per field, 0 to 122 cm deep, ranged from 0.4 to 63 nematodes/g soil, and in 15 fields more than half of the R. reniformis present were below 30.5 cm, which is the greatest depth usually plowed by farmers or sampled by consultants. In 11 fields measured, root density was greatest in the top 15 cm of soil; however, roots consistently penetrated 92 to 122 cm deep by midseason, and in five fields in Texas and Louisiana the ratio of nematodes to root-length density within soil increased with depth. Repeated sampling during the year in Texas indicated that up to 20% of the nematodes in soil below 60 cm in the fall survived the winter. Differences between Baermann funnel and sugar flotation extraction methods were not important when compared with field-to-field differences in nematode populations and field-specific vertical distribution patterns. The results support the interpretation that R. reniformis below plow depth can significantly impact diagnosis and treatment of cotton fields infested with R. reniformis.

Ontogeny of androgen receptor immunoreactivity in lumbar motoneurons and in the sexually dimorphic levator ani muscle of male rats.

We documented the ontogeny of androgen receptor (AR) immunoreactivity for rat lumbar motoneurons of the sexually dimorphic motor pools, the spinal nucleus of the bulbocavernosus (SNB) and the dorsolateral nucleus (DLN), and for the sexually monomorphic retrodorsolateral nucleus (RDLN). We also assessed the ontogeny of AR immunoreactivity in the rat sexually dimorphic levator ani (LA), which is a target muscle for SNB motoneurons. Lumbar spinal cords and LA muscles from gonadally intact males at ages postnatal days (P)7, P10, and P14 and as adults were incubated with the rabbit antiserum PG-21. Half of the prepubertal males (P7-P14) received 200 micrograms of testosterone propionate (TP) 2 hours prior to death to enhance immunodetection of ARs. We found that SNB motoneurons developed AR immunoreactivity at first and achieved adult levels by P10. In contrast, the number of RDLN motoneurons with AR-immunopositive nuclei during development remained well below the adult number. Development of AR immunoreactivity in the DLN shared characteristics with both the SNB and the RDLN. AR immunoreactivity developed in some DLN motoneurons by P10, although the percentage of labelled motoneurons remained below that in adulthood. Acute TP treatment significantly increased the number of SNB motoneurons with AR-positive nuclei at P7. The LA showed a robust pattern of AR immunostaining from P7 to adulthood. Immunostaining was present only in nuclei and constituted only a subpopulation of the nuclei present in muscle. The present results confirm and extend previous results based on steroid autoradiography and steroid binding assays regarding regional and developmental differences in the expression of ARs.

Progressive multifocal leukoencephalopathy (PML): in vitro cell-mediated immune responses to mitogens and JC virus.

Cell-mediated immunity was studied by in vitro tests in seven patients with progressive multifocal leukoencephalopathy (PML). Lymphocyte proliferation in response to mitogenic stimulation was reduced to a variable degree in all patients, indicating a general impairment of cell-mediated immune responsiveness, although mitogen-induced production of the lymphokine migration inhibitory factor (LIF) was normal in most cases. Cell-mediated immunity to JC virus (JCV) was assessed by LIF production in response to JCV antigen. In the six PML patients tested, LIF production with JCV antigen was absent despite the presence of antibody to JCV in serum. This contrasted with positive LIF production in seropositive normal individuals and patients and patients with other diseases. These results provide the first in vitro evidence of a depressed cell-mediated immune response to JCV in patients with PML, and support the hypothesis that PML is accompanied by a selective marked deficiency in cellular response to this virus in association with a general depression of cell-mediated immunity of variable severity.

Effect of persistent fibroma virus infection on susceptibility of cells to other viruses.

