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MICR 420
Emerging and Re-Emerging
Infectious Diseases
Lecture 8: Influenza Viruses
Dr. Nancy McQueen & Dr. Edith Porter
Overview
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RNA viruses
Influenza viruses
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Brief history
Nomenclature
Morphology and nature of the genome
Viral replication cycle
Genetic variability
Pathogenesis and clinical symptoms
Diagnosis
Treatment
Prevention
Threat
What a virus?
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Not a living cell
Acellular particle
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No cell membrane
Consist of nucleic acid and protein only
 RNA or DNA, not both
 Protein coat : capsid
 Some are enveloped: membrane from host cell
Infects living host cells to replicate
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All forms of cells can be infected by a virus
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Bacteria, archaea, eukaryotes
Virus depends on host metabolism
Viral genome subverts host cell’s machinery to
reproduce
Estimate of 1032 viruses on earth
Transcription and replication
of RNA viruses
RNA viruses
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Why are so many of the newly emerging
infectious diseases caused by RNA viruses?
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All must bring in their own RNA dependent RNA
polymerases (replicases)
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Are error-prone - error rate of 10-3- 10-5
Have no proof reading function
Many quasi-species found in viral infections
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Nitric oxide (NO) production by host accelerates
viral mutations
Rapid
evolution
What is influenza?
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“Influenza (the flu) is a contagious respiratory
illness caused by influenza viruses. It can
cause mild to severe illness, and at times can
lead to death.”
(http://www.cdc.gov/flu/about/disease/index.htm)
Influenza Lung
Normal Lung
Figure 1. Gross and microscopic lesions from dog infected with highly pathogenic
avian influenza (HPAI) H5N1. A) Severe congestion and edema in the lung. B) Lung
histopathologic results showing severe pulmonary edema and hemorrhage with
black-brown particles (hemosiderin) (magnification ×100). (cdc web site).
http://www.biologyofhumanaging.com/s
lides/lng120d1.jpg
Brief history and epidemiology
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Influenza appears to have afflicted humans since
ancient times.
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Epidemics of influenza
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Hippocrates in 412 BC
Numerous epidemics in the middle ages
Every winter
> 20,000 deaths/year
Elderly or immunocompromised individuals.
Pandemics
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Irregular 10-50 year intervals.
The Spanish flu (1918-1920) - killed 20-40 million people
worldwide
The Asian flu (1956-1957) - 60,000 deaths in North
America.
Spread of the Asian Influenza
pandemic in 1957
Nomenclature
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Family Orthomyxoviridae
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Myxo = mucus - virons bind to sialic acid residues in
mucoproteins
Genus: Influenza Virus
•
Three groups which share a common structure and mode of
replication, but differ serologically based on M and NP antigens
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Type A infect humans and animals; epidemics and pandemics
Type B infects humans only; epidemics
Type C infects humans and pigs; mild disease
Classification of human
influenza viruses
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Type A or B
Geographic source
Isolate number
Year of isolation
Four HA: H0, H1, H2, H3
Two NA: N1, N2
More on the significance of HA
and NA will be discussed later on
World Health Organization
Influenza Nomenclature
Hemagglutinin subtype
Influenza
type
Year of isolation
A/Panama/2007/99 (H3N2)
Geographic source
Isolate
number
Neuraminidase subtype
Morphology and nature of the
genome
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SS (-) RNA genome
Segmented genome (8 segments encode
11 proteins)
Virions may be spherical or filamentous
Influenza Virus
Replication of Influenza Virus
Why do we continue to have
Influenza Virus Epidemics?
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Genetic variability
Influenza virus keeps changing its structure via two different
mechanisms:
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Antigenic drift - changes in the antigenic determinants of the HA
and NA that accumulate with time. (result in variants of the SAME
NA or HA type)
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Viral RNA polymerase
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Error prone
No proofreading
Rapid evolution
Provides a selective advantage
Antigenic shift - major changes due to a re-assortment of genes
that occurs when two different influenza viruses infect the same
host.
Recombination by template switching
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Between homologous segments of two different strains
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More common in avian isolates
Antigenic Shift
Two different viruses infect the same pig
and through re-assortment of the gene
segments, a new virus is generated
Human influenza virus
Avian influenza virus
New human influenza
virus
How do we acquire viruses
from pigs?
