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Polyomaviruses
Chapter 20
Polyomaviruses The prototype member of the family Polyomaviridae is the polyoma virus of mice. This virus induces many different types of malignant tumors when artificially injected into infant rodents, such as hamsters, although it causes only harmless inapparent infections in mice when spread by natural routes. Another polyomavirus, simian virus 40 (SV40), causes subclinical infection in monkeys but induces tumors after inoculation into newborn rodents. During the 1960s and 1970s these two viruses were important models for the biochemical investigation of virus-induced malignancy, in both cultured cells and experimental animals (see Chapter 9: Mechanisms of Viral Oncogenesis). Subsequently two human polyomaviruses, BK and JC, were discovered in 1971. BK virus was recovered from the urine of a renal transplant recipient (with the initials “BK”), and JC virus from the brain of a patient with a rare demyelinating condition, progressive multifocal leukoencephalopathy (PML). As with SV40, BK and JC viruses are oncogenic for newborn hamsters and transform mammalian cells in vitro, but evidence that these viruses cause human cancer is lacking. Both BK and JC viruses are ubiquitous in humans, producing inapparent infections that persist for many years in the urinary tract, and undergo intermittent reactivation particularly following immunosuppression. Since 2007, this field has been re-invigorated by the discovery of a further nine new polyomaviruses from humans and several from non-human primates, revealing that this family is in fact larger and more divergent than previously recognized. These findings raise the possibility of a plethora of new polyomaviruses that remain to be discovered, as well as raising new questions about the evolution, tropism, latency, reactivation, and pathogenicity of this virus family. With the exception of Merkel cell carcinoma-associated polyomavirus (MCPyV) and trichodysplasia spinulosaassociated polyomavirus (TSPyV), any association between these more recently described polyomaviruses and human disease remains to be clarified.
Fenner and White’s Medical Virology. DOI: http://dx.doi.org/10.1016/B978-0-12-375156-0.00020-5 © 2017 2012 Elsevier Inc. All rights reserved.
CLASSIFICATION, PROPERTIES, AND REPLICATION Historically, papillomaviruses and polyomaviruses were classified within a single family Papovaviridae because both possess a double-stranded circular DNA genome and a similar virion capsid structure, as well as replicating and assembling in the host cell nucleus. However the seventh report of ICTV (2000) designated Polyomaviridae as a separate family from Papillomaviridae, on the basis of distinct molecular differences including a lack of major sequence homology, different genome organization, and the fact that polyoma transcription is bidirectional whereas papillomavirus transcription occurs in one direction only and from a single DNA strand. At least 12 different polyomavirus species infecting mammals and one infecting birds are recognized within the single genus Polyomavirus. Additional unclassified human polyomaviruses have been identified (Table 20.1) using polymerase chain reaction (PCR) with random primers on concentrated patient samples, characterizing cDNAs made from cell RNA transcripts, or rolling circle amplification of skin swab material. A number of unclassified polyomaviruses have also been isolated from bats. A proposal to recognize three separate genera (Orthopolyomavirus, Wukipolyomavirus, and Avipolyomavirus) is currently being considered. Polyomavirus virions are approximately 40 to 45 nm in diameter (cf. 55 nm for papillomaviruses), and do not have an envelope, the icosahedral capsid being composed of 72 capsomers (Fig. 20.1). The genome is a single molecule of closed circular, double-stranded DNA of approximately 5 kbp, which exists in the mature virion as a supercoiled, chromatin-like structure in association with host cell histone proteins. The genome includes a non-coding control region (NCCR), in which are located the origin of DNA replication (ORI) and also promoter and enhancer elements with binding sites for different DNA-binding proteins and transcription factors (Fig. 20.2). Replication and transcription of the viral genomes as well as virion maturation, collectively take place in the nucleus,
283
284 PART | II Specific Virus Diseases of Humans
TABLE 20.1 Polyomaviruses Isolated from Humans Virus
Site of Isolation/Excretion
Year Described
Disease
BKV
Urine
1971
Nephropathy/graft rejection in renal transplant recipients; hemorrhagic cystitis in stem cell transplant recipients
JCV
Urine
1971
PML in HIV/AIDS, or in patients treated with humanized mcAbs, e.g., natalizumab
KIPyV
Nasopharyngeal aspirate
2007
None known
WUPyV
Nasopharyngeal aspirate
2007
None known
MCPyV
Merkel cell carcinoma tissue
2008
Merkel cell carcinoma, a rare neuroendocrine skin tumor
HPyV6
Normal skin swabs
2010
None known
HPyV7
Skin swabs
2010
None known
TSPyV
Trichodysplasia spinulosa (TS) skin spicules
2010
TS (skin papules, spicules, alopecia in immunosuppressed patients)
HPyV9
Serum, urine, skin
2011
None known
MWPyV
Stools
2012
None known
STLPyV
Stools
2012
None known
and, given the limited coding capacity of the polyoma virus genome, the virus makes extensive use of host cell functions. Early viral proteins are produced from a series of RNAs generated by differential splicing from a single main transcript originating near the non-coding control region. These proteins are known as T (tumor) antigens, for example large T, middle (m) T (not found in polyomaviruses of primates), small t antigens, and other intermediates; these proteins interact with cell cycle regulatory proteins including p53 and Rb, and the viral proteins can induce cell transformation, de-repression of some host enzymes, and stimulation of cellular DNA synthesis. After initiation of viral DNA synthesis, RNA transcription then begins from the NCCR in the opposite direction to produce late mRNAs for three structural proteins VP1, VP2, and VP3, and in SV40, VP4. The structural proteins eventually selfassemble in the nucleus to form full and empty capsids, the major component of the outer shell being VP-1. Viral DNA synthesis is initiated by binding of the nonstructural viral T antigen to the origin of viral DNA replication (ORI) site. DNA replication proceeds bidirectionally with involvement of the host DNA polymerase and host nuclear transcription factors, and terminates approximately 180° from this point.
