Antiviral drugs face several challenges due to viruses replicating inside host cells. Acyclovir is a nucleoside analog effective against herpes viruses that requires activation by viral kinases. Foscarnet directly inhibits viral DNA polymerase. Amantadine and rimantadine target influenza A M2 protein while oseltamivir and zanamivir inhibit neuraminidase. Lamivudine and entecavir are used to treat hepatitis B as nucleoside analogs. Interferons and ribavirin are used against hepatitis C. Phenotypic and genotypic tests determine antiviral susceptibility.

1. ANTIVIRAL AGENTS
DR A CHAUDHURY

2. Antiviral Drugs-Problems
• Need knowledge of replication at molecular level
to define targets
 Viruses as intracellular parasites make targeting more
difficult to avoid host toxicity.
• Lack of culture systems for some agents hinders
development.
• Resistance due to rapid mutation of many viruses
• Intracellular – can’t use non-permeable drugs
• Symptoms usually occur at height of viral replication
• Latency or non-replicating phases hard to target
• Limited capability for rational drug development; Labour
intensive and expensive.

3. History
• Early works focused on the use of the
current bacterial antibiotics, including
sulphonamides. The futility of these early
attempts led to the dogma that viruses are
not susceptible to ‘antibiotics’.
• Virologists were taught that selective
toxicity for these obligate intracellular
parasites was unattainable

4. History
• In 1957 came the famous first description
by Isaacs and Lindenmann of interferon.
However, the discovery of the interferons
in the late 1950s was something of a false
dawn.
• Idoxuridine: the first useful antiviral .The
description of 5-iodo-2′-deoxyuridine by Dr
Bill Prusoff in 1959 and the realization of
its antiviral properties.

5. History
• The first publications on this and similar
nucleoside analogues appeared in cancer
journals.
• The development of IDU from laboratory
inhibitor to useful antiviral drug was driven
by several notable pioneers, especially the
ophthalmologist Dr Herbert Kaufman who
proved its clinical value in 1962 for treating
viral keratitis.

6. History
• Antiviral activity of adenine arabinoside
(vidarabine, ara-A) by M. Privat de Garilhe
and J. De Rudder also dates from the year 1964.
• By providing treatment early in the disease, it
was possible to curtail herpes zoster in the
immunosuppressed and reverse the potentially
lethal progression of herpes encephalitis and the
overwhelming herpes infections that
occasionally occur in the newborn.

7. History
• β-thiosemicarbazone was first reported to be aninhibitor
of a related poxvirus, vaccinia virus,in 1951 by D.
Hamre, K.A. Brownlee and R. Donovick.
• Dr John Bauer at the then Wellcome Foundation
Laboratories in Beckenham, UK, led the team which
developed the drug marboran, a β-thiosemicarbazone
derivative.
• Marboran was shown in several trials to have some
clinical efficacy both for the treatment of smallpox and
the complications of vaccinia following vaccination

8. Many well-known antiviral
compounds are
nucleoside and nucleotide
analogs:
• Acyclovir is a nucleoside
analog similar to
guanosine, but contains
an acyclic sugar group
• AZT is a nucleoside
analog similar to
thymidine, but contains an
azide group

9. Antivirals other than anti-HIV
agents
A. Herpesviruses
B. Respiratory viruses
C. Hepatitis viruses

10. A. Agents for Herpesvirus: HSV,
CMV, VZV.
• Acyclovir : HSV, VZV
• Valacyclovir: HSV, VZV
• Famciclovir: HSV ,VZV
• Ganciclovir: HSV, CMV, VZV
• Valganciclovir; CMV
• Foscarnet : , HSV,CMV
• Cidofovir : HSV, CMV
• Trifluridine: Topical eye HSV Keratitis
• Idoxuridine: HSV Keratitis

11. ACYCLOVIR
• Close to a perfect antiviral drug (specific, nontoxic).
• Highly effective against herpes simplex virus (HSV),
less so against varicella-zoster virus (VZV).
• Highly selective and extremely safe.
• Acyclic guanine derivative (nucleoside analogue )
that inhibits viral DNA synthesis.
• It is a pro-drug, a precursor of the antiviral
compound.
• Activation of the drug requires three kinase activities
to convert acyclovir to a triphosphate derivative, the
actual antiviral drug.

