Antiviral Drugs

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Antiviral Drugs
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General Characteristics of Viruses

• Depending on one's viewpoint, viruses may be
  regarded as exceptionally complex aggregations of
  nonliving chemicals or as exceptionally simple
  living microbes.

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• Viruses contain a single type of nucleic acid
  (DNA or RNA) and a protein coat, sometimes
  enclosed by an envelope composed of lipids,
  proteins, and carbohydrates.

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• Viruses are obligatory intracellular parasites.
  They multiply by using the host cell's
  synthesizing machinery to cause the synthesis of
  specialized elements that can transfer the viral
  nucleic acid to other cells.

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Host Range

• Host range refers to the spectrum of host cells
  in which a virus can multiply. (narrow vs. broad)
• Most viruses infect only specific types of cells
  in one host species, so they do not generally
  cross species barriers.
• Host range is determined by the specific
  attachment site on the host cell's surface and
  the availability of host cellular factors.

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Viral Structure

• A virion is a complete, fully developed viral
  particle composed of nucleic acid surrounded by a
  coat.
• Helical viruses (for example, Ebola virus)
  resemble long rods and their capsids are hollow
  cylinders surrounding the nucleic acid.

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• Polyhedral viruses (for example, adenovirus) are
  many-sided. Usually the capsid is an icosahedron.

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• Enveloped viruses are covered by an envelope and
  are roughly spherical but highly pleomorphic (for
  example, Poxvirus). There are also enveloped
  helical viruses (for example, Influenzavirus) and
  enveloped polyhedral viruses (for example,
  Herpesvirus). Pleomorphic Many-formed. A tumor
  may be pleomorphic.

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• Complex viruses have complex structures. For
  example, many bacteriophages have a polyhedral
  capsid with a helical tail attached.
  Bacteriophage A virus that infects and lyses
  certain bacteria.

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Schematic of Influenza Virus
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Influenza Virus
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Nucleic Acid

• Viruses contain either DNA or RNA, never both,
  and the nucleic acid may be single- or
  double-stranded, linear or circular, or divided
  into several separate molecules.

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• The proportion of nucleic acid in relation to
  protein in viruses ranges from about 1 to about
  50.

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DNA viruses

• gene expression is much like that of the host
  cell
• DNA-dependent RNA polymerase synthesizes mRNA
• Host cell ribosomes and tRNAs used to translate
  viral mRNA
• Unique viral proteins include structural proteins
  and replication enzymes for viral DNA.
• Example-Herpesvirus, Epstein-Barr (mononucleosis)
  

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HBV Hepatitis B virus, a DNA virus
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RNA viruses

• Cells cannot make copies of RNA. Three kinds of
  strategies for RNA viruses

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Positive -strand RNA viruses

• the genome is also a mRNA
• The first task of the virus is to translate
  viral-specific proteins including RNA-dependent
  RNA polymerase (viral transciption/repliction
  enzyme) from viral RNA. The enzyme makes more
  mRNA and new RNA for viruses.

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Positive-stranded RNA genome is a molecule of
single-stranded "sense" RNA

• polioviruses
• rhinoviruses (frequent cause of the common
  "cold")
• coronaviruses (includes the agent of Severe Acute
  Respiratory Syndrome (SARS)
• rubella (causes "German" measles)
• yellow fever virus
• West Nile virus
• dengue fever viruses
• equine encephalitis viruses
• hepatitis A ("infectious hepatitis") and
  hepatitis C viruses
• tobacco mosaic virus (TMV)

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Hepatitis C

• Link
• Hepatitis C attacks the liver
• Hepatitis C is transmitted by direct blood to
  blood contact
• Injection drug use
• Blood transfusion
• Occupational exposure to blood

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Negative-strand RNA viruses

• the genome is the complement of mRNA
• First task of the virus is to make mRNA.
  Therefore, the virus imports RNA polymerase or
  transcriptase as a part of the virus structure.