PATHOGENESIS Primary infection of mammals is normally asymptomatic, followed by a prolonged persistent infection. In humans, BK and JC viruses are known to remain latent in the tonsils, other lymphoid tissues, bone marrow, and urinary tract.
Using virus-like particles (VLPs), ELISA assays specific for the different human polyomaviruses have been used to map the prevalence of these infections. Seroprevalence rates for the various human polyomaviruses range from 35 to 99% of the population. However, despite the ubiquitous nature of these viruses, disease is a rare consequence of infection with only some of these viruses, and usually only in immunosuppressed individuals, with no evidence of human disease being associated with the remaining viruses (Table 20.1). Different pathological mechanisms have been proposed in polyomavirus infections in different settings (see Box 20.1). These include (1) direct cytopathic effect accompanying high-level virus replication, as with the loss of oligodendrocytes infected with JC virus in the brains of patients with progressive multifocal leukoencephalopathy (PML), (2) immune reconstitution inflammatory syndrome (IRIS), an inflammatory response against polyoma virus antigens seen following a partial restoration cell-mediated immunity, (3) a combination of viral cytopathology and inflammation, (4) induction of autoantibodies, and (5) cell transformation and oncogenesis. Mammalian polyomaviruses usually grow best in vitro in cells derived from a native host species. This restriction is thought to be determined at two levels: both by the presence or absence of appropriate receptors at the cell surface, and by the presence or absence of intracellular factors allowing full gene expression and virus replication. The finding that polyomavirus infection of cells of different species from the natural host can induce cell transformation without a full virus replication cycle has led to polyomaviruses
Polyomaviruses Chapter | 20 285
FIGURE 20.1 Polyomavirus morphology and structure. (A) Negative contrast electron microscopy of a polyomavirus. (B) Thin-section electron microscopy of masses of JC virions in the nucleus of an oligodendrocyte in brain of an immunosuppressed patient with progressive multifocal leukoencephalopathy (PML). (C) Graphic rendering of murine polyomavirus capsid assembly, composed of 72 pentamers of protein VP1—the capsid is colored according to distinct bonding environments of various VP1 proteins, showing how a single protein can be assembled into a very complex structure. (D) Reconstruction of an SV40 virion from cryoelectron microscopy images, showing vertex penton (pentomer) capsomers (blue) and hexon (hexomer) capsomers (green). (A) From Erskine Palmer, U.S. Centers for Disease Control and Prevention; (B) from Shigeki Takeda and Hitoshi Takahashi, Niigata Neurosurgical Hospital, Japan; (C) from Research Collaboratory for Structural Bioinformatics (RCSB), Protein Data Bank (PDB); (D) from Jean-Yves Sgro, University of Wisconsin. All with permission.
being extensively used as models for studying oncogenesis. Cells transformed by polyomaviruses express early viral proteins and usually contain integrated viral DNA. However, despite considerable research, very little evidence exists for the involvement of polyomaviruses in human cancers. In particular, many individuals were inadvertently exposed to SV40 virus as a contaminant of early batches of polio vaccine. Detailed follow-up studies of these populations have found no evidence for increased rates of cancer or a significant increase in mortality among the vaccine recipients.
BK DNA has been reported in some human tumors from a number of sites, but the significance of this is controversial because widespread asymptomatic carriage of virus occurs. Nevertheless, a WHO working group recently classified BK and JC viruses as “possibly carcinogenic for humans.” The one clear exception is MCPyV and Merkel cell carcinoma; integrated MCPyV DNA can be found within tumor cells, and RNA interference has shown an essential role for T-antigen expression, thereby suggesting at least a major etiological role for this virus in this form of cancer.
