Human Papillomaviruses: Natural History and Virology Elizabeth R. Unger Ph.D., M.D. Acting Chief,...
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Transcript of Human Papillomaviruses: Natural History and Virology Elizabeth R. Unger Ph.D., M.D. Acting Chief,...
Human Papillomaviruses: Natural History and Virology
Elizabeth R. Unger Ph.D., M.D.
Acting Chief, Papillomavirus Section
Centers for Disease Control and Prevention
November 29, 2001
Papillomaviruses
Non-enveloped dsDNA viruses Circular genome ~8 kb 55-nm spherical capsid coat
Widely distributed in higher vertebrates Tight species specificity
Tropism for squamous epithelium Associated with warts and papillomas
Genome Organization
Similar for all papillomaviruses Only one strand transcribed Open reading frames (ORFs) named in
relation to bovine papillomavirus genes “Early” genes E1-E7 (but no E3 in HPV) “Late” genes L1 and L2, coding for major and
minor capsid proteins
HPV Genome: Episome
E6
E7
E2
E4
E5
L1
L2
E1
URR transformation
transformation
episomereplication
late cytoplasmicprotein
transcriptionalregulator
capsidproteins
P97
Simple Genome
Dependent on host cell for replication, transcription and translation
Viral functions tightly linked to cellular differentiation
Poly-cistronic viral transcripts Multiple promoters, multiple splice patterns Promoter usage linked to differentiation
HPV URR
Upstream regulatory region Also called long control region (LCR) or non-
coding region (NCR) Contains transcriptional and replication
regulatory elements
Late genes
Region of greatest genetic conservation L1 is major capsid protein
Capsid is 72 pentamers of L1 Expressed L1 assembles into viral
conformation, viral-like particles (VLPs) L2 is minor capsid protein
Required for encapsidation of viral genome
Early genes
E1: Viral replication; maintains episome E2: Transcriptional regulation, co-factor for
viral replication E4: Disrupts cytokeratins E5: Interacts with growth factor receptors E6: Transforming protein; p53 degradation E7: Transforming protein; Rb binding
Viral Replication
Replication and assembly in nucleus Infection initiated in basal epithelial cells
Steady-state viral replication, some early-region transcription
Presumed site of latent infection High-copy viral replication, late gene
transcription and virion production limited to differentiating cells
Viral Integration
Not part of normal viral life-cycle Occurs randomly in host chromosomes Characteristic breakpoint in viral genome
E1-E2 disruption Abnormally regulated E6/E7 expression
Associated with oncogenesis but not required
Immune response
Non-lytic infection minimizes exposure to immune system Virus released with desquamating cells
Immune system influences outcome of HPV infection
Humoral and cellular responses identified Not all infected hosts have detectable
immune response
Human Papillomaviruses
More than 100 types, >80 fully sequenced Typing based on nucleic acid sequence
>10% sequence variation = new type; 2-10% = subtype, <2% = variant
Types assigned sequential number based on order of discovery No relation to phylogeny
HPV Types
Two major phylogenetic branches, differing affinities for site of infection
Cutaneous: Keratinized squamous epithelium Mucosal: Non-keratinized squamous epithelium
HPV Mucosal Types and Variants
More than 30 types found in anogenital tract “Low risk” types: rarely found in cancers “High risk” types: frequently found in cancers or
similar to types found in cancer High risk types most prevalent in population,
regardless of disease status Variants best characterized for HPV 16
E6/E7 polymorphisms could modify oncogenicity Cross-reactive in ELISA assays
Unique features of HPV
No simple in vitro culture method Antibody methods lack sensitivity Diagnosing infection requires detection of
HPV genetic information Corollary: requires cellular sample from the
site of infection Only current infections identified
HPV Detection
“Infection” monitored by DNA detection Sample and assay frame view of disease Complicates definition of latent, occult,
persistent or recurrent infection
Tissue Samples
Biopsies provide direct correlation between pathology and virus Includes basal layer of epithelium Limited area sampled Not suitable for screening
Exfoliated Cytology Samples
Noninvasive approach for population screen Sampling not directed at “lesion” Quality dependent on collection device and
anatomic site sampled Swabs, brushes, scrapes, washings
Basal epithelium not commonly included