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Negative-stranded RNA viruses genome consists of
one or more molecules of single-stranded
"antisense" RNA

• Examples
• measles
• mumps
• respiratory syncytial virus (RSV), parainfluenza
  viruses (PIV), and human metapneumovirus. (In the
  U.S., these close relatives account for hundreds
  of thousands of hospital visits each year, mostly
  by children.)
• rabies
• Ebola
• influenza

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Retroviruses

• Virus has the enzyme reverse transcriptase as a
  part of the viral structure.
• A double-stranded DNA copy of the viral genome is
  produced.
• This copy can integrate into the host cell
  chromosome.
• Some retroviruses can cause tumors in animals
  oncogenes
• Human immunodeficiency virus (HIV) is a
  retrovirus. This is the causative agent of AIDS.

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Virus Genome Polarity Segments Morphology Enveloped Diseases
Picorna RNA ss 1 Icosahedral No Polio, Hepatitis A, Colds
Toga RNA ss 1 Icosahedral Yes Encephalitis, Rubella
Retro RNA ss 11 Icosahedral Yes AIDS
Orthomyxo RNA -ss 6-8 Helical Yes Influenza
Rhabdo RNA -ss 1 Helical Yes Rabies
Paramyxo RNA -ss 1 Helical Yes Parainfluenza, Mumps, Measles
Papova DNA ds 1 Icosahedral No Warts
Adeno DNA ds 1 Icosahedral No Respiratory Infections
Herpes DNA ds 1 Icosahedral Yes HS, VZ, Mononucleosis, Cancer
Pox DNA ds 1 Complex Yes Smallpox
Hepatitis B DNA ds 1 Icosahedral Yes Serum Hepatitis
             
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Capsid and Envelope

• The protein coat surrounding the nucleic acid of
  a virus is called the capsid.
• The capsid is composed of subunits, capsomeres,
  which can be a single type of protein or several
  types.

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• The capsid of some viruses is enclosed by an
  envelope consisting of lipids, proteins, and
  carbohydrates.
• Some envelopes are covered with
  carbohydrate-protein complexes called spikes.

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Viruses and Cancer

• The earliest relationship between cancer and
  viruses was demonstrated in the early 1900s, when
  chicken leukemia and chicken sarcoma were
  transferred to healthy animals by cell-free
  filtrates.
• Transformation of Normal Cells into Tumor Cells
• When activated, oncogenes transform normal cells
  into cancerous cells.
• Viruses capable of producing tumors are called
  oncogenic viruses.
• Several DNA viruses and retroviruses are
  oncogenic.
• The genetic material of oncogenic viruses becomes
  integrated into the host cell's DNA.
• Transformed cells lose contact inhibition,
  contain virus-specific antigens (TSTA and T
  antigen), exhibit chromosomal abnormalities, and
  can produce tumors when injected into susceptible
  animals.

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Causes of the Common Cold

• More than 200 different viruses are known to
  cause the symptoms of the common cold. Some, such
  as the rhinoviruses, seldom produce serious
  illnesses.
• Others, such as parainfluenza and respiratory
  syncytial virus, produce mild infections in
  adults but can precipitate severe lower
  respiratory infections in young children.

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• Rhinoviruses (from the Greek rhin, meaning
  "nose") cause an estimated 30 to 35 percent of
  all adult colds, and are most active in early
  fall, spring, and summer. More than 110 distinct
  rhinovirus types have been identified. These
  agents grow best at temperatures of about 91
  degrees Fahrenheit, the temperature inside the
  human nose.

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• Scientists think coronaviruses cause a large
  percentage of all adult colds. They bring on
  colds primarily in the winter and early spring.
  Of the more than 30 kinds, three or four infect
  humans. The importance of coronaviruses as a
  cause of colds is hard to assess because, unlike
  rhinoviruses, they are difficult to grow in the
  laboratory.

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• Approximately 10 to 15 percent of adult colds are
  caused by viruses also responsible for other,
  more severe illnesses adenoviruses,
  coxsackieviruses, echoviruses, orthomyxoviruses
  (including influenza A and B viruses, which cause
  flu), paramyxoviruses (including several
  parainfluenza viruses), respiratory syncytial
  virus, and enteroviruses.
• The causes of 30 to 50 percent of adult colds,
  presumed to be viral, remain unidentified. The
  same viruses that produce colds in adults appear
  to cause colds in children. The relative
  importance of various viruses in pediatric colds,
  however, is unclear because it's difficult to
  isolate the precise cause of symptoms in studies
  of children with colds.