12. Acyclovir: MOA
– Preferentially taken up by virally infected cells
– Monophosphorylated by virally encoded thymidine
kinases
– Di- and triphosphorylation completed by cellular
kinases
– ACV-TP is the active moiety
• Competitive inhibitor of viral DNA polymerase
– Cellular DNA polymerases much less susceptible to
inhibition
• Leads to viral DNA chain termination

13. Acyclovir-MOA
• Inhibition of HSV DNA Polymerase is a
three step process:
1. ACV-TP competitively inhibits dGTP
incorporation.
2. Next ACV-TP acts as a substrate and is
incorporated in the growing DNA chain
3. Polymerase translocates to the next position on
the template, but cannot add a new dNTP
because there is no 3’-hydroxyl on ACV-TP→
Dead end complex→Enzyme inactivation.

15. Acyclovir :Resistance
– Mediated by mutations in viral thymidine
kinase and/or viral DNA polymerase
genes
• TK-deficient and TK partial virus can be
produced. These viruses fail to phosphorylate
acyclovir.
• Acyclovir resistance mutations can also alter pol
to be less inhibited by the drug.
– Clinically significant infections can be caused by
drug resistant HSV and VZV.

16. CIDOFOVIR
• It is converted to the diphosphate form by
cellular kinases. Initial
monophosphorylation by viral TK or other
kinases not needed.
• Acts as an alternate substrate of dCTP
and an inhibitor of DNA polymerase.

17. FOSCARNET
• Foscarnet (trisodium phosphonoformate)
blocks, through non-competitive inhibition,
of the pyrophosphate binding site of viral
DNA polymerase, thereby preventing the
cleavage of pyrophosphate from
deoxynucleoside triphosphate and
elongation of the viral DNA chain.
• Foscarnet does not require viral/cellular
thymidine kinase for activation

18. Foscarnet
• Foscarnet selectively inhibits viral
polymerase; inhibition of mammalian DNA
polymerase requires a 100-fold greater
concentration of foscarnet than that
required to block viral replication.

19. Foscarnet
• Since foscarnet, unlike acyclovir and
ganciclovir, does not require intracellular
phosphorylation for antiviral activity,
thymidine kinase mutations in HSV or VZV
and UL97 phosphotransferase mutations
in CMV do not confer resistance.
• Foscarnet resistance in CMV has been
associated with mutations in the viral DNA
polymerase gene in patients receiving prolonged
therapy for AIDS-associated CMV retinitis.

20. CLINICAL USES
ACYCLOVIR:
 Intravenous therapy for HSV encephalitis,
neonatal herpes, severe VZV infection
 Prophylaxis for HSV and VZV in
transplant patients.
 Oral agent for Primary or recurrent HSV 1
and 2, and uncomplicated VZV infections.

21. Clinical Uses
• Ganciclovir is the first line agent against
CMV infections in immunocompromised.
• Clinically, foscarnet is employed almost
exclusively to treat infections with CMV,
particularly when ganciclovir resistant, and
acyclovir-resistant HSV and VZV.
• Cidofovir is also used for CMV infections
(administration every 1 or 2 weeks).

22. B. Agents for Respiratory Virus:
Influenza Virus
• Amantadine
• Rimantadine
• Zanamivir
• Oseltamivir
• Ribavirin

23. Amantadine and Rimantadine
• Tricyclic amines
• Active against influenza A only at clinically achievable
concentrations
• Mechanism of action:
• Viral M2 protein acts as an ion channel which
normally facilitate the hydrogen ion mediated
dissociation of the matrix protein from the nucleocapsid.
– Interference with function of viral M2 protein
This prevents viral uncoating and release of viral RNA into
the cytoplasm and transport of ribonucleoprotein
complex into the nucleus.
• Resistance mediated by mutations in M2 coding region:
single AA substitution within the M2 transmembrane
domain.

