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From the Divisions of Comparative Pathology*
and
Microbiology,
New England Regional Primate
Research Center, Harvard Medical School, Southborough, Massachusetts,
and the Pathology Department,
Tulane Regional
Primate Research Center, Covington, Louisiana
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Previous studies indicated that SV40 causes interstitial nephritis in primary infections and PML upon reactivation of a latent infection in SIV-infected macaques with AIDS.10 We now describe a second CNS manifestation of SV40: a meningoencephalitis affecting cerebral gray matter, without demyelination, distinct from PML. This lesion can be a primary manifestation of SV40 in conjunction with SV40-induced interstitial nephritis and pneumonitis. In this report we describe this new CNS lesion and characterize it with respect to disease already associated with SV40 in immunosuppressed macaques.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The SV40 cases described herein were selected from macaques dying with SIV-induced AIDS during the period June 1990 through October 1997 (n = 229) at the New England Regional Primate Research Center (NERPRC). SV40-associated meningoencephalitis (ME) was first identified in an SIV-infected rhesus monkey (7NE) that died with SV40-associated interstitial nephritis. Once we recognized a lesion in the gray matter associated with SV40-infected cells, but differing from PML, we carefully re-examined earlier cases. We found that two animals previously diagnosed with renal and/or pulmonary SV40 (1NE and 6NE)13 additionally had mild ME, confirmed by in situ hybridization for SV40, and one more case (9NE) was later identified. Eleven animals housed at the NERPRC (designated 1NE to 11NE) diagnosed with SV40 infection after postmortem examination were selected for further study. Four additional cases from the Tulane Regional Primate Research Center (designated 12T to 15T) were included in the study. Of the 15 animals with SV40-associated lesions, 14 were rhesus monkeys (Macaca mulatta) infected with SIV and euthanized with terminal AIDS. One additional animal, a Formosan rock macaque (Macaca cyclopis; 1NE) died with immunodeficiency of undetermined origin (possibly SIV or type D retrovirus).12 Formalin-fixed, paraffin-embedded tissues were sectioned and stained with hematoxylin and eosin (H&E) for routine histological examination. In addition, a Luxol fast blue stain for myelin was performed on selected sections of brain. Selected tissues were snap-frozen in optimal cutting temperature (OCT) compound (Miles Scientific, Elkhart, IN) for immunohistochemical labeling.
In Situ Hybridization
In situ hybridization for SV40 or SIV was performed as previously described.14 The SV40 probe contained the entire genome in plasmid pUC19.13 The SIV probe was a combination of two plasmids, which together make up essentially the entire SIVmac239 genome.15,16 Briefly, the probes were labeled with digoxigenin-dUTP by random priming. The hybridization was performed under denaturing conditions to identify both DNA and RNA. After hybridization, tissue sections were immunostained to localize the digoxigenin-labeled nucleic acids using nickel-enhanced diaminobenzidine (DAB) as the chromogen.
Immunohistochemistry
Immunohistochemistry for SV40 was performed on formalin-fixed,
paraffin-embedded or frozen sections using a monoclonal antibody
directed against SV40 T antigen (Table 1)
, as previously
described10,17
with DAB as the chromogen, and
counterstained with Mayer's hematoxylin. As accumulation of the tumor
suppressor gene p53 in nuclei in JCV infection has been demonstrated by
several groups,18-20
and SV40 T-antigen is known to bind
p53, we also immunostained selected tissues for p53 as an additional
marker for SV40 infection. Sections of brain with significant
inflammation noted on histopathological examination were immunostained
with antibodies to identify B or T lymphocytes and monocyte/macrophages
(Table 1)
.
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Selected tissues were labeled by immunohistochemistry with
antibodies to identify astrocytes (GFAP), oligodendrocytes, macrophages
(HAM-56), or neurons (NSE; Table 1
), followed or preceded by in
situ hybridization or immunohistochemistry for SV40. Briefly,
after immunostaining with the first antibody, slides were immunostained
with the second antibody, or in situ hybridization for SV40
was performed. The chromogen was also changed for the second procedure,
eg, standard DAB (brown) followed by nickel-enhanced DAB (black) or
Vector Red (Vector Laboratories, Burlingame, CA) to allow
differentiation of the two labels.