286 PART | II Specific Virus Diseases of Humans
FIGURE 20.2 Scheme of a prototype murine polyomavirus genome showing closed circular, double-stranded DNA with three main regions: the noncoding control region (top) containing the early and late promoters, their transcription start sites and the origin of replication; an early region encoding large T antigen (LT) and small t antigen (ST), and an alternatively spliced LT (LT′); and a late region encoding the viral capsid proteins VP1, VP2, VP3, and VP4. VP2, VP3, and VP4 are translated in the same reading frame and they terminate at the same site, but translation starts at successive initiating AUG codons to generate the different proteins. VP4 has been confirmed only in the primate virus SV40. VP1 is read in a different reading frame. Agnoprotein (Agno) is encoded by a late transcript in JC and BK polyomaviruses, but has yet to be confirmed as present in the newly described polyomaviruses. Also, a microRNA present in the late transcripts, that overlaps and hence targets the large T-antigen transcript, is shown. Reproduced from DeCaprio, J.A., Garcea, R.L., 2013. Nat. Rev. Microb. 11, 264–276, with permission.
BOX 20.1 Proposed Pathological Mechanisms in Polyomavirus-Induced Disease Mechanism
Example
1. Direct cytopathic effect associated with high-level virus replication
Oligodendrocytes infected with JC virus in PML
2. Immune-reconstitution inflammatory syndrome (IRIS)
(i) BK associated hemorrhagic cystitis after stem cell transplantation (ii) Worsening PML in AIDS patients after starting antiretroviral treatment, or after removal of natalizumab by plasmapheresis in multiple sclerosis
3. Cytopathic-inflammatory: high-level virus replication and inflammatory response
Polyomavirus-associated nephropathy in renal grafts
4. Auto-immune disease
T antigens complexed to DNA and nucleosomes inducing autoantibodies
5. Oncogenesis: Early gene expression leading to cell transformation
Merkel cell carcinoma
BK POLYOMAVIRUS BK virus infects most children before the age of ten, often subclinically although sometimes it is associated with mild upper respiratory symptoms, suggesting that transmission may occur via the respiratory route. The viral genome persists for life in a number of organs, including the kidney and lower urinary tract, the tonsils, lymphoid tissue, and bone marrow, without any apparent ill effects. Reactivation occurs during the last trimester of about 3% of pregnancies, causing asymptomatic shedding of virus intermittently in the urine. Approximately 20% of renal transplant recipients show BK viremia within one year of transplantation, and BK virus nephritis is a significant and increasing problem leading to renal graft failure in a majority of cases. Among bone marrow transplant recipients BK virus infection is associated with hemorrhagic cystitis, and systemic infection leading to meningitis, retinitis, pneumonia, or vasculopathy has also been reported. However, the virus has not been associated with PML. Diagnosis of BK virus infection is usually made by PCR for virus DNA in the blood and/or urine. However since asymptomatic urinary shedding and viremia are not uncommon in immunosuppressed patients, quantitative measurements of virus load are used to assess clinical significance. Other investigations are urine cytology to
Polyomaviruses Chapter | 20 287
detect “decoy” cells containing viral inclusions, and renal biopsy. Isolation of BK virus from urine in cultured human diploid fibroblasts, or JC virus from urine or brain in human fetal glial cells, are not often done.
JC POLYOMAVIRUS JC virus has a similar natural history to BK virus, although primary infections may occur somewhat later in childhood and only about 75% of older healthy adults have antibody. Again, lifelong persistence is established in the kidney, and virus is shed in the urine sporadically throughout life, more frequently during pregnancy or immunosuppression. JC virus causes progressive multifocal leukoencephalopathy (PML), an uncommon subacute demyelinating disease of the CNS that is invariably fatal. PML occurs as a complication of advanced disseminated malignant conditions such as Hodgkin’s disease or chronic lymphocytic leukemia, and also in primary or secondary immunodeficiency syndromes, or following immunosuppression for organ transplantation. JC virus became more common in the early years of the AIDS pandemic, affecting around 5% of AIDS patients. Indeed, the HIV-1 Tat protein has been shown to transactivate transcription of the late JC viral genes. However, AIDS-related PML has since become rare with the advent of improved antiretroviral therapy. The target cell is the oligodendrocyte, in which the virus undergoes a lytic productive infection; neurons are unaffected. Histologically, the disease is characterized by multiple foci of demyelination
in the brain, accompanied by proliferation of giant bizarre astrocytes. The surrounding oligodendrocytes are enlarged, with swollen nuclei occupied by a prominent inclusion body, which in fact contains a crystalline aggregate of many thousands of virions. Clinically, PML presents as focal neurological defects, usually insidious in onset and slowly progressing as the regions of demyelination expand (Fig. 20.3). Initial diagnosis relies on clinical findings and neuro-imaging including MRI, and demonstration of JC virus DNA in the CSF by PCR provides strong confirmation. Occasionally a brain biopsy is required, and this may show characteristic tissue cytopathology, including oligodendrocytes with intranuclear inclusions, bizarre astrocytes, and lipid-laden macrophages. The presence of JC virus can be detected by immunohistochemistry, in situ nucleic acid hybridization, or electron microscopy. Treatments including antiviral, immunomodulatory, and psychotropic drugs have been investigated but are of no proven benefit. However PML in patients with AIDS can be arrested and in some cases show some improvement following initiation of appropriate antiretroviral therapy. JC virus variants isolated from the brain of PML patients usually differ from the “archetype” found in the urine of asymptomatic carriers, showing extensive deletions and duplications in the nucleotide sequences within the promoter/enhancer region of the genome. These modifications may alter cell tropism and affect the switch between lytic and latent infection.