Genetic variability
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The 1957 Asian influenza pandemic- antigenic shift
(new HA, NA, and PB1)
The 1968 Hong Kong pandemic - antigenic shift
(new HA and PB1)
1918 Spanish influenza pandemic was not due to
shift- new studies indicate that it arose from an avian
virus by drift.
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Enhanced cleavability of HA due to NA changes!
Changes in NS1 made it a potent inducer of proinflammatory responses
A single mutation in HA resulted in a virus that had gained
the ability to bind to sialic acid residues present in the
human respiratory tract.
Changes in PB1 enhanced viral replication
PB1-F2 - promotes apoptosis
Pathogenesis and clinical
symptoms
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Aerosol transmission
3 day incubation (influenza A)
Virus initially infects epithelial cells in the upper
respiratory tract
Loss of the ciliated epithelium
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Direct effect of virus multiplication and release
Due to toxic oxygen radical formation (host cell response)
Due to apoptosis
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dsRNA and NA may trigger host cell responses that contribute to
apoptosis
PB1-F2 sensitizes cells to apoptosis (not produced by all strains)
Pathogenesis and clinical
symptoms
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With loss of ciliated epithelium:
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Loss in the ability of the respiratory tract to clear
viruses or bacteria by mucociliary flow
Death
Secondary bacterial infections
Virus replication induces interferons and
other cytokines (IL-6, IL-8, TNF-) leading to
local and systemic inflammatory responses.
This results in the symptoms that define the
“flu” syndrome:
Pathogenesis and clinical
symptoms
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Fever
Headache
Chills
Malaise
Muscle aches
As the fever declines
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runny nose
coughing
Selected virulence
characteristics
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HA for attachment
Inhibition of host mRNA translation (establishing control of the
host)
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Cap snatching
Viral mRNAs compete more effectively for initiation factors.
Inactivation of the cap binding reaction by removing the required
phosphate from eIF-4E, reducing available initiation factors
NS1 interferes with host cell mRNA splicing, polyadenylation,
and transport to the cytoplasm
Inactivation of eIF-4
Summary of virulence
characteristics
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Evasion of host defenses
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NS1 binds to dsRNA to inhibit activation of IFN
Damage
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Induction of apoptosis - dsRNA, NA, and Pb1-F2 all play a
role
NO and O2 NO enhances development of more quasi-species
Induction of cytokines
Strain dependent differences
in pathogenesis
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Strain differences may result in differences in the severity of
the disease for both human and avian viruses.
Aquatic birds are the natural reservoir for avian influenza A
viruses
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Is usually asymptomatic in feral birds
Highly pathogenic strains may cause serious systemic infections
in domestic poultry
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Due to the presence of a polybasic cleavage site in HA
(Cleavage of HA at a basic residue by host cell proteases is
required for viral infectivity)
• For human viruses, systemic spread has not been documented.
This may be due to:
Strain dependent differences
in pathogenesis
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In humans variations in pathogenicity may be due to
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Lack, in other organs, of proteases that capable of cleaving
the HA
Interferon activity
Differences in the effectiveness of NS1 to antagonize IFN
/ production
Differences in NA that allow binding of host proteases that
assist in HA cleavage activation or activation of apoptosis
Those most likely to succumb to the disease are
usually the elderly and the very young. Why?
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The 1918 strain was an exception to this rule - it caused
more severe symptoms in those who were the most
immunocompetent!
Due to an overdeveloped immune response (“cytokine
storm”) of the host against the virus!
Cytokine storm
Diagnosis
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Nasopharyngeal swabs, washes, or aspirates
taken early in the course of the disease are
the best specimens
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The virus can be grown in the amniotic or allantoic
cavity of embryonated chicken eggs, or in tissue
culture cells with trypsin added to cleave HA.
Diagnosis
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May assay directly for the virus (direct assay)
May assay for antibodies, produced in the host,
against the virus (indirect assay)
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Hemagglutination assay-a direct method to identify
the presence of the virus and to get a rough titer of the
virus.
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Is based on the ability of influenza viruses to agglutinate
RBCs.
Virus is titered by making serial two-fold dilutions of the
virus and determining the highest dilution of virus that
causes agglutination of the RBCs.