Molecular Cloning and Sequencing
SV40 was isolated from frozen tissues (6 animals, 1NE to 6NE), or DNA was isolated from frozen or formalin-fixed, paraffin-embedded tissues (6 animals, 7NE to 9NE; 12T, 13T, and 15T, for a total of 12 of the 15 cases) for PCR, cloning, and sequencing. All routine cloning procedures were performed essentially as described earlier.21,22 Epicurian Coli XL1-Blue MRF' Supercompetent Cells (Stratagene Cloning Systems, La Jolla, CA) were used for the transformation and plasmid growth. Each of the resulting plasmids was sequenced at least twice through the viral insert. PCR-amplified viral fragments from infected tissues were treated with polynucleotide kinase and cloned into pUC18/SmaI-cut vector (Pharmacia, Piscataway, NJ) or PCRScript (Stratagene) according to the manufacturer's recommendations.
All nucleotide primers were custom-made by GIBCO BRL (Grand Island, NY). Primers 5'-GGTTTTTCAGTTAACCTTTCTGG-3' (513 to 491), 5'-GTATCTTCCCCTTCACAAAATTG-3' (468 to 446), 5'-ATACACAAACAATTAGAATCAGTAG-3' (4577 to 4601), and 5'-GCTTTAAATCTCTGTAGGTAG-3' (4644 to 4664) were used for PCR amplification of the SV40 origin/enhancer region. Primers 5'-GAGGAGGTTAGGGTTTATGAGG-3' (2492 to 2513), 5'-ACACAGAGGAGCTTCCTGGG-3' (2514 to 2533), 5'-ACAGGCTCTGCTGACATAGAAG-3' (3683 to 3662), and 5'-TGGATGGCTGGAGTTGCTTGG-3' (3659 to 3639) were used for PCR amplification of the carboxyl-terminal part of SV40 T-antigen. Universal and reverse M13/pUC19 primers were used for sequencing through the amplified region.
PCR amplification and DNA sequencing were performed essentially as described earlier.21,23 Each sequence was confirmed by two or more independent PCR reactions. A thermal cycler produced by MJ Research (Watertown, MA) was used for PCR amplification. DNA sequencing was performed using an ABI Prizm automated sequencer. Sequencing reactions were performed with the help of AmpliTaq FS sequencing kit from Perkin-Elmer Cetus (Norwalk, CT), which was used according to the manufacturer's recommendations on either MJ Research or Hybaid Omnigene (SunBioscience, Branford, CT) thermal cyclers.
Nucleotide sequences were analyzed and aligned using MacVector version 5.0.1. software (International Biotechnologies, New Haven, CT). The sequence of SV40 was taken from Genebank24 (accession number JO2400).
Serology
Serum samples were assayed for the presence of antibodies to SV40 by a dot-immunobinding technique (Virus Reference Laboratory, San Antonio, TX), as previously described.25
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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SV40-associated disease was recognized only in immunocompromised
animals.
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Two CNS lesions associated with SV40 were identified in
immunosuppressed macaques: progressive multifocal leukoencephalopathy
(PML)8,10,26,27
and meningoencephalitis (ME). PML is a
demyelinating lesion primarily of the white matter, due to infection of
oligodendrocytes (Figure 1, A, C, and E)
.
In contrast, SV40-associated ME is a lesion of the meninges and
superficial gray matter, without significant demyelination (Figure 1, B, D, and F)
. The meninges are variably thickened by edema and
infiltrates of lymphocytes, macrophages, and eosinophils, largely
around vessels. The inflammation extends into the gray matter along
blood vessels, which have hypertrophic endothelium. As in PML,
distinctive features of ME include cells with enlarged nuclei with
smudgy amphophilic intranuclear inclusions and large, bizarre,
gemistocytic astrocytes, with enlarged and sometimes multiple nuclei.
The mildest ME lesions may consist entirely of a few gemistocytic
astrocytes and occasional intranuclear viral inclusions. ME can be
focal in mild cases or diffusely involve the superficial cerebral
cortex and periventricular gray matter. In one case with severe ME
(9NE), lesions focally extend into white matter, and those foci have
associated demyelination.