24. Amantadine and Rimantadine
• Useful for treatment and prophylaxis of influenza
A infections
– Should not be used when amantadine
resistant strains are in circulation
Cross resistance.
• Can reduce severity & duration of illness if
started within 48 hrs of onset of symptoms

25. Zanamivir, Oseltamivir
• Mechanism of action
Viral neuraminidase catalyzes cleavage of
terminal sialic acid residues attached to
glycoproteins and glycolipids, a process
necessary for release of virus from host
cell surfaces
Neuraminidase inhibitors thus prevent
release of virions from infected cell

26. NA Inhibitors
• These agents are Sialic acid analogues,
and they block the active sites of the
enzyme NA. So sialic acid cannot be
cleaved and progeny viruses are not
released from infected cells.
• NA mutation results in an enzyme which
is less inhibited by the drug.
• No cross rersistance.

27. Oseltamivir, Zanamivir
• Indications
– Treament of influenza A and B within 24-48
hrs of symptom onset
– Prophylaxis
– N.B.: Neither drug interferes with antibody
response to influenza vaccination
More than 300 cases of oseltamivir-resistant
(H1N1) carrying the H275Y mutation have been
detected, up to October 2010.

29. C. Hepatitis Virus:
Hepatitis B Virus
• Interferon-alpha (pegylated)
• Lamivudine :
– Nucleoside analog first developed for HIV
– Lower dose used for HBV (100 mg/day)
• Adefovir dipivoxil
– Nucleotide analog first developed for HIV but nephrotoxic at higher
doses
– Approved for HBV at lower dose (10 mg/day)
• Entecavir
– Nucleoside analog with activity originally thought limited to HBV
but has anti-HIV-1 activity.
– Approved for use at dose of 0.5-1.0 mg/day
• Telbivudine
– Nucleoside (thymidine) analog with activity against HBV but not HIV
– Recently approved at a dose of 600 mg/day

30. Lemivudine,Telbivudine
• Dideoxy analogue of cytidine.
• Phosphorylated intracellularly to the
triphosphate form.
• Competitive inhibitor of dCTP
• Incorporation of the triphosphate form into
viral DNA by HBV DNA Polymerase
results in chain termination.
• Resistance: Polymerase substitution.

31. Hepatitis Virus
Hepatitis C
• Approved
– Interferon-alpha (pegylated)
– Ribavirine
• In development
– Protease inhibitors
– Polymerase inhibitors

32. RIBAVIRINE
• Synthetic nucleoside analog
• Active against broad range of RNA and DNA viruses
– Flavi-, paramyxo-, bunya-, arena-, retro-, herpes-,
adeno-, and poxviruses
• Mechanism of action complex
– Triphosphorylated by host cell enzymes
• For influenza
– Ribavirin-TP interferes with capping and elongation of
mRNA and may inhibit viral RNA polymerase
• For other agents
– Ribavirin-MP inhibits inosine-5’-monophosphate
dehydrogenase depleting intracellular nucleotide pools,
particularly GTP.

33. Ribavirine
• Indications
– Aerosol treatment of RSV in children
Effectiveness debated
– Oral treatment of HCV (in combination
with pegylated IFN alpha).

34. Interferons
• Types
– Alpha/Beta (leukocyte/fibroblast)
• Coding genes located on chromosome 9
• At least 24 subtypes of alpha, 1 of beta
– Gamma
• Coding gene located on chromosome 12
• 1 subtype

35. Interferons: MOA
• Act by inducing an antiviral state within cells
• Bind to specific receptors on cell surface
• Receptor associated tyrosine kinases activated
– Tyk2 and JAK 1 for alpha and beta
– JAK1 and JAK2 for gamma
• Induction of a phosphodiesterase with inhibition of
peptide chain elongation
• Synthesis of MxA protein which can bind to cytoskeletal
proteins and inhibit viral transcriptases
• Induction of nitric oxide by gamma IFN in macrophages
• Viral penetration, uncoating, viral mRNA transcription,
viral protein synthesis, replication of viral genome,
assembly and release of progeny virus are inhibited.

37. Interferons: Use
• Antiviral indications
– IFN-alpha 2b (pegylated) for HCV (in
combination with ribavirin)
– Intralesional for condyloma acuminata.

38. Antiviral Drug Susceptibility
Tests.
• For optimal patient management.
• Used mainly for HSV, VZV, CMV,
Influenza virus, and HIV.
• Phenotypic , Genotypic.
Uses: 1. Defining mechanism of resistance
2. Determine the frequency with which drug resistant
viral mutants emerge.
3. To test for cross resistance
4. Evaluation of a new agent.