Shope fibroma virus establishes a persistent cytoplasmic infection in primary (RK) and serially cultivated (DRK(3)) rabbit kidney cells which is accompanied by a morphological alteration of the cells. The response of such cells to superinfection by other viruses was compared with that of control cells by determining plaque production and virus yield of superinfecting viruses. It was found that the growth of other poxviruses, myxoma and vaccinia, was greatly inhibited in the fibroma virus-infected cells, but that of pseudorabies and herpes simplex viruses, which are unrelated deoxyribonucleic acid viruses, was virtually unaffected. The ribonucleic acid (RNA) viruses, poliovirus 1 and coxsackievirus B1, did not produce plaques on either RK or fibroma virus-infected (F-RK) monolayers. However, the growth of several other RNA viruses, vesicular stomatitis virus, encephalomyocarditis virus, Sindbis virus, and Newcastle disease virus, was enhanced in F-RK cells. None of these latter RNA viruses produced any infectious progeny in DRK(3) cells, but they all plaqued on and produced good yields in DRK(3) cells persistently infected with fibroma virus. This phenomenon is termed facilitation. Facilitation results from the infection of DRK(3) cells by fibroma virus. Neither interference nor facilitation were due to changes in the adsorption or eclipse of the superinfecting virus.

Virologic and serologic studies of progressive multifocal leukoencephalopathy.

The human polyomavirus JCV has been associated with diseased brain tissue from 54 cases of PML. The disease occurred in persons of all ages--the youngest being 5 years old--whose immune mechanisms were depressed for any of a variety of reasons. In six patients the disease ceased progressing and they stabilized or improved. The brains of two of these, who died months or years later, were found to be free of active PML at autopsy. Sera from 21 patients had HAI antibodies against JCV, but none had antibodies against T or the common internal antigens, and only one had IgM antibodies detectable by immunofluorescent staining. Nine CSF were devoid of JCV-related antibodies except for one which gave an HAI titer of 8. Virus has been isolated from 15 PML brains, and preliminary studies indicate that one isolate, while clearly of JCV type, is serologically distinguishable from prototype Mad 1 JCV.

JC virus, a human polyomavirus associated with progressive multifocal leukoencephalopathy: additional biological characteristics and antigenic relationships.

JC virus, a human polyomavirus, failed to grow or produce cytopathic effects in any of a variety of cells tested other than primary human fetal glial (PHFG) cells. Cells tested included other primary human cells and glial cells from other animals. Only a rare cell in inoculated insusceptible human cell cultures produced T or virion antigen. In PHFG cell cultures JC virus produced subtle cytopathic effects, and the majority of progeny remained cell associated. Only a few cells in the heterogenous PHFG cell cultures contained T antigen at 24 h postinoculation, and virion antigen was not detected until 48 h postinoculation. The infectivity of JC virus was resistant to inactivation by ether and by heating at 50 degrees C for 1 h. A three-way minor antigenic relationship was demonstrated among the virion antigens of JC virus, BK virus, and simian virus 40 by neurtralization and/or hemagglutination inhibition tests. Serological evidence is presented for the existence of JC virus as a distinct entity before the use of simian virus 40-contaminated poliovirus vaccines and for the nonexistence of an animal reservoir for JC virus infection.

Distribution of nonintegrated DNA from JC papovavirus in organs of patients with progressive multifocal leukoencephalopathy.

Tissues from 10 patients with progressive multifocal leukoencephalopathy (PML) and 14 patients without PML who were serologically positive for JC papovavirus were examined by molecular hybridization for human polyomavirus DNA sequences. Although viral proteins were not identified by fluorescent antibody methods, viral DNA was found in the kidneys of seven of nine patients with PML by hybridization, at 0.2-10 viral genome copies per cell genome equivalent, compared with 0.6-4 X 10(3) copies per cell in diseased areas of the brain. Examination of viral DNA from brains and kidneys of patients with PML by blot hybridization yielded no evidence of integration into the host cell genome. In three of the patients with PML, viral DNA was also found in liver, lung, lymph node, and spleen. Two of these patients, with widely disseminated JC virus, were children. In tissues from patients without PML, no evidence of JC virus infection was found, but BK papovavirus DNA was detected in two of 14 kidneys tested.

Characterization of tissue culture-induced heterogeneity in DNAs of independent isolates of JC virus.