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http//www.commoncold.org/undrstnd.htm
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Influenza

• Influenza is a disease caused by a member of the
  Orthomyxoviridae. Many features are common with
  those of the paramyxovirus infections of the
  respiratory tract.

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CLINICAL FEATURES

• Influenza is characterized by fever, myalgia,
  headache and pharyngitis. In addition there may
  be cough and in severe cases, prostration. There
  is usually not coryza (runny nose) which
  characterizes common cold infections. Infection
  may be very mild, even asymptomatic, moderate or
  very severe.

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• Source The reservoir is acute infection in other
  human beings.
• Spread Is rapid via aerial droplets and fomites
  with inhalation into the pharynx or lower
  respiratory tract.
• Incubation Is short 1-3 days. Rapid spread leads
  to epidemics

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Complications

• Tend to occur in the young, elderly, and persons
  with chronic cardio-pulmonary diseases
• Consist of?
• 1. Pneumonia caused by influenza itself
  Pneumonia an inflammatory condition of the lungs
  in which they become obstructed with fluid,
  causing difficult breathing and possibly
  suffocation. Pneumonia may be caused by bacteria,
  viruses, fungi, or chemical agents.
• 2. Pneumonia caused by bacteria- Haemophilus
  influenzae- Staphylococcus aureus- Streptococcus
  pneuminiae
• 3. Other viral superinfection, eg.
  Adenovirus.Overall death rates increase in times
  of influenza epidemics.

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The virion is generally rounded but may be long
and filamentous.A single-stranded RNA genome is
closely associated with a helical nucleoprotein
(NP), and is present in eight separate segments
of ribonucleoprotein (RNP), each of which has to
be present for successful replication. The
segmented genome is enclosed within an outer
lipoprotein envelope. An antigenic protein called
the matrix protein (MP 1) lines the inside of the
envelope and and is chemically bound to the RNP.
The envelope carries two types of protruding
spikes. One is a box-shaped protein, called the
neuraminidase (NA) (pink rectangles on the
surface), of which there are nine major antigenic
types, and which has enzymic properties as the
name implies.
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The other type of envelope spike is a trimeric
protein called the haemagglutinin (HA)
(illustrated on the left)of which there are 13
major antigenic types. The haemagglutinin
functions during attachment of the virus particle
to the cell membrane, and can combine with
specific receptors on a variety of cells
including red blood cells.The lipoprotein
envelope makes the virion rather labile -
susceptible to heat, drying, detergents and
solvents. Haemagglutinin A substance, such as
an antibody, that causes agglutination of red
blood cells. Agglutination The clumping together
of red blood cells or bacteria.
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The Life Cycle of Influenza Virus
Receptor-bound viruses are taken into the cell by
endocytosis. In the low pH environment of the
endosome, RNP is released from MP1, and the viral
lipoprotein envelope fuses with the lipid-bilayer
of the vesicle, releasing viral RNP into the cell
cytoplasm, from where it is transported into the
nucleus. New viral proteins are translated from
transcribed messenger RNA (mRNA). New viral RNA
is encased in the capsid protein, and together
with new matrix protein is then transported to
sites at the cell surface where envelope
haemagglutinin and neuraminadase components have
been incorporated into the cell membrane. Progeny
virions are formed and released by budding. The
cell does not die (at least not initially).
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Influenza Overview
Link Link
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Flu is one of a rare few viruses that has its
genome in separate segments (eight). - This
increases the potential for recombinants to form
(by interchange of gene segments if two different
viruses infect the same cell), and may contribute
to the rapid development of new flu strains in
nature - can also be duplicated in the laboratory
(used for making vaccine strains). Avian and
human strains recombining in pigs in the Far East
may permit virulent human strains to evolve.
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CLASSIFICATION of virus STRAINSIs done on the
basis of antigenicity of NP (nucleoprotein) and
MP (matrix protein) into three main groups
Influenza A -HA undergoes minor and occasional
major changes.- NA some variation.Influenza B)
Undergoes relatively slow change in HA with time.
Known only in man.Influenza C) Uncommon strain,
known only in man.
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Nomenclature of Viruses
A Singapore 6 86 (H1N1)

Type of Influenza Town where first isolated Number of isolates Year of isolation Major Type of HA and NA
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Influenza A virus is essentially an avian virus
that has "recently" crossed into mammals. Birds
have the greatest number and range of influenza
strains. Avian haemagglutinins sometimes appear
in pig human and horse influenza strains.
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• Every now and then (10 - 15 years) a major new
  pandemic strain appears in man, with a totally
  new HA and sometimes a new NA as well (antigenic
  shift). This variant causes a major epidemic
  around the world (pandemic).
• Over the subsequent years this strain undergoes
  minor changes (antigenic drift) every two to
  three years, probably driven by selective
  antibody pressure in the populations of humans
  infected.

