viral diagnosis and treatment

Question 1

NAT

Answer 1

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.

Question 2

PCR modifications

Answer 2

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

Question 3

real time PCR

Answer 3

 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

Question 4

Indirect detection of infection:
Serological diagnosis

Answer 4

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

Question 5

serological diagnosis: acute infection

Answer 5

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

Question 6

control of viral infections

Answer 6

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

Question 7

can latent viruses be eradicated?

Answer 7

NO

Question 8

antivirals problems

Answer 8

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

Question 9

therapeutic index of antivirals

Answer 9

at least 10, preferably 100-1000

Question 10

treatment or drug failure?

Answer 10

treatment failure = non compliance, pharmacokinetic

drug failure = viral resistance

Question 11

antiviral therapeutic strategies

INHIBI

Answer 11

INHIBIT virus replication at:

attachment

entry/uncoating

NUCLEIC ACID SYNTHESIS

Assembly/maturation

Release

Question 12

targets of licensed antivirals for DNA VIRUSES

Answer 12

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)

Question 13

targets of licensed antivirals for RNA viruses

Answer 13

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

Question 14

ACICLOVIR

Answer 14

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)

Question 15

ACV actions outline

Answer 15

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

Question 16

selectivity of aciclovir

Answer 16

 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

Question 17

issues with aciclovir

Answer 17

1. Oral Bioavailability only 15 – 30% & Poor oral absorption
2.  Plasma Half-life 3 hour
3.  Frequent oral dosing required; 5 x per day
4.  85% of drug excreted unchanged in urine
5.  Problem if renal impaired
6.  Nephrotoxicity due to precipitation in renal tubules
7.  CNS toxicity

Question 18

aciclovis modification for absorption

Answer 18

added valine ester to aciclovir => VALACICLOVIR

1g tds of Valaciclovir comparable blood level as iv Aciclovir 5mg/kg tds

Question 19

resistance to aciclovir

Answer 19

Common: Virus thymidine kinase absent
or
altered substrate specificity

Rarely: Virus DNA polymerase altered substrate specificity

Question 20

GANCICLOVIR mode of action

Answer 20

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%

Question 21

Retroviruses treatments.

Answer 21

Reverse Transcriptase synthesises DNA from HIV RNA genome

RT inhibitors:

• *Nucleoside (NRTI)**
  e. g. AZT (azidothymidine aka zidovudine)
• *Non-nucleoside (NNRTI)**
  e. g. nevirapine

Question 22

Zidovudine/Azidothymidine (AZT)

Answer 22

AZT

                               ↓ CELLULAR kinases

AZT-P

                          ↓cellular kinases

AZT-PPP

         ↓viral RT

Viral DNA

↓

Chain termination

Question 23

resistance to antiretrovirals

Answer 23

 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