39. Phenotypic Test
• Directly measure the effect of
antiviral drug in viral growth.
• Can be measured by
infectivity, viral antigen
production, or viral nucleic acid
production.
• Labour intensive, slow.
• Difficult to standardize
• Result is method dependent.
• Herpes group, Influenza, HIV-
1.
Genotypic Tests
• Test for genetic
basis(mutation) for resistant
phenotype.
• RFLP, differential
hybridization assays, or
sequencing.
• Useful only if the genetic basis
or resistance is known.
• Most useful if there are only
limited number of genetic
changes.
• CMV, HIV-1

40. Variables Affecting the Testing
No existing standards regarding
• Cell line
• Viral inoculum titre
• Incubation time
• Concentration range for antivirals
• Reference strains
• Assay method
• End point criteria
• Calculation and interpretation of end point

41. PHENOTYPIC TESTS
PLAQUE REDUCTION ASSAY:
 Considered the “gold standard”
 CLSI has developed a standard for PRA
testing of HSV.
 Inhibition of viral plaque formation in the
presence of antiviral agent.
 The concentration of the agent which
inhibits plaque formation by 50% is IC50.

42. PRA
• Cell lines are grown on wells or plates.
• A standardized viral stock is inoculated
into multiple identical cell cultures.
• The viral titre of the stock is previously
determined and adjusted to yield
countable number of plaques in each well
( 50-100PFU/60 mm wide tissue culture
plates)

43. PRA
• A solidifying agent (agarose) is added to
the cultures to minimize spread of virus
through media.
• Increasing concentration of the test drug
is incorporated into a series of wells.
• IC50 : Lowest concn. of drug that results in
a 50% decrease in the number of plaques
compared with the control well with no
antiviral drug.

45. Dye Uptake Assay
• Used mainly for HSV
• The vital dye Neutral red is taken up by
viable cells , but not by nonviable cells.
• The extent of viral lytic activity is
measured by the relative amount of the
dye bound to viable cells after infection
with HSV compared with the amount
bound to uninfected cells.

46. DU Test
• The dye bound by viable cells is eluted by
ethanol and measured in a colorimeter.
• Drug concentration which inhibits viral
lytic activity by 50% is the IC50.
• It gives IC50 of acyclovir 3-5 times greater
than that given by PRA
− Higher inoculum ( 500PFU/ml) used
− Use of a liquid overlay which allows drug resistant viruses to
‘amplify’ resulting in more sensitive detection of small amounts of
drug resistant viruses.

47. DU Test
Advantages:
 Semi-automated
 Efficient testing of large number of isolates
 Ability to detect small number of resistant virus
Disadvantages:
 Relatively high cost of automated equipment
 Overseeding of cells into culture wells
 Precipitation of neutral red onto the monolayer.

48. OTHER PHENOTYPIC TESTS
• EIA: Concentration of antiviral agent
which reduces the absorbance to 50% of
the control.
• Flow Cytometry
• NI Assay: Assay for Neuraminidase
inhibitors of influenza virus.
• Yield Reduction Assay: Ability of the
agent to inhibit the production of
infectious virus

49. GENOTYPIC TESTS
• It has been used to screen CMV isolates
for mutation associated with ganciclovir
resistance.
• UL 97(Phosphotransferase) and UL54
( DNA Polymerase) mutations can be
detected.
• PCR amplification of short fragment of
UL97 followed by restriction endonuclease
digestion to detect mutations.

50. Genotypic Tests
• Can detect 78% of Ganciclovir resistant
isolates.
• Can recognize mutant virus when present
at 10% of the total virus population.
• Every mutation is not a cause of
resistance. MARKER TRANSFER
EXPERIMENT must be performed to
definitely define that a particular mutation
is the cause of resistance.

51. Genotypic Tests
• Marker Transfer Resistance: PCR
amplified UL 97 and UL54 fragments
containing the resistance mutation are co-
transfected with CMV strain AD169 (Drug
Susceptible). The resulting recombinant
plaques are assayed for antiviral
susceptibility by PRA.

52. Genotypic Tests
Influenza Antiviral Susceptibility: H275Y
mutation causes oseltamivir resistance in
H1N1 viruses.
1. Searching for specific mutations by real-
time PCR or pyrosequencing technology.
2. Full length neuraminidase gene
sequencing .