Cervical sample most commonly used in women Appropriate sample in males is not clear
Estimates of HPV Associated Disease in the US
Genital warts: 1%, 1.4 million Colposcopic (sub-clinical) changes: 4%, 5
million DNA positive, no lesions: 10%, 14 million HPV antibody positive but no DNA or
lesions: 60%, 81 million OVERALL 75% of population exposed
Natural History Overview
HPV infection is very prevalent in the population
Genital HPV is acquired around the time of sexual debut
Infection is usually transient and not associated with symptoms
Persistent infection is more likely to be associated with potential for neoplasia
HPV and Cervical Cancer
Consistent epidemiologic association of HPV with cervical cancer precursor lesions
Plausible biologic mechanisms for HPV oncogenesis
HPV oncogenesis is a rare event with long interval between infection and cancer Infection alone is insufficient to cause cancer Additional factors required for neoplasia
Questions about HPV Infection
Is HPV eliminated from the host? HPV “clearing” is monitored by DNA detection
in cytology samples Negative results indicate shedding below limit
of detection but basal compartment of epithelium not sampled
HPV can be detected in histologically normal margins surrounding gross lesion
Duration of HPV Infection
HPV Types Months, median (95% CI)
Franco (1999) “Oncogenic” 8.1 (6.0 -12.6)
Woodman (2001)
HPV 16 10.3 (6.8 - 17.3)
HPV 18 7.8 (6.0 – 12.6)
Persistent Infection?
No consensus on definition Requires detection of same HPV type on
more than one occasion Time interval varies: 3-6 months
Long intervals: re-infection not excluded Consistent detection on each occasion versus
intermittent detection
Latent Infection?
Formal definition: Presence of HPV DNA in the absence of virion production
Practical definition: Detectable HPV DNA in the absence of identifiable lesion HPV DNA positive, normal cytology Equated with occult infection
HPV DNA Assays
Multiple HPV types complicate assays Sensitivity and type-specificity vary Inter-assay comparisons difficult
Direct hybridization Southern blot, dot blot, in situ, HybridCapture
Amplification (PCR) Type specific, versus consensus
HPV Hybrid Capture Assay
Current FDA approved test 1995 tube format; 1999 micro-titer format
Liquid hybridization technique Chemiluminescent detection
Semi-quantitative signal, but no control for amount of input DNA
RNA probes react with DNA targets RNA-DNA hybrids captured and detected with
monoclonal antibody to hybrids
Hybrid Capture II Assay
Low risk probe mix HPV types 6, 11, 42, 43, 44
High risk probe mix HPV types 16, 18, 31, 33, 35, 39, 45, 51, 52,
56, 58, 59 and 68 Good inter-laboratory comparison Results not type-specific
Hybrid Capture II Assay
Designed to work with exfoliated cervical sample
Recommended collection kit includes brush and sample transport media Collects endo- and ectocervical cells
5% of total specimen assayed for each probe group
HPV PCR Assays
Small portion of genome targeted Allows testing of samples with poor quality DNA Small changes in virus (variants or integration)
may give false negative results Amount of DNA assayed varies (limits
number of cells sampled)
HPV PCR Assays
Type specific assays Generally target E6/E7 region
Consensus assays Generally target L1 region Type(s) determined by type specific
hybridization, restriction digestion or sequencing
Typing Consensus PCR Product: Roche Line Blot Assay
HPV Quantitation
“ Viral load” difficult to estimate because of uneven tissue distribution and variation in sampling
Requires some measure of number of cells in assay (denominator)
Quantitative PCR assays, usually type-specific
HPV In Situ Hybridization
Only method permitting direct visualization of virus in morphologic context
Applicable to formalin-fixed paraffin-embedded tissues
Type specificity is good, but cross-hybridization cannot be totally avoided
Results are very technique dependent Integration status can be determined
HPV Serology
ELISA-based detection of antibodies against L1-VLPs Serum or mucosal; IgG or IgA
Type-specific, at least at low titers Reaction indicates past or current infection
Less than 70% of HPV positive subjects develop detectable antibodies; lag-time of several months
L1-VLP Assays
Formats vary (direct vs. indirect) VLP production not standardized
Different expression systems, preparative methods, QC approach
No gold-standard for setting threshold for positive result
Few inter-laboratory comparisons