viral diagnosis and treatment Flashcards
NAT
detection of gene sequences
Viral DNA or RNA can be detected in samples by
Nucleic Acid Amplification Techniques (NAT)
e.g. polymerase chain reaction (PCR)
These techniques harness the specificity of the
genetic code, using specific primers to anneal to
target. NAT involve amplification of signal
generated by labelled reagents
Due to speed, increased sensitivity, detect
unculturable agents, automatability NAT has
largely replaced other methods of direct
detection.
PCR modifications
Modifications:
Multiplex – simultaneous amplification of different
targets
Nested PCR – enhanced sensitivity
RNA detection - initial Reverse transcriptase step
Quantitative PCR – measures amount of NA
detected
Real Time – sophisticated adaption detects and
quantifies target in real time without the need to
open the reaction tube
real time PCR
Conventional PCR requires gels, blots etc for
amplicon evaluation
Real time PCR – fluorescent labels on
primers, probes or amplicons
Products can be read during amplification
step
Can be quantitative
Faster
Suits high-throughput lab
Indirect detection of infection:
Serological diagnosis
Infers infection by the detection of microbe-specific
antibody
Serological tests detect IgG, IgM or total antibody.
Serum is the usual sample tested – hence
“serology”
Other body fluids may be convenient/useful to test
e.g. saliva, CSF
Tests based on exactly the same principles as
antigen detection
serological diagnosis: acute infection
In many acute or primary infections
e.g. childhood viral exanthems, virus-specific IgM
appears within days of the clinical presentation
and persists for weeks or months.
eg measles, parvovirus
Acute infection can also be demonstrated by
seroconversion – with the appearance of specific
antibody (IgG or total antibody) in convalescent
samples. (4-fold rise in titre in older tests)
IgM not detectable during the initial non-specific febrile ilness but eg rubella IgM appears >7d post rash-appearance
control of viral infections
Public health
– Sanitation, drinking water, food supply, pest control
• Immunisation
– Smallpox polio measles mumps rubella,
– Influenza, hepatitis A, B
– Rabies, yellow fever, japanese encephalitis
– Varicella zoster virus
– Human papilloma virus
• Antiviral therapies
– Virucides – directly inactivate viruses but cytotoxic: detergent, organic solvents laser, uv, cry
– Directly acting antiviral agents
– Immunomodulators – IFN, Ig, Monoclonals, adoptive T-cell Rx
– Host modifiers
can latent viruses be eradicated?
NO
antivirals problems
antiviral drugs must inhibit virus replication but viral replication is dependent on HOST metabolic pathways, thus necessary to SPECIFICALLY inhibit virus encoded proteins with essential functions
therapeutic index of antivirals
at least 10, preferably 100-1000
treatment or drug failure?
treatment failure = non compliance, pharmacokinetic
drug failure = viral resistance
antiviral therapeutic strategies
INHIBI
INHIBIT virus replication at:
attachment
entry/uncoating
NUCLEIC ACID SYNTHESIS
Assembly/maturation
Release
targets of licensed antivirals for DNA VIRUSES
Viral DNA Replicative Enzymes
Nucleoside Analogues
Aciclovir, ganciclovir against Herpes virus DNA
polymerase
Nucleotide Analogue
Cidofovir against CMV & HHVs, adenovirus, HPV
Non-nucloside DNA polymerase inhibitor
Foscarnet (pyrophosphate analogue)
targets of licensed antivirals for RNA viruses
Viral Proteases
e.g. protease Inhibitors
Lopinavir against HIV protease
Viral surface proteins
e.g.Neuraminidase Inhibitors
Oseltamivir against Influenza neuraminidase
- *Multiple viral and cellular targets**
- Ribavirin*
Complex mechanisms including immunomodulation
Interferon in viral hepatitis
ACICLOVIR
for VZV, herpes simplex
ACV-PPP inhibits viral DNA polymerase
ACV is selectively activated by thymidine kinase > it adds Pi: ACV-P
ACV-P >> ACV-PPP (phosphates addedby cellular enzymes)
ACV actions outline
Nucleoside analogue
Active in triphosphate form
Requires viral thymidine kinase activity to addfirst PO4
2- group
Competitive substrate for viral DNA polymerase
Preferentially, irreversibly incorporated into growing DNA chain
Inhibits viral DNA polymerase
Obligatory chain terminator
selectivity of aciclovir
Preferential monophosphorylation catalysed by
viral thymidine kinase
Infected vs uninfected cell - 40-50 fold difference in phosphorylation
_Aciclovir tri-phosphate is a competitive inhibitor
of viral DNA polymerase_
Cellular DNA less susceptible to inhibition by
10 -30 fold
Effective chain termination due to the lack of 3’
OH group
issues with aciclovir
- Oral Bioavailability only 15 – 30% & Poor oral absorption
- Plasma Half-life 3 hour
- Frequent oral dosing required; 5 x per day
- 85% of drug excreted unchanged in urine
- Problem if renal impaired
- Nephrotoxicity due to precipitation in renal tubules
- CNS toxicity
aciclovis modification for absorption
added valine ester to aciclovir => VALACICLOVIR
1g tds of Valaciclovir comparable blood level as iv Aciclovir 5mg/kg tds
resistance to aciclovir
Common: Virus thymidine kinase absent
or
altered substrate specificity
Rarely: Virus DNA polymerase altered substrate specificity
GANCICLOVIR mode of action
Active form phosphorylated (GCV-P)
CMV UL97 gene product >> GCV-P
GCV-P >> GCV-PPP by cell
oral absorption only 5%
with added valine ir increases to 60%
Retroviruses treatments.
Reverse Transcriptase synthesises DNA from HIV RNA genome
RT inhibitors:
- *Nucleoside (NRTI)**
e. g. AZT (azidothymidine aka zidovudine) - *Non-nucleoside (NNRTI)**
e. g. nevirapine
Zidovudine/Azidothymidine (AZT)
AZT
↓ CELLULAR kinases
AZT-P
↓cellular kinases
AZT-PPP
↓viral RT
Viral DNA
↓
Chain termination
resistance to antiretrovirals
Error Rate of HIV Reverse Transcriptase:: 1 per 10,000 base copied
Viral genome size = 9,200 bp
Viral replication = 107 - 109 per day
Quasispecies = Swarm of related viruses around a predominant strain
Selective pressure exerted by the use of anti-retroviral
therapy
>> A new predominant strain with the best survival
selected
>> Emergence of Resistance
Removal of selective pressure - e.g. treatment interruption
>>Loss of selective advantage
>> Predominant strain revert to wild type
>> Resistance strain archived as latent
virus in infected PBMC.
Resistance test must be done while on
treatment
When selective pressure is re-applied, the
archived resistance strains will be rapidly reselected