FIGURE 20.3 Progressive multifocal leukoencephalopathy (PML), CT images, Axial FLAIR (A), and Axial T2 (B) formats. Extensive right frontoparietal white matter abnormality extending to involve the subcortical white matter but sparing the cortex. There is also a focal site of infection in the left hemisphere. The patient was confirmed as having high viral loads of JC virus, consistent with the diagnosis of PML. From Radiopedia.org, with permission.
288 PART | II Specific Virus Diseases of Humans
OTHER RECENTLY DESCRIBED HUMAN POLYOMAVIRUSES KI and WU polyomaviruses (named after Karolinska Institute and Washington University respectively) were detected in 2007 in the nasopharyngeal aspirates from children with respiratory infections using PCR and random
FIGURE 20.4 Merkel cell virus (MCV) was identified in Merkel cell carcinomas (MCCs) using digital transcriptome subtraction. Immunohistochemistry of lesions has clearly implicated the virus in the etiology of MCC, a rare but aggressive form of skin cancer that is increasing in incidence. (A) MCC infiltrating dermal layers of skin; H&E. (B) MCC in subepithelial layers of skin; immunohistochemistry using antibody to MCV large T protein. Reproduced from Feng, H., et al., 2008. Clonal integration of a polyomavirus in human Merkel cell carcinoma [MC polyomavirus]. Science 319, 1096–1100, with permission.
primers. Serological population surveys indicate that half to more than 90% of adults have been infected, with most infections occurring in childhood. At the present time no firm evidence has been found for a pathogenic role for these viruses in either human respiratory disease or cancers. In 2008 a polyomavirus was identified using cDNAs of RNA transcripts isolated from cells from Merkel cell carcinoma, a rare aggressive neuroendocrine skin tumor occurring in the elderly or immunosuppressed (Fig. 20.4). Antibodies to MCPyV are also widespread in human populations, and around 80% of cases of Merkel cell carcinoma contain integrated MCPyV DNA. The integrated DNA is frequently truncated in the LT antigen and VP1 regions (Fig. 20.2), supporting a view that the integrated DNA may have lost its replicative ability whilst retaining the property of transformation. Extensive studies of other skin tumors and malignancies from many other sites have not revealed any association with other forms of cancer. The full mechanism of the oncogenic role of this common infection in this one specific rare form of cancer remains to be better understood. The finding of MCPyV stimulated further molecular searches for additional polyomaviruses in skin samples, with the result that HPyV6 and HPyV7 were discovered in 2010: neither appears to have any disease association at the present time. Trichodysplasia spinulosa is a rare skin disease of immunosuppressed patients characterized by facial spines, papules, and alopecia, and patient samples had been reported in 1999 to contain intracellular virus particles. In 2010, extracts of patients’ plucked facial spines were shown using rolling circle amplification to contain polyomavirus DNA fragments. TSPyV has not been found in a limited range of other tissues, and its role in human disease remains to be clarified. Similarly the significance of other polyomaviruses (HPyV9 and MWPyV) recently identified in human samples is unknown.
FURTHER READING Dalianis, T., Hirsch, H.H., 2013. Human polyomaviruses in disease and cancer. Virology 437, 63–72. DeCaprio, J.A., Garcea, R.L., 2013. A cornucopia of human polyomaviruses. Nature Rev. 11, 264–275. White, M.K., Gordon, J., Khalili, K., 2013. The rapidly expanding family of human polyomaviruses: recent developments in understanding their life cycle and role in human pathology. PLoS Pathogens 9, e1003206.