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Camp Devens is near Boston, and has about 50,000
men, or did have before this epidemic broke
loose. It also has the Base Hospital for the Div.
of the N. East. This epidemic started about four
weeks ago, and has developed so rapidly that the
camp is demoralized and all ordinary work is held
up till it has passed. All assembleges of
soldiers taboo.These men start with what appears
to be an ordinary attack of LaGrippe or
Influenza, and when brought to the Hosp. they
very rapidly develop the most viscous type of
Pneumonia that has ever been seen. Two hours
after admission they have the Mahogany spots over
the cheek bones, and a few hours later you can
begin to see the Cyanosis extending from their
ears and spreading all over the face, until it is
hard to distinguish the coloured men from the
white. It is only a matter of a few hours then
until death comes, and it is simply a struggle
for air until they suffocate. It is horrible. One
can stand it to see one, two or twenty men die,
but to see these poor devils dropping like flies
sort of gets on your nerves. We have been
averaging about 100 deaths per day, and still
keeping it up. There is no doubt in my mind that
there is a new mixed infection here, but what I
dont know.
Copy of original letter found in Detroit in 1959
Camp Devens, Mass.Surgical Ward No 1629
September 1918(Base Hospital)
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• When viruses infect the body, the frequently
  result in the production of large amounts of
  cytokines
• Cytokines signal immune cells, like T cells and
  macrophages to respond to the infection, but can
  also result in the production of still more
  cytokines.
• If this immune system loop becomes uncontrolled,
  it can lead to a cytokine storm, resulting in
  extremely large amounts of inflammation and fluid
  production.

http//www.cytokinestorm.com/cytokine_storm.html
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This constant antigenic change down the years
means that new vaccines have to be made on a
regular basis.
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Anti-Viral Medications
The relative simplicity of the virus machinery,
coupled with the rapid mutation of strains, makes
it challenging to design effective anti-viral
medications
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Anti-influenza Agents Amantadine  Oseltamivir 
Peramivir  Rimantadine  Zanamivir Anti-herpesvi
rus agents   Aciclovir  Cidofovir  Docosanol 
Famciclovir  Foscarnet  Fomivirsen 
Ganciclovir  Idoxuridine  Penciclovir 
Trifluridine  Tromantadine  Valaciclovir 
Valganciclovir  Vidarabine Antiretroviral
Agents  NRTIsZidovudine  Didanosine 
Stavudine  Zalcitabine  Lamivudine  Abacavir 
Tenofovir NNTIs Nevirapine  Efavirenz 
Delavirdine PIsSaquinavir  Indinavir 
Atazanavir  Ritonavir  Nelfinavir 
Amprenavir  Lopinavir  Tipranavir Other
antiviral agents Fomivirsen  Enfuvirtide 
Imiquimod  Interferon  Ribavirin  Viramidine
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The final stage in the life cycle of a virus is
the release of completed viruses from the host
cell, and this step has also been targeted by
antiviral drug developers. Two drugs named
zanamivir and oseltamivir that have been recently
introduced to treat influenza prevent the release
of viral particles by blocking a molecule named
neuraminidase that is found on the surface of flu
viruses, and also seems to be constant across a
wide range of flu strains.
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Both these drugs are effective against the known
strains of H5N1 in mouse models. Tamiflu has been
disappointing in recent real world use in human
H5N1 infection due to 1. delays in treatment
and 2. the emergence of resistance. Relenza
has not yet been tried in human H5N1 infection.
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• Most attention has been given to oseltamivir
  (Tamiflu) because it is a tablet, which is easy
  to administer.
• Zanamavir (relenza) is administered as a dry
  powder inhaler much like some asthma inhalers. An
  intravenous version of Relenza has been
  administered to volunteers under study conditions
  but it is not yet approved or in production.
• Both drugs can be used to treat influenza they
  are also both approved for the prevention of
  influenza.
• These drugs are also effective against all
  strains of influenza A, unlike vaccines which are
  specific only to the strain for which they were
  designed.
• Both medications are well tolerated with few
  side effects, although there is concern over the
  possibility of psychological effects of Tamiflu
  and there may be occasional problems with
  asthmatics who use Relenza.