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. A more diffuse
infiltrate of macrophages is seen in ME, increasing with the severity
of the lesion. The morphology of the macrophages varies, with those in
the demyelinating PML lesions more ovoid with fewer cytoplasmic
processes than those in the ME lesions (Figure 2, A and B)
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indicates the lesion(s) associated with SV40 in the 15 affected
animals and the tissues in which SV40 was identified. Eight animals had
PML, six had ME, and in one animal (3NE) SV40 was localized to the
kidney only, both by histopathology and in situ
hybridization. In the remaining cases, pulmonary and/or renal SV40 was
associated exclusively with ME. Of the 10 cases for which we have
epidemiological data, 2NE to 11NE, the incidence of SV40-associated
lesions was 4.4% of AIDS cases (10/229). Among these 10 animals with
SV40-associated lesions, PML was most common (6/229, 2.6%) followed by
ME (3 cases, 1.3%), nephritis (1.3%), and pneumonia (0.4%) (Table 2)
. These 10 animals comprise all of the cases of SV40-associated
disease that we have recognized in SIV-infected macaques at the New
England Regional Primate Research Center (NERPRC) through October 1997.
Of the six cases of ME, five, including all of the cases of ME at the
NERPRC, had SV40-associated lesions in two or more locations. Serology
Serology for SV40 was performed on 14 of the 15 animals at
multiple times (Table 3)
, including time
of inoculation with SIV, date of death, and one or more times in
between. All animals with PML were seropositive for SV40 at the time of
SIV infection, with two stipulations. Animal 2NE was spontaneously
SIV-infected, so date of inoculation was unknown. This animal was
seropositive for SV40 at least 4.5 years before death. Animal 10NE was
given an attenuated strain of SIV and then challenged 3.5 years later
with virulent SIV, 2 years before death. This animal was seronegative
for SV40 at the time of the original SIV infection but seroconverted at
some time in the next 16 months, so was seropositive at the time of
challenge with pathogenic SIV and for 4 years before death. All animals
with ME, nephritis, and/or SV40 pneumonia seroconverted to SV40 after
SIV infection. Once an animal seroconverted to SV40, every additional
sample tested was also positive. The average survival of those animals
was 2.01 years after SIV infection (Table 3)
. All of the animals with
PML were SV40 infected before SIV infection, or for more than 4 years
before death, and they survived an average of 2.53 years after SIV
infection. The ME/renal cases averaged 3.53 years old when they died
(range, 2.06 to 5.66 years; only one animal was over 5 years), and the
PML cases averaged >7.5 years (range, 4.8 to 12 years; only one animal
was under 5 years).
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To confirm that the lesions were due to SV40, in situ
hybridization or immunohistochemistry was performed. SV40 nucleic acid
and/or T antigen was found in multiple nuclei of oligodendrocytes and
astrocytes in the brain in both ME and PML (Figure 2, C and D)
.
SV40-infected neurons were not seen in any case. Immunostaining for p53
protein labeled the nuclei of similar numbers of cells with a similar
distribution in brain and kidney as in situ hybridization
and immunohistochemistry for SV40. This finding confirms that SV40,
like JCV, binds p53 protein in vivo.
In addition to oligodendrocyte and astrocyte infection, SV40 antigen
and/or nucleic acid was found in mononuclear cells within the cellular
infiltrates. SV40-infected cells were also found in the meninges and
rarely in choroid plexus, and in one animal with severe ME (9NE), in
cells in the wall of meningeal and parenchymal vessels (Figure 2B)
.
Macrophages occasionally contained SV40 nucleic acid in their
cytoplasm, and in one case of ME, nuclear labeling with SV40
co-localized with cytoplasmic labeling for macrophages (Figure 2B)
. In
several animals, prominent perivascular cuffs of mononuclear cells were
seen in association with both PML and ME. CD3+ T
lymphocytes were observed in the perivascular cuffs, as well as in
aggregates in areas of demyelination in PML (Figure 2, E and F)
; in ME
they were in the meninges and around vessels, generally in lower
numbers. Using an antibody directed against CD20, B lymphocytes were
observed in low numbers in only one of five PML cases examined and were
not found in the one case of ME with significant perivascular
inflammation (Figure 2F)
. Antibodies recognizing CD79a and 
or 
light chains identified a larger number of B cells than CD20, but the
number relative to T cells remained quite small.
In situ hybridization for SIV was performed on sections with SV40 lesions in 13 of 15 animals. None of the sections from any animal had SIV and SV40 in the same focus of inflammation. One animal, 6NE, had SIV encephalitis28,29 with SIV nucleic acid in multiple cells within the encephalitis but none in the regions of ME. An occasional SIV-containing cell was found in a section of lung from one other case with SV40 pneumonia, 9NE, but again, SIV was not present within the SV40 lesion.