Polyomaviruses The prototype member of the family Polyomaviridae is the polyoma virus of mice. This virus induces many different types of malignant tumors when artificially injected into infant rodents, such as hamsters, although it causes only harmless inapparent infections in mice when spread by natural routes. Another polyomavirus, simian virus 40 (SV40), causes subclinical infection in monkeys but induces tumors after inoculation into newborn rodents. During the 1960s and 1970s these two viruses were important models for the biochemical investigation of virus-induced malignancy, in both cultured cells and experimental animals (see Chapter 9: Mechanisms of Viral Oncogenesis). Subsequently two human polyomaviruses, BK and JC, were discovered in 1971. BK virus was recovered from the urine of a renal transplant recipient (with the initials “BK”), and JC virus from the brain of a patient with a rare demyelinating condition, progressive multifocal leukoencephalopathy (PML). As with SV40, BK and JC viruses are oncogenic for newborn hamsters and transform mammalian cells in vitro, but evidence that these viruses cause human cancer is lacking. Both BK and JC viruses are ubiquitous in humans, producing inapparent infections that persist for many years in the urinary tract, and undergo intermittent reactivation particularly following immunosuppression. Since 2007, this field has been re-invigorated by the discovery of a further nine new polyomaviruses from humans and several from non-human primates, revealing that this family is in fact larger and more divergent than previously recognized. These findings raise the possibility of a plethora of new polyomaviruses that remain to be discovered, as well as raising new questions about the evolution, tropism, latency, reactivation, and pathogenicity of this virus family. With the exception of Merkel cell carcinoma-associated polyomavirus (MCPyV) and trichodysplasia spinulosaassociated polyomavirus (TSPyV), any association between these more recently described polyomaviruses and human disease remains to be clarified.
Fenner and White’s Medical Virology. DOI: http://dx.doi.org/10.1016/B978-0-12-375156-0.00020-5 © 2017 2012 Elsevier Inc. All rights reserved.
CLASSIFICATION, PROPERTIES, AND REPLICATION Historically, papillomaviruses and polyomaviruses were classified within a single family Papovaviridae because both possess a double-stranded circular DNA genome and a similar virion capsid structure, as well as replicating and assembling in the host cell nucleus. However the seventh report of ICTV (2000) designated Polyomaviridae as a separate family from Papillomaviridae, on the basis of distinct molecular differences including a lack of major sequence homology, different genome organization, and the fact that polyoma transcription is bidirectional whereas papillomavirus transcription occurs in one direction only and from a single DNA strand. At least 12 different polyomavirus species infecting mammals and one infecting birds are recognized within the single genus Polyomavirus. Additional unclassified human polyomaviruses have been identified (Table 20.1) using polymerase chain reaction (PCR) with random primers on concentrated patient samples, characterizing cDNAs made from cell RNA transcripts, or rolling circle amplification of skin swab material. A number of unclassified polyomaviruses have also been isolated from bats. A proposal to recognize three separate genera (Orthopolyomavirus, Wukipolyomavirus, and Avipolyomavirus) is currently being considered. Polyomavirus virions are approximately 40 to 45 nm in diameter (cf. 55 nm for papillomaviruses), and do not have an envelope, the icosahedral capsid being composed of 72 capsomers (Fig. 20.1). The genome is a single molecule of closed circular, double-stranded DNA of approximately 5 kbp, which exists in the mature virion as a supercoiled, chromatin-like structure in association with host cell histone proteins. The genome includes a non-coding control region (NCCR), in which are located the origin of DNA replication (ORI) and also promoter and enhancer elements with binding sites for different DNA-binding proteins and transcription factors (Fig. 20.2). Replication and transcription of the viral genomes as well as virion maturation, collectively take place in the nucleus,
283
284 PART | II Specific Virus Diseases of Humans
TABLE 20.1 Polyomaviruses Isolated from Humans Virus
Site of Isolation/Excretion
Year Described
Disease
BKV
Urine
1971
Nephropathy/graft rejection in renal transplant recipients; hemorrhagic cystitis in stem cell transplant recipients
JCV
Urine
1971
PML in HIV/AIDS, or in patients treated with humanized mcAbs, e.g., natalizumab
KIPyV
Nasopharyngeal aspirate
2007
None known
WUPyV
Nasopharyngeal aspirate
2007
None known
MCPyV
Merkel cell carcinoma tissue
2008
Merkel cell carcinoma, a rare neuroendocrine skin tumor
HPyV6
Normal skin swabs
2010
None known
HPyV7
Skin swabs
2010
None known
TSPyV
Trichodysplasia spinulosa (TS) skin spicules
2010
TS (skin papules, spicules, alopecia in immunosuppressed patients)
HPyV9
Serum, urine, skin
2011
None known
MWPyV
Stools
2012
None known
STLPyV
Stools
2012
None known
and, given the limited coding capacity of the polyoma virus genome, the virus makes extensive use of host cell functions. Early viral proteins are produced from a series of RNAs generated by differential splicing from a single main transcript originating near the non-coding control region. These proteins are known as T (tumor) antigens, for example large T, middle (m) T (not found in polyomaviruses of primates), small t antigens, and other intermediates; these proteins interact with cell cycle regulatory proteins including p53 and Rb, and the viral proteins can induce cell transformation, de-repression of some host enzymes, and stimulation of cellular DNA synthesis. After initiation of viral DNA synthesis, RNA transcription then begins from the NCCR in the opposite direction to produce late mRNAs for three structural proteins VP1, VP2, and VP3, and in SV40, VP4. The structural proteins eventually selfassemble in the nucleus to form full and empty capsids, the major component of the outer shell being VP-1. Viral DNA synthesis is initiated by binding of the nonstructural viral T antigen to the origin of viral DNA replication (ORI) site. DNA replication proceeds bidirectionally with involvement of the host DNA polymerase and host nuclear transcription factors, and terminates approximately 180° from this point.
