Polyomavirus Lecture

Polyomaviruses

History•Research dates to the 1950s1)

•Murine leukemia virus passaged through mice, developed salivary gland tumors and not leukemia

•‘agent’ different from MLV, caused multiple tumor types in newborn mice ->

•polyomavirus Greek poly (many) and -oma, denoting cancer

1) Gross, J. Exp Med, 1953, 414-21

History• Next member Simian virus 40 (SV40), 1960

• Screening samples from polio vaccine production (rhesus monkey kidney cells)

• no cytopathic effects, virus resistant to formalin inactivation

• 1971, human polyomaviruses from immunocompromised patients

• JC , brain from patient with PML1)

• BK from Urine (renal tx patient) 2)

1) Padgett, 1971, Lancet, 1257-60

2) Gardner, 1971, Lancet, 1253-57

Polyomavirus infection of different hosts

Host Virus

HumanBK virusJC virus

MonkeySimian virus (SV40)

Simian agent 12Lymphotropic papovavirus

CattleBovine

polyomavirus

RabbitRabbit kidney

vacuolating virus

Host Virus

Mouse Mouse polyomavirusK virus

Hamster

Hamster

papovavirusAthymic Rat

Rat polyomavirus

Parakeet

Budgerigar fledgling disease

virus

SV40, model for basic eukaryotic cell processes

•For many years SV40 used in basic research to study basic eukaryotic processes (transcription, splicing)

•SV40 most heavily mutagenized DNA molecule per base pair

•Polyomavirus in humans studied with the HIV epidemic and progress in transplantation

Classification

Family Genus

Polyomaviridae Polyomavirus

Virion Structure

180000x

• small, nonenveloped

• three capsid proteins VP1,VP2 and VP3

• double-stranded covalently closed circular DNA

• 5kbp genome length

• icosahedral symmetry

• relative resistant to heat formalin and lipid solvent

• only VP1 exposed on surface of capsid

EM magnification 152k and 280k, UrineK Bienz, Basel

Genome Structure

Chapter 61, Polyomaviruses, Fields Virology, 5th ed 2007

Genome divided in 3 parts

• early region - expressed before onset of replication

• late region - expressed after replication

• regulatory region - origin of replication, promoters for early and late transcription

• non overlapping late/early, transcribed from opposite strand

• large T antigen binds to tumor suppressor proteins Rb and p53, initiate cell cycle

SV40, BK and JC, 70-5% homology

Imperiale et al, Fields Virology 5th Edition, Vol2,p2266

Viral entry and trafficking

Dugan, A.S., Eash, S. & Atwood, W.J.Transplant Infectious Disease 8 (2), 62-67

BK JC SV40

Receptors

α(2,3)-linked sialic acid

Terminal α(2,6)-linked sialic acid

Sialic acid independent

Co-Receptor

sUnknown

Serotonin receptor (5HT-2a)

MHC class I

Epidemiology

•Seroconversion for BK virus beween age 5-7

• JC virus later

•High seroprevalence >80%

•Antibodies to both viruses remain lifelong

•But fluctuate in titer

•Unclear if this is due to re-infection or reactivation

Imperiale et al, Fields Virology 5th Edition, Vol2,p2284

Clinical Features of Polyomavirus infection

• Progressive Multifocal Leukoencephalopathy (PML)

•Haemorhagic Cystitis (post HSCT)

•BK Nephropathy or Polyomavirus-associated nephropathy (PVAN)

• rare, encephalitis, pneumonitis, retinitis and vasculopathies

Progressive Multifocal Leukoencephalopathy

FeaturesFeatures

An end-stage complication of HIV, caused by the JC virus

 PML is rare in the general community, but

relatively common in HIV infection (affecting 4% of all AIDS patients). Routine testing for HIV should be considered for any patient with PML

 Evolution occurs over weeks to months

CD4<100

Progressive mulltifocal leukoencephalopathy (PML) Presenting Signs and Symptoms

§ Afebrile, alert, no headache

§ Progressively impaired speech, vision, motor function

§ Cranial nerve deficit and cortical blindness

§ Cognition affected relatively late

“Classical” AIDS-associated PML in the pre-HAART era

• CD4+ T cell counts < 200 μl

• non-enhancing white matter lesions on MRI, without edema and mass effect

• detectable JCV DNA by PCR in CSF

• Absence of inflammatory infiltrates in brain biopsy

• Progressive evolution and fatal outcome in few months

• PML can be contained if• Sufficient number of CD4+ T cells

• presence of JCV-specific CD8+ cytotoxic T lymphocytes in the blood

• these CD8+ T cells infiltrate the brain lesions and destroy JCV-infected cells

• Inflammatory reaction• causes a break-down of the blood-brain-barrier

• decreases viral replication in the brain and JCV VL in CSF

• CT brain scan may be normal or remarkable for areas of diminished density or demyelination (deterioration of the covering of the nerve)