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How do these drugs work?
Current anti-influenza drugs target an enzyme
used in release of the virus from the cell.
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What is Sialic Acid?
Sialic acid-rich oligosaccharides on the
glycoconjugates found on surface membranes help
keep water at the surface of cells. The sialic
acid-rich regions contribute to creating a
negative charge on the cells surface. Since water
is a polar molecule, it has a partial positive
charge on both hydrogen molecules, it is
attracted to cell surfaces and membranes. This
also contribues to cellular fluid uptake.
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It seems that the Hemagluttinin subtypes in avian
influenza strains bind preferentially to a2,3
linked sialic acid residues, while the human
influenza isolates prefer a2,6 linked sialic acid
residues.
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Sialic acid binding site of human influenza A
hemagglutinin subtype H3. Sialic acid bound in
the pocket appears in gray. The listing below
shows the binding affinities for sialic acid when
particular amino acids are changed experimentally
by site-directed mutagenesis (Martín et al.
1998). The number on the left defines the amino
acid site in HA1, x ? y shows the original and
new amino acid, and the number on the right is
the binding affinity of the mutant as a
percentage of the affinity of the wild type.
Dashes show cases in which the receptor site is
not properly expressed.
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What is neuraminidase?

• Neuraminidase has functions that aid in the
  efficiency of virus release from cells.
• Neuraminidase cleaves terminal sialic acid
  residues from carbohydrate moieties on the
  surfaces of infected cells.
• This promotes the release of progeny viruses from
  infected cells.
• Neuraminidase also cleaves sialic acid residues
  from viral proteins, preventing aggregation of
  viruses.
• Inhibiting neuraminidase slows the release of the
  virus from the host cell and commercial drugs
  target this enzyme

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Currently utilized inhibitors of neuraminidase
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How were these drugs designed?
Question Knowing the reactant and the product
of an enzyme-catalyzed Reaction, how do you
design an inhibitor to bind more tightly than
either one? Recall An enzyme speeds a reaction
by lowering the energy of The transition
state. Therefore the overall DG at the
transition state must be greater (I.e. more
negative) than at either starting material or
product. Therefore If you design an inhibitor
to resemble the transition state, It should be
very tightly bound at the active site.
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Lets look at the reaction mechanistically
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So it is logical that the inhibitors were
designed to be sp2 hybridized flat at the
carbon adjacent to the CO2H group
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Assigned Reading
Wang, Taia T. Palese, Peter. Unraveling the
mystery of swine influenza virus. Cell
(Cambridge, MA, United States) (2009), 137(6),
983-985. Salomon, Rachelle Webster, Robert G.
The influenza virus enigma. Cell (Cambridge,
MA, United States) (2009), 136(3),
402-410 Tscherne, D. M. Garcia-Sastre, A.
Virulence determinants of pandemic influenza
viruses. The Journal of Clinical Investigation
(2011), 121 (1) 6-13.
Optional Reading
Nelson, Martha I. Holmes, Edward C. The
evolution of epidemic influenza. Nature
Reviews Genetics (2007), 8(3),
196-205. Lehmann, F. Tiralongo, E. Tiralongo,
J. Sialic acid-specific lectins occurrence,
specificity and function. Cellular and
Molecular Life Sciences (2006), 63(12),
1331-1354.
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Homework Questions

1. Describe three markers of viral virulence
   (pathogenicity).
2. Summarize the characteristics of both the 2009
   H1N1 virus and the avian H5N1 virus with respect
   to these markers of pathogenicity.
3. What species is believed to be the primary
   reservoir for the influenza virus?
4. The haemaglutannin surface glycoprotein of human
   and avian influenza virus isolates seem to have
   slightly different binding affinities for
   structurally different forms of sialic acid
   linked to galactose. Explain.
5. Since resistance to current antiviral arises
   rapidly, it is important to find new therapeutic
   targets. List a few such targets.