PATHOGENESIS Primary infection of mammals is normally asymptomatic, followed by a prolonged persistent infection. In humans, BK and JC viruses are known to remain latent in the tonsils, other lymphoid tissues, bone marrow, and urinary tract.
Using virus-like particles (VLPs), ELISA assays specific for the different human polyomaviruses have been used to map the prevalence of these infections. Seroprevalence rates for the various human polyomaviruses range from 35 to 99% of the population. However, despite the ubiquitous nature of these viruses, disease is a rare consequence of infection with only some of these viruses, and usually only in immunosuppressed individuals, with no evidence of human disease being associated with the remaining viruses (Table 20.1). Different pathological mechanisms have been proposed in polyomavirus infections in different settings (see Box 20.1). These include (1) direct cytopathic effect accompanying high-level virus replication, as with the loss of oligodendrocytes infected with JC virus in the brains of patients with progressive multifocal leukoencephalopathy (PML), (2) immune reconstitution inflammatory syndrome (IRIS), an inflammatory response against polyoma virus antigens seen following a partial restoration cell-mediated immunity, (3) a combination of viral cytopathology and inflammation, (4) induction of autoantibodies, and (5) cell transformation and oncogenesis. Mammalian polyomaviruses usually grow best in vitro in cells derived from a native host species. This restriction is thought to be determined at two levels: both by the presence or absence of appropriate receptors at the cell surface, and by the presence or absence of intracellular factors allowing full gene expression and virus replication. The finding that polyomavirus infection of cells of different species from the natural host can induce cell transformation without a full virus replication cycle has led to polyomaviruses
Polyomaviruses Chapter | 20 285
FIGURE 20.1 Polyomavirus morphology and structure. (A) Negative contrast electron microscopy of a polyomavirus. (B) Thin-section electron microscopy of masses of JC virions in the nucleus of an oligodendrocyte in brain of an immunosuppressed patient with progressive multifocal leukoencephalopathy (PML). (C) Graphic rendering of murine polyomavirus capsid assembly, composed of 72 pentamers of protein VP1—the capsid is colored according to distinct bonding environments of various VP1 proteins, showing how a single protein can be assembled into a very complex structure. (D) Reconstruction of an SV40 virion from cryoelectron microscopy images, showing vertex penton (pentomer) capsomers (blue) and hexon (hexomer) capsomers (green). (A) From Erskine Palmer, U.S. Centers for Disease Control and Prevention; (B) from Shigeki Takeda and Hitoshi Takahashi, Niigata Neurosurgical Hospital, Japan; (C) from Research Collaboratory for Structural Bioinformatics (RCSB), Protein Data Bank (PDB); (D) from Jean-Yves Sgro, University of Wisconsin. All with permission.
being extensively used as models for studying oncogenesis. Cells transformed by polyomaviruses express early viral proteins and usually contain integrated viral DNA. However, despite considerable research, very little evidence exists for the involvement of polyomaviruses in human cancers. In particular, many individuals were inadvertently exposed to SV40 virus as a contaminant of early batches of polio vaccine. Detailed follow-up studies of these populations have found no evidence for increased rates of cancer or a significant increase in mortality among the vaccine recipients.
BK DNA has been reported in some human tumors from a number of sites, but the significance of this is controversial because widespread asymptomatic carriage of virus occurs. Nevertheless, a WHO working group recently classified BK and JC viruses as “possibly carcinogenic for humans.” The one clear exception is MCPyV and Merkel cell carcinoma; integrated MCPyV DNA can be found within tumor cells, and RNA interference has shown an essential role for T-antigen expression, thereby suggesting at least a major etiological role for this virus in this form of cancer.