After several serial passages at low multiplicities of infection in primary human foetal glial cells at 37 degrees C, the DNA of prototype (MAD-1) JC virus and that of MAD-2 and MAD-3 are typically heterogeneous in size, but DNAs of MAD-4 and MAD-6 are relatively homogeneous. A similar dichotomy was observed in the DNAs of six isolates propagated more recently in glial cultures at 39 degrees C under similar conditions of brief passage in vitro at low multiplicities of infection: the DNAs of two (MAD-9 and -10) were heterogeneous, but the DNAs of four others (MAD-8, -11, -12 and -14) were homogeneous. Therefore, the propensity of the viral genome to sustain deletions was an intrinsic property of each isolate. However, actual induction and maintenance of the presumably defective DNAs was influenced by the relative proportions of permissive spongioblasts and semi-permissive astrocytes in the glial cultures and by the multiplicity of infection. Deletions in MAD-1 DNA were confined to the presumptive early region and spanned the BamHI cleavage site (map position 0.505). The heterogeneity was more complex in the DNAs of MAD-2 and MAD-3, but again most of the deletions, which ranged up to 12% of full-length DNA, spanned the BamHI site. We propose that the differential susceptibility to deletion among isolates is a consequence of natural genetic variation in JC virus.

Propagation and primary isolation of papovavirus JC in epithelial cells derived from human urine.

Human papovavirus JC, previously passaged in amnion cells or in primary human fetal glial cells, replicated efficiently in urine-derived epithelial cells. Primary isolation of the virus from brain extracts was possible in urine-derived cells, but these cells were not as sensitive as primary human fetal glial cells for this purpose. Primary isolations of human papovavirus JC from urine sediments of renal transplant patients were made in urine-derived cells.

Comparison of infectious JC virus DNAs cloned from human brain.

We cloned JC virus DNA obtained directly from brain tissue of 10 cases of progressive multifocal leukoencephalopathy and compared DNAs by restriction endonuclease mapping. Before cloning, each DNA preparation was homogeneous with respect to restriction patterns, but with the cloned DNAs we found variability in three regions of the genome among DNAs from different cases. There was a region of hypervariability between 0.67 and 0.725 map units; no two DNAs were exactly alike in this region. We determined that the origin of DNA replication also was in this region at 0.69 +/- 0.02 map units. In 4 of the 10 DNAs examined there was a deletion of approximately 75 base pairs between 0.14 and 0.235 map units, the region presumed to contain the codons for the C-terminal ends of the structural protein Vpl and for T antigen. JC virus DNA from these same four cases had an additional HincII-HpaI site at 0.895 map units in the presumptive Vp3 and Vp2 coding regions. Overall, no two JC virus genomes were identical although all were from fatal central nervous system infections and were infectious in vitro. Our restriction patterns suggest that there are two subtypes of JC virus circulating in the population.

Specificity of the Tumor-specific transplantation antigen induced by JC virus, a human polyomavirus.

The specificity of the tumor-specific transplantation antigen (TSTA) induced by JC virus was investigated by cross protection tests in weanling hamsters. Hamsters immunized with JC virus, BK virus, or SV40 were challenged 5 weeks later with known numbers of JC virus- or SV40-induced hamster tumor cells. Both the JC virus-immune and the SV40-immune hamsters showed resistance to challenge with homologous but not heterologous tumor cells, and the BK virus-immune hamsters were not resistant to either heterologous tumor cell. The TSTA induced by JC virus did not cross-react with that induced by SV40.

Distribution of androgen receptor immunoreactivity in the spinal cord of wild-type, androgen-insensitive and gonadectomized male rats.

The polyclonal antiserum PG21 was used to detect androgen receptor (AR) protein in three motoneuronal pools of the male rat lumbar spinal cord. In gonadally intact, wild-type males, the spinal nucleus of the bulbocavernosus (SNB), dorsolateral nucleus (DLN), and retrodorsolateral nucleus (RDLN) all displayed immunolabeling of cell nuclei. The percentage of motoneurons displaying such labeling was highest in the SNB and lowest in the RDLN. This pattern of AR immunocytochemical labeling agrees well with previous steroid autoradiographic studies of androgen accumulation in the rat spinal cord. In contrast, virtually no motoneurons in any of the three pools displayed nuclear AR immunostaining in long-term gonadectomized males or in gonadally intact males carrying the Tfm mutation, which renders the AR incompetent. In gonadectomized males, labeling was restored in the SNB and DLN, but not the RDLN, 30 min after an injection of replacement testosterone. Eight hours of testosterone exposure restored immunolabeling in all three motor nuclei. Apparent cytoplasmic staining was seen in SNB motoneurons of untreated castrates and Tfm rats, but not intact rats, suggesting that AR residing in the cytoplasm translocates to the nucleus on binding to androgen in these motoneurons.