SV40 DNA Sequencing
Ilyinskii et al13
reported previously on the sequences
of the ori-enhancer and T-antigen carboxy terminus of SV40 isolated
from six of the animals (1NE to 6NE). We now have sequences from an
additional 6 animals, for a total of 12 of the 15 cases (Table 2)
.
We examined an additional 17 clones of the ori-enhancer region from 6
animals (Figure 3)
and confirmed our
previous finding of a single 72-bp element in all of the animal
isolates, in contrast to the laboratory strain of SV40, 776, which
contains a tandem repeat of the 72-bp element. The nucleotide sequences
of the new clones were essentially identical to those previously
reported, with one exception. All of the clones from the brain, lung,
and kidney of animal 15T lacked one of the usual complement of three
21-bp repeats seen in strain 776 and all other clones examined from the
animals.
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than in the
ori-enhancer. SV40 from one animal, 12T, had the same sequence as
laboratory strain 776 over the 200-bp region examined. Unlike previous
findings,13
in which kidney-derived SV40 had a consensus
sequence in this region and the various brain isolates had more
variation, sequences of the additional clones examined contained more
varied sequences, with no identifiable relationship to the tissue from
which the SV40 was derived (brain, kidney, or lung). In animals with
SV40 derived from two or three tissues (4NE and 15T), one sequence was
predominant in all tissues, although both animals had a minor variant
in brain-derived SV40.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The critical difference in SV40 lesion type appears to be the temporal relationship of SV40 infection to SIV infection and the age of the affected animals. All of the animals with ME, renal, and/or pulmonary SV40 lesions were infected with SIV before SV40 infection. They survived an average of 2 years after SIV infection and averaged 3.5 years old at death. Animals with PML survived longer after SIV infection (2.5 years) and were older when they died (>7.5 years on average). Using a different serological technique, virus neutralization, for antibodies to SV40 on animals 12T to 15T, another study30 had similar results, with two exceptions: 13T was found to be SV40 positive at the time of SIV inoculation, whereas 15T was never seropositive for SV40. Our data suggest that 1) PML is a reactivation of SV40, whereas ME, along with SV40-associated nephritis and pneumonia, is a manifestation of primary infection, and 2) older animals with perhaps a more indolent course of SIV infection are more likely to have SV40-associated PML.
Data from humans are consistent with our findings in macaques. PML patients have IgG antibody to JCV, evidence that PML results from reactivation of latent JCV infection.31 And despite seroepidemiological studies suggesting that the majority of individuals are infected with JCV during childhood, PML is rare in children with immunocompromise, including HIV infection.27,32
Lesions in gray matter in AIDS patients with PML have been reported,33 and we observe them as well, particularly by extension at the junction of gray and white matter and adjacent to ependyma. However, the gray matter lesions seen in cases of PML are not seen isolated from foci of demyelination, and the meningeal involvement seen in ME is never present. We are aware of one description34 of an AIDS patient with disseminated BKV infection. This patient had BKV-associated pulmonary and renal lesions and meningoencephalitis without white matter involvement identical to the lesions seen in ME caused by SV40 in macaques, with the exception that BKV-infected oligodendrocytes were not identified.34 This is in contrast to our cases of SV40-associated ME, as well as PML, in which many of the infected cells are oligodendrocytes.
If the report of Vallbracht et al34 represents a true difference in pathogenicity for the CNS between JCV and BKV, several explanations are possible. Although both BKV and JCV can be found in kidney and are shed in the urine, only BKV has been associated with disease of the kidney and urinary tract.8 Despite apparent efficient replication in multiple cell types in vivo, growth of JCV is highly restricted in vitro, compared with SV40 and BKV,35,36 although recent studies have identified JCV-susceptible primary and established cell lines.37,38 This restricted cell type specificity is thought to be due to tissue-specific transcription factors that interact with the regulatory region of the JCV genome.37,39-41 JCV DNAs from different sites in the same patient show sequence variations in their regulatory regions, suggesting organ-specific adaptation of the virus.42,43 JCV has a regulatory region very different from those of BKV and SV40,44,45 and greater variation in this region has been identified in patient isolates.36,46 Possibly SV40 is playing the role of both BKV and JCV in different scenarios.