286 PART | II Specific Virus Diseases of Humans
FIGURE 20.2 Scheme of a prototype murine polyomavirus genome showing closed circular, double-stranded DNA with three main regions: the noncoding control region (top) containing the early and late promoters, their transcription start sites and the origin of replication; an early region encoding large T antigen (LT) and small t antigen (ST), and an alternatively spliced LT (LT′); and a late region encoding the viral capsid proteins VP1, VP2, VP3, and VP4. VP2, VP3, and VP4 are translated in the same reading frame and they terminate at the same site, but translation starts at successive initiating AUG codons to generate the different proteins. VP4 has been confirmed only in the primate virus SV40. VP1 is read in a different reading frame. Agnoprotein (Agno) is encoded by a late transcript in JC and BK polyomaviruses, but has yet to be confirmed as present in the newly described polyomaviruses. Also, a microRNA present in the late transcripts, that overlaps and hence targets the large T-antigen transcript, is shown. Reproduced from DeCaprio, J.A., Garcea, R.L., 2013. Nat. Rev. Microb. 11, 264–276, with permission.
BOX 20.1 Proposed Pathological Mechanisms in Polyomavirus-Induced Disease Mechanism
Example
1. Direct cytopathic effect associated with high-level virus replication
Oligodendrocytes infected with JC virus in PML
2. Immune-reconstitution inflammatory syndrome (IRIS)
(i) BK associated hemorrhagic cystitis after stem cell transplantation (ii) Worsening PML in AIDS patients after starting antiretroviral treatment, or after removal of natalizumab by plasmapheresis in multiple sclerosis
3. Cytopathic-inflammatory: high-level virus replication and inflammatory response
Polyomavirus-associated nephropathy in renal grafts
4. Auto-immune disease
T antigens complexed to DNA and nucleosomes inducing autoantibodies
5. Oncogenesis: Early gene expression leading to cell transformation
Merkel cell carcinoma
BK POLYOMAVIRUS BK virus infects most children before the age of ten, often subclinically although sometimes it is associated with mild upper respiratory symptoms, suggesting that transmission may occur via the respiratory route. The viral genome persists for life in a number of organs, including the kidney and lower urinary tract, the tonsils, lymphoid tissue, and bone marrow, without any apparent ill effects. Reactivation occurs during the last trimester of about 3% of pregnancies, causing asymptomatic shedding of virus intermittently in the urine. Approximately 20% of renal transplant recipients show BK viremia within one year of transplantation, and BK virus nephritis is a significant and increasing problem leading to renal graft failure in a majority of cases. Among bone marrow transplant recipients BK virus infection is associated with hemorrhagic cystitis, and systemic infection leading to meningitis, retinitis, pneumonia, or vasculopathy has also been reported. However, the virus has not been associated with PML. Diagnosis of BK virus infection is usually made by PCR for virus DNA in the blood and/or urine. However since asymptomatic urinary shedding and viremia are not uncommon in immunosuppressed patients, quantitative measurements of virus load are used to assess clinical significance. Other investigations are urine cytology to
Polyomaviruses Chapter | 20 287
detect “decoy” cells containing viral inclusions, and renal biopsy. Isolation of BK virus from urine in cultured human diploid fibroblasts, or JC virus from urine or brain in human fetal glial cells, are not often done.
JC POLYOMAVIRUS JC virus has a similar natural history to BK virus, although primary infections may occur somewhat later in childhood and only about 75% of older healthy adults have antibody. Again, lifelong persistence is established in the kidney, and virus is shed in the urine sporadically throughout life, more frequently during pregnancy or immunosuppression. JC virus causes progressive multifocal leukoencephalopathy (PML), an uncommon subacute demyelinating disease of the CNS that is invariably fatal. PML occurs as a complication of advanced disseminated malignant conditions such as Hodgkin’s disease or chronic lymphocytic leukemia, and also in primary or secondary immunodeficiency syndromes, or following immunosuppression for organ transplantation. JC virus became more common in the early years of the AIDS pandemic, affecting around 5% of AIDS patients. Indeed, the HIV-1 Tat protein has been shown to transactivate transcription of the late JC viral genes. However, AIDS-related PML has since become rare with the advent of improved antiretroviral therapy. The target cell is the oligodendrocyte, in which the virus undergoes a lytic productive infection; neurons are unaffected. Histologically, the disease is characterized by multiple foci of demyelination
in the brain, accompanied by proliferation of giant bizarre astrocytes. The surrounding oligodendrocytes are enlarged, with swollen nuclei occupied by a prominent inclusion body, which in fact contains a crystalline aggregate of many thousands of virions. Clinically, PML presents as focal neurological defects, usually insidious in onset and slowly progressing as the regions of demyelination expand (Fig. 20.3). Initial diagnosis relies on clinical findings and neuro-imaging including MRI, and demonstration of JC virus DNA in the CSF by PCR provides strong confirmation. Occasionally a brain biopsy is required, and this may show characteristic tissue cytopathology, including oligodendrocytes with intranuclear inclusions, bizarre astrocytes, and lipid-laden macrophages. The presence of JC virus can be detected by immunohistochemistry, in situ nucleic acid hybridization, or electron microscopy. Treatments including antiviral, immunomodulatory, and psychotropic drugs have been investigated but are of no proven benefit. However PML in patients with AIDS can be arrested and in some cases show some improvement following initiation of appropriate antiretroviral therapy. JC virus variants isolated from the brain of PML patients usually differ from the “archetype” found in the urine of asymptomatic carriers, showing extensive deletions and duplications in the nucleotide sequences within the promoter/enhancer region of the genome. These modifications may alter cell tropism and affect the switch between lytic and latent infection.
