Molecular Genetics - HPV and HBV (II) Flashcards
Structure of HPV
Non-enveloped dsDNA virus with supercoiled genome
List 2 high risk and 2 low risk HPV genotypes.
Associated diseases?
High risk: 16,18
Oncogenic, HSIL, Cervical cancer
Low risk: 6,11
Anogenital warts, LSIL
2 oncogenes commonly detected in cervical cancer.
Activity in low risk and high risk HPV types?
Function of these oncogenes?
HPV oncogene E6 and E7
high transforming activity in high risk HPV
Interact with growth-regulating targets in host cells.
E6»_space; p53, E7»_space; pRb
> > Maintain malignant phenotype of cervical carcinoma cells
Primary prevention against cervical cancer?
- HPV vaccination
- Avoid risk factors
Secondary prevention against cervical cancer?
- Cervical cytology/ HPV test screening
- Detect pre-invasive lesion of uterine cervix (HSIL)
3 reasons HPV infection persists?
- Evades normal immune mechanisms
- Do not produce humoral immune response
- Diagnostic serology test not widely available
3 main types of HPV DNA test
1) HPV pooled DNA test
2) HPV genotyping
3) HPV pooled test + High risk HPV 16/18 genotyping
3 purposes of HPV genotyping test
- Epidemiology
- Assess clinical behavior of subtypes
- Design targeted therapy and vaccine
Most important factors that validate HPV molecular tests
1) Quality control and clinical validation in diagnostic settings
2) Adequate data to correlate between cut-off point and detection of cervical cancer and precursor lesion
In situ hybridization.
used for which type of HPV test?
Early HPV detection
PCR sequencing
- used for which type of HPV test?
HPV detection
HPV genotyping
(+/- Detect multiple HPV)
Describe HPV DNA chips function
PCR products of L1 and E6, E7 gene sequences are hybridized to arrayed probes on the chip»_space; DNA sample with target genes bind to probes
HPV genotypes identified by fluorescence scanner
List techniques for HPV testing *****
- HPV DNA chip: Genotype by gene products
- Qualitative multiplex assay and RT-PCR
HPV detection
HPV high risk genotype 16,18
Identify multiple HPV infection - RNA transcription detection
Persistent HPV infection
Qualitative multiplex assay
- used for which type of HPV test?
HPV detection
HPV high risk genotype 16,18
Identify multiple HPV infection
RT-PCR
- used for which type of HPV test?
HPV detection
HPV high risk genotype 16,18
Identify multiple HPV infection
Which targets are tested in extended HPV genotyping (check)
E6,E7 DNA regions
Screen for all 14 high-risk HPV types
Best test for persistent HPV infection?
RNA test:
RNA transcript detection
more specific for detection of persistent infection and detection of HSIL than DNA test
Invasive cervical cancer and high grade lesion is suspected based on clinical exam
Order 2 tests for confirmation
Co-testing:
Pap-smear + HPV DNA testing
Define ASC-US and associated risks
Atypical squamous cells of undetermined significance
- Cytological abnormalities, higher risk of LSIL or HSIL
Triage of ASCUS
- Next step after detection of ASCUS through cytology testing
Cytology and HPV co-testing
1) Repeat cytology again at 6 months and 12 months
a) Positive for ASCUS or LSIL/ HSIL in both tests = colposcopy
b) Both Normal results = repeat cytology again in 3 years
2) HPV testing
a) High risk HPV positive = Colposcopy
b) High risk HPV negative = Repeat cytology or co-testing at 3 years
Purpose of ASCUS triage?
Reduce patient anxiety
Decrease cost of repeat smears
Earlier Dx of HSIL
One other cancer associated with HPV
Testing method?
HPV 16»_space; Oropharyngeal squamous cell carcinoma
Increase incidence in men with age
IHC for p16 as surrogate marker from tissue specimen
3 un-recommended use of HPV tests
Low risk test
Test under 25
Reflexive HPV test after Pap smear of HSIL
HPV +ve and HPV -ve cervical cancer
- Associated with which risk factors
- Which has better prognosis
HPV +ve = sexual behavior
HPV -ve= smoking and tobacco
HPV +ve has better prognosis
Method for test of cure after excision of high grade cervical carcinoma in situ?