• PCR of CSF for detection of JC virus

•PCR is positive in about 60-80% of the cases

• Differential diagnosis:

•Toxoplasmosis

•Primary CNS lymphoma

Diagnostics

• Definitive diagnosis is by brain biopsy

Pathologic characteristics of JC infection

Detection of JC DNA by in-situ hybridisation

Luxol fast stain for myelin with lesions caused by JC lytic infection

(d=demyelination)

Crystaline array of assembled virion particles in the nucleus of infected oligodentrocytes

PML Radiological findings

• CNS imaging may reveal changes typical of PML

• is not specific

• MRI more sensitive than CT scans

• single or multiple hypo-dense lesions in the subcortical white matter

• with or without surrounding oedema

• MRI shows increased T2 signal with liitle/no enhancement with gadolinium

• CT generally nonenhancing

• Inflammatory response (enhancement) sometimes seen in patient on ART

Progressive Multifocal Leukoencephalopathy

MRI showing subcortical white matter demyelination

Text

Role of antivirals in treatment of PML

Cytarabine in Progressive Multifocal Leukoencephalopathy Associated with HIV

Infection

• Pre HART area clinical trial

• 57 patient randomized to

• anti-retroviral drug

• or antiretroviral drug + Cyto-Ara ivorCyto-Ara intrathecal

Median survival 8 wks Cyt-Ara15 wks intrathecal Cyt-Ara, 11 anti-retroviralonly

Hall, NEJM,1998, 1345

6/05

PML: Treatment

• No effective therapy

• ART with increase in CD4 count may improve neurologic function

• Clinical trial data do not support use of vidarabine or cidofovir

Natalizumab-associated

PM• Natalizumab humanized monoclonal antibody

that binds to the a4 chain of the a4β1 and a4β7 integrins

• reduce the migration of leukocytes from the peripheral blood into tissues

• In MS, natalizumab reduction of leukocyte extravasation into the CNS

• After approval by FDA, 3 patient developed PML

• Risk of PML after natalizumab treatment 0.1%

BK virus• Immunocompetent:

–Asymptomatic shedding in the urine (0.3%)• Bone marrow transplant patient

–Haemorrhagic cystitis• Renal transplant recipients

–Ureteral stenosis (~ 3%)–BK nephropathy (BKVN)

Term Definition

BK infection•Serological/virological exposure•Including replicxative/non rep.

state

BK replication•Virus multiplication (lytic

infection) in non latency sites

BK disease•Histological evidence of BK mediated organ pathology

Terminology

BKVN a new disease?

• 1-5% of renal transplant recipients

– Allograft loss in 45%

• Virtually nonexistant prior to 1995

– 0/616 between 1985-95 vs 5/70 between 1995-97 (Binet et al. Transplantation, 99)

Nickeleit et al, NEJM,342:1309,2000

9 patients with BKN and 41 withoutOnly 2 of control in plasma

Text

Time Course of Detection of BK Virus DNA in the Plasma of Representative Renal-Allograft Recipients with BK

Virus Nephropathy

neg plasma PCR

pos plasma PCR

X biopsy proofen+ clinical signs

Clinical manifestation of BKVN

• Typical manifests as–Slowly decrease of GFR (increase in creatinin)

–Failure to respond to anti-rejection therapy

• Onset 28-40 weeks (6-150)

• Histology:–Early noninflamatory foci, progression to inflammatory

changes with necrosis and fibrosis (-> graft loss 5 month)

• No extra renal sites or native kidney involved

Study Prevalence FK506 MMF Graft loss

Mengel et al. 2003 1.1 57 87 71

Trofe et al. 2003 2.1 77 54 54

Buehrig et al. 2.7 100 89 38

2003 Ginevri et al. 2003 3.0 66 66 33

Rahaminov et al. 2003 3.8 100 87 14

Kang et al. 2003 3.9 100 33 100

Ramos et al. 2002 5.1 91 97 82

Hirsch et al. 2002 6.0 60 40 0

Li et al. 2002 7.0 100 50 33

Maiza et al. 2002 7.1 50 50 50

Moriyama et al. 2003 10.3 36 NA 22

Natural history of BK nephropathy

Quant PCR

Qual PCR

Creatinine

Diagnosis of PVN

• Histology–Intranuclear polyomavirus inclusion bodies in

tubular or glomerular parietal cells–Cell necrosis F tubular injury / dysfunction–May be very focal–Alterations are NOT pathognomonic PVN

• Immuno histochemistry (large T-Ag) or in-situ hybridisation

Polyomavirus-associated nephropathy (PVAN) - Histology

Drachenberg 2005, Human Pathology, 1245-55

Nickeleit, Transplant International 19 (12), 960-973

Drachenberg, 2006, American Journal of Transplantation 4 (12), 2082-2092

mild severe

Polyomavirus BK Versus JC Replication and Nephropathy in Renal Transplant Recipients: A Prospective Evaluation.Drachenberg, Cinthia; Hirsch, Hans; Papadimitriou, John; Gosert, Rainer; Wali, Ravinder; Munivenkatappa, Raghava; Nogueira, Joseph; Cangro, Charles; Haririan, Abdolreza; Mendley, Susan; Ramos, EmilioTransplantation. 84(3):323-330, August 15, 2007.