FIGURE 20.3 Progressive multifocal leukoencephalopathy (PML), CT images, Axial FLAIR (A), and Axial T2 (B) formats. Extensive right frontoparietal white matter abnormality extending to involve the subcortical white matter but sparing the cortex. There is also a focal site of infection in the left hemisphere. The patient was confirmed as having high viral loads of JC virus, consistent with the diagnosis of PML. From Radiopedia.org, with permission.
288 PART | II Specific Virus Diseases of Humans
OTHER RECENTLY DESCRIBED HUMAN POLYOMAVIRUSES KI and WU polyomaviruses (named after Karolinska Institute and Washington University respectively) were detected in 2007 in the nasopharyngeal aspirates from children with respiratory infections using PCR and random
FIGURE 20.4 Merkel cell virus (MCV) was identified in Merkel cell carcinomas (MCCs) using digital transcriptome subtraction. Immunohistochemistry of lesions has clearly implicated the virus in the etiology of MCC, a rare but aggressive form of skin cancer that is increasing in incidence. (A) MCC infiltrating dermal layers of skin; H&E. (B) MCC in subepithelial layers of skin; immunohistochemistry using antibody to MCV large T protein. Reproduced from Feng, H., et al., 2008. Clonal integration of a polyomavirus in human Merkel cell carcinoma [MC polyomavirus]. Science 319, 1096–1100, with permission.
primers. Serological population surveys indicate that half to more than 90% of adults have been infected, with most infections occurring in childhood. At the present time no firm evidence has been found for a pathogenic role for these viruses in either human respiratory disease or cancers. In 2008 a polyomavirus was identified using cDNAs of RNA transcripts isolated from cells from Merkel cell carcinoma, a rare aggressive neuroendocrine skin tumor occurring in the elderly or immunosuppressed (Fig. 20.4). Antibodies to MCPyV are also widespread in human populations, and around 80% of cases of Merkel cell carcinoma contain integrated MCPyV DNA. The integrated DNA is frequently truncated in the LT antigen and VP1 regions (Fig. 20.2), supporting a view that the integrated DNA may have lost its replicative ability whilst retaining the property of transformation. Extensive studies of other skin tumors and malignancies from many other sites have not revealed any association with other forms of cancer. The full mechanism of the oncogenic role of this common infection in this one specific rare form of cancer remains to be better understood. The finding of MCPyV stimulated further molecular searches for additional polyomaviruses in skin samples, with the result that HPyV6 and HPyV7 were discovered in 2010: neither appears to have any disease association at the present time. Trichodysplasia spinulosa is a rare skin disease of immunosuppressed patients characterized by facial spines, papules, and alopecia, and patient samples had been reported in 1999 to contain intracellular virus particles. In 2010, extracts of patients’ plucked facial spines were shown using rolling circle amplification to contain polyomavirus DNA fragments. TSPyV has not been found in a limited range of other tissues, and its role in human disease remains to be clarified. Similarly the significance of other polyomaviruses (HPyV9 and MWPyV) recently identified in human samples is unknown.
FURTHER READING Dalianis, T., Hirsch, H.H., 2013. Human polyomaviruses in disease and cancer. Virology 437, 63–72. DeCaprio, J.A., Garcea, R.L., 2013. A cornucopia of human polyomaviruses. Nature Rev. 11, 264–275. White, M.K., Gordon, J., Khalili, K., 2013. The rapidly expanding family of human polyomaviruses: recent developments in understanding their life cycle and role in human pathology. PLoS Pathogens 9, e1003206.