We examined two regions of the SV40 genome, the ori-enhancer region and the T-antigen carboxy terminus, regions previously identified by us and others13,30,42,47,48 to be potential sites for tissue-specific polyomavirus replication. Sequence differences in the ori-enhancer (regulatory) region appear not to be responsible for tissue-specific SV40 replication as sequences were virtually identical from brain and kidney isolates. As we and others have noted previously,13,30,48-50 all animal isolates contain a single 72-bp enhancer, not the two copies found in the laboratory strain 776. Only one variation was seen in the ori-enhancer region: a loss of one of the usual complement of three 21-bp repeats, seen in all clones from brain, kidney and lung from a single animal in the current study, a variation also noted by others30 in the same animal.
The T-antigen carboxy terminus of SV40 is a variable region and, as
such, a potential site to distinguish and identify viral
strains.13,30
Although sequence variations in the SV40
T-antigen carboxy terminus were more widespread than in the
ori-enhancer, no tissue- or lesion-specific changes were noted in this
study. Thirteen of sixteen clones from the three Tulane cases had two
unique deletions not seen in any of the NE clones, and 29 of 30
sequences from NE clones contained a deletion not seen in any of the
Tulane clones (Figure 4)
. Lednicky and colleagues30
found
the same sequence in 12T (identical to strain 776 over this region) and
15T but some variation in the T-antigen sequence from 13T. We obtained
two clones of the T-antigen region from the brain of 13T and also
sequenced the amplified product directly from the PCR mixture; all
approaches gave the same sequence. Lednicky et al30
reported minor variants in the ori-enhancer sequences from this animal,
so the T-antigen sequence discrepancy may reflect a real SV40
heterogeneity in animal 13T, as we have observed in other cases.
Polyomaviruses probably spread to the CNS via the circulation. The multifocal distribution of early PML lesions and the vascular and perivascular infected cells in ME and PML34,51 support a hematogenous entry. Multiple studies have identified peripheral blood mononuclear cells as sites of persistence of BKV and JCV5,52-55 and, in the case of JCV, B lymphocytes specifically.38,51,56-58 Rhesus monkeys become viremic during primary SV40 infection.59 Although the infected circulating cells in primary SV40 infection have not been identified, SV40 can be isolated from peripheral blood mononuclear cells from SIV-infected macaques with SV40-associated disease.30 JCV-infected lymphocytes36,51 and macrophages60 have been identified in the brains of patients with PML by some investigators but not others.26,61 B lymphocytes are rare in both SV40 and SIV-associated CNS inflammation in macaques10,62 and in some cases of PML and HIV encephalitis in AIDS patients.26,61 We have not observed SV40 co-localized to lymphocytes or in perivascular cuffs of inflammatory cells in PML. This may be due, at least in part, to the lack in SV40 of sequences in the JCV regulatory region that interact with B cells.41,44,45
Multiple studies have identified cytoplasmic JCV in macrophages after phagocytosis of cellular debris60,63-65 in PML, and we have observed this rarely as well (data not shown). We have identified apparent macrophage infection in severe ME, and infected macrophages were found in the reported case of disseminated BKV.34
AIDS patients with PML frequently have HIV-infected cells among the inflammatory cells in the demyelinating lesion.26,61,63,66 In situ hybridization failed to identify SIV in the SV40-associated lesions in any animal, although one case with SIV encephalitis (SIVE) had abundant SIV nucleic acid in macrophages and multinucleated cells in the SIVE lesions, and other cases had SIV localized to lymphoid tissue. This confirms previous findings in our laboratory that SIV does not co-localize to sites of inflammation due to opportunistic infections in simian AIDS67-71 and may represent a difference in pathogenesis of the two lentiviruses, particularly in the terminal stages of disease.
In conclusion, SV40 is associated with both primary (ME) and recrudescent (PML) CNS disease in macaques with AIDS. Sequence analysis of the regulatory region and T antigen from different lesions does not support a tissue- or lesion-specific sequence. Instead, age of the animal, time of SIV infection, and possibly immunological status relative to time of SV40 infection may be the most important factors in determining the SV40-associated lesions of macaques with simian AIDS.
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Acknowledgements |
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Footnotes |
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Supported in part by Public Health Service grants RR00168, RR00164, NS30769, and NS35732. A. Lackner is the recipient of an Elizabeth Glaser Scientist Award.
Accepted for publication November 9, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B and Sp1 binding elements. J Virol 1996, 70:3118-3126[Abstract]
B and SP1 binding elements. J Virol 1997, 71:1880-1887[Abstract]
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