FIGURE 3. Correlation between BKV viremia and biopsy findings in 75 patients shedding BKV (see Table 1, footnotes). BKV viremia levels above 10,000 copies/mL were strongly associated with a concurrent biopsy showing PVAN (PP=0.0001). In contrast, JCV viremia was uncommon and of low level (mean 2.0E+03) correlating with the scarce numbers of tubules with positive SV40 staining in the biopsies with JCV PVAN (mean 5%).

BK viremia

Risk Factors for PVAN

• Immunosuppressive drugs and combinations–Combinations containing MMF or FK506

• Drug concentration–no clear drug level identified

• Antilymphocyte preparations–For steroid-resistant graft rejection

• Steroids–Only if with unaltered immunosuppression

Other factors increasing the risk

• Age

• Male gender

• White ethnicity

• Negative BK serostatus (children)–Positive serostatus not protective

• Rare in other transplant settings

• No risk factors:–Cold ischemia, cadaveric, delayed graft function

Surrogate markers for PVN

• Heterogeneity of risk factors

• Polyomavirus replication single common features for PVN–Urine cytology (Decoy cells)–Quantification (Urine) BK DNA / VP1 RNA–EM

• Problems– Inhibition, degradation, cell pellets vs supernatans

Prevention/Treatment of BKVN

Cytology/UrinePCRBK viremia

BK nephropathy4 wks

12 wks

Prognostic values of diagnostic tests for BKVN

Viscount et al, Transplantation. 2007 Aug 15;84(3):340-5.

Viscount et al, Transplantation. 2007 Aug 15;84(3):340-5.

Relationship between the levels of BKV viruria and viremia in 204 kidney transplant recipients

Managment of PVAN

• Modification of Immunosuppression– Graft loss (10 to 80%)– No randomized study comparing protocols

Rejection risk 1st year >> 2nd year

Does Reduction in Immunosuppression in Viremic

Patients Prevent BK Virus Nephropathy

•prospective study, 2005/6

•single centre, France

•139 renal tx patients, 123 analysed

•Recommandation followed, screen for active infection at 1,3,6,9 and 12 month

•positive urine -> qPCR in blood

•pos blood -> biopsy and reduction in IS

Study design

Alméras et al, Transplantation, 2008:1099-14

Calineurin -20%

MMF -50%qPCR in blood4wk and 6wk

Alméras et al, Transplantation, 2008:1099-14

•72% of patient with presumptive BKVN did not progress to BKVN and cleared infection in blood

•28%(3/11) did not clear viremia despite reduction in IS

•1/11 (0.9%) subsequently developed BKVN

•50% (1//2) of patients with BKVN resolved BK nephropathy after reduction of IS

Alméras et al, Transplantation, 2008:1099-14

Alméras et al, Transplantation, 2008:1099-14

-1.5 log10 from baseline

Cidofovir

•very broad spectrum of antiviral activity (Herpes viruses, Adenovirus, Poxviruses, polyomaviruses)

•poor oral bioavailability

•nephrotoxic

•mechanism by which cidofovir mediate anti-BKV activity not clear (lack of polymerase)

•cidofovir can inhibit BK in-vitro

• lipid ester of cidofovir have superior anti BK activity

Potential antivirals for treatment of PVAN

Josephson, M.A., 2006 Transplant Infectious Disease 8 (2), 95-101.

Response of PVAN after cidofovir treatment

Kadambi, 2003,American Journal of Transplantation 3 (2), 186-191

Leflunomide

• FDA approved for treatment of rheumatoid arthritis

• family of drugs called lonitrilamides

• substantial immune suppressive activity

• antiviral activity in-vitro against CMV, BK and HSV

• mechanism not clear

Leflunomide

Williams J, NEJM 2005, 1157

• 17 patient, biopsy proven BKN• loading (100mg), followed 20-60mg• blood level of 50-100 micog/mLOutcome• all patient reduction or viral clearance blood+urine (if >40microg/mL• 7 blood and 5 from blood + urine clearance• serum creatinine stabilized

58

Guidelines for prevention diagnosis and management of

BKVN in renal transplant patients

59

Screening for2 years

60

• We suggest screening all KTRs for BKV with quantitative plasma NAT (2C) at least:

• monthly for the first 3–6 months after transplantation (2D);

• then every 3 months until the end of the first post-transplant year (2D);

• whenever there is an unexplained rise in serum creatinine (2D); and

• after treatment for acute rejection. (2D) • We suggest reducing immunosuppressive medications

when BKV plasma NAT is persistently greater than 10 000 copies/mL (107 copies/L). (2D)