High risk HPV test + cytology to monitor recurrence
12 months and 24 months after excision
Both negative = return to normal screening interval
Method for testing for HPV infection in uterine cervix SCC and Adenocarcinomas.
IHC for p16 surrogate marker + HPV DNA testing
Genome structure and method of genetic replication in HBV
4 overlapping reading frames to make protein
Partially dsDNA virus
P - polymerase/ reverse transcriptase
S - HBsAg
C - HBcAg and HBeAg
X - HBx
Replicates via reverse transcription
Form of HBV DNA that can reactivate inside hepatocytes and indicate treatment?
cccDNA
Covalently closed circular DNA
Remains inside hepatocyte nucleus even after treatment
Describe the time course of anti-HBc and anti-HBs antibody development after infection.
1-2 months after: Generate Anti-HBc IgG and Anti-HBc IgM
IgM level drops after few months
IgG level persists
3 months after: Generate Anti-HBs
Level slightly decreases over decades
First phase of HBV infection:
- HBV DNA level? HBsAg and HBeAg?
- Liver enzyme level?
- Host reaction?
HbeAg- positive infection (Immune tolerance)
- High HBV DNA, HBsAg present, HBeAg present
- Normal AST, ALT (small spike up to 1 month)
- No host reaction
Second phase of HBV infection
- HBV DNA level? HBsAg and HBeAg?
- Liver enzyme level?
- Host reaction?
HBeAg- positive hepatitis (Immune clearance) —> HBeAg negative hepatitis
- HBV DNA fluctuate, HBsAg present, HBeAg present +/- Anti-HBe
- AST/ ALT fluctuate
- Active hepatitis > symptomatic
- Cytotoxic T cell kill infected hepatocytes
Third phase of HBV infection
- HBV DNA level? HBsAg and HBeAg?
- Liver enzyme level?
- Host reaction?
HbeAg- negative infection (inactive carrier)
- HBV DNA low HBsAg present, Anti-HBe present
- Low AST/ALT
- Frequent cirrhosis/ HCC
Fourth phase of HBV infection
- HBV DNA level? HBsAg and HBeAg?
- Liver enzyme level?
- Host reaction?
HBsAg-negative
- Cannot detect HBV DNA, Anti-HBs and Anti-HBe present
- Normal liver enzyme
- HCC/ Cirrhosis possible
Role of HBV DNA testing (4)
- Indicate infectivity/ disease activity
- Find HBV in HBsAg-negative cases
- Indicate risk of progression to HCC/ Cirrhosis
- Predict antiviral treatment outcome and drug-resistance
Process of HBV DNA testing?
- Sample?
- Steps?
Whole blood (EDTA or clotted blood)
1) Centrifuge for plasma/ serum
2) Lysis/ extraction of HBV DNA
3) Signal/ target amplification (e.g. PCR)
4) Fluorometric or colorimetric measurement
Advantage of new HBV assays over old assays?
Higher sensitivity (can detect at very low conc.) Much shorter time
Over entire course of HBV infection, when to do HBV DNA test?
1) Test newly infected patient - indicate disease activity, plan treatment
2) During antiviral therapy - predict drug response or resistance
3) After stopping antivirals - check persistent infection
MoA of antivirals for HBV infection.
Name 4
Nucleoside analogues = NRTI
Chain terminator of DNA synthesis and inhibits reverse transcription
Nucleoside analogues:
Lamivudine
Entecavir
Nucleotide analogues
Tenofovir: Tenofovir alafenamide or Tenofovir disoproxil fumurate (for WT and Lamivudine resistant HBV)
Interferon-a therapy +/- steroid for immune rebound enhancement
1 gene sequence that confers resistance to antivirals in HBV. What type of mutation?
Reverse transcriptase gene mutation confers resistance
» Polymerase/ Reverse transcriptase mutation
YMDD gene mutation
> > YVDD»_space; Methionine to valine SNP
Which Antiviral for HBV has highest resistance rate?
Lamivudine
List 3 HBV markers
cccDNA
HBsAg
HBeAg
How to assess amount of cccDNA transcription in HBV infection?
Quantitative HBsAg assay
Reflects transcription activity of cccDNA