Hemagglutination assay
Hemagglutination assay
www.medicine.mcgill.ca/.../hemag_plate.jpg
Serological/Immunological
Methods
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Hemagglutination-Inhibition Assay – an indirect
test for antibody against specific influenza virus
types -
Hemagglutination Inhibition
www.cdc.gov/.../16/2/images/09-1733-Ft.gif
Serological/Immunological
Methods
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Immunofluorescence
Enzyme immunoassay (EIA)
Optical immunoassay
Treatment
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Amantidine and rimantidine – targets the M2 protein,
blocking the ion channel it forms and preventing
uncoating of the virus.
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Only effective against Group A influenza viruses
Treatment
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Zanamivar (Relenza) and Oseltamivar
(Tamiflu) – target the neuraminidase,
inhibiting its activity and, therefore, inhibiting
release of the virus.
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Effective against both Groups A and B
Prevention by vaccination
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Need a new vaccine every year because of shift and drift of
the virus
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Whole inactivated virus - flu shot
Live, attenuated cold adapted virus (LAIV or FluMist)
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Made by combining the HA and NA genes of the targeted virus
strain with the six other gene segments from mutant viruses
known to have restricted growth at 370C
Nasal-spray inoculation
The reassortment viruses cannot replicate in the lung at core
body temperature, but grow well in the cooler nasal mucosa
where they stimulate an excellent immune response.
PB2
PB2
PB1
PB1
PA
PA
HA
HA
NA
NA
NP
PB2
NP
M
PB1
M
NS
PA
NS
HA
Attenuated
Influenza
Vaccine Virus
NA
NP
M
NS
Attenuated
Vaccine Virus To
New Virus Type
Virulent Wild
Type Influenza
Virus
Vaccination in development
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Subunit vaccines
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Poxvirus recombinants expressing single viral
proteins
Oligopeptides corresponding to the antigenic
components of the HA protein
DNA-based vaccines
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Target epitopes that are highly conserved in all
influenza A viruses
WINTER - 2007
Should we be afraid of the avian (bird) flu?
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Starting in 1997, a highly virulent avian form of influenza (H5N1)
spread through the commercial poultry farms in Hong Kong.
It has now been found in many sites in Southeast Asia and a
number of humans have been infected, with several resulting deaths
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Due to apoptosis of alveolar epithelial cells and leukocytes
Due to enhanced proinflammatory cytokine response
Due to pneumonia that progresses to ARDS and multi-organ failure
Fortunately, these strains have not yet shown signs of spreading
efficiently among humans……….
Avian influenza
Cycle of Avian Influenza
Viruses in Animals & Humans
Domestic birds
Natural avian
influenza cycle
Shore birds
Direct bird to human
transmission is also
possible.
Pandemic
disease cycle
Water fowl
Mammals
(primarily swine)
No human-human spread
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Avian HA proteins preferentially recognize and bind to
sialoligosaccharides terminated by N-acetylsialic acid linked
to galactose by an α2,3 linkage.
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found on the respiratory epithelium of birds
found on lower respiratory tract of humans along with α2,6
linkage
only α2,6 linkage is found on the upper respiratory epithelium of
humans
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Pneumonia more common
Shift of drift could change this…….
Attachment of influenza
viruses
Genes involved in H5N1
pathogenesis
Avian Influenza pathogenesis
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Changes in polymerase allow for replication at lower
temperatures of humans
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The avian viruses normally do not replicate well in the
upper respiratory tract of humans (33º C); normally
replicate in intestinal tract of birds (41º C)
PB1-F2 causes apoptosis in macrophages which
delays immune response; also enhances proinflammatory response
NS1 is a potent inducer of pro-inflammatory cytokines
(TNF) through PDZ domain
HA binds to sialic acid residues with α2,3 linkage
Nations With Confirmed Cases H5N1 Avian
Influenza (February 2007)
Cumulative Number of Confirmed Human Cases of
Avian Influenza A/(H5N1 ) Reported to WHO
6 May 2010
Country
2003
2004
2005
2006
2007
2008
2009
2010
Total
cases deaths cases deaths cases deaths cases deaths cases deaths cases deaths cases deaths cases deaths cases deaths
Azerbaij an 0
0
0
0
0
0
8
5
0
0
0
0
0
0
0
0
8
5
Bangladesh 0
0
0
0
0
0
0
0
0
0
1
0
0
0
0
0
1
0
Cambodia
0
0
0
0
4
4
2
2
1
1
1
0
1
0
1
1
10
8
China
1
1
0
0
8
5
13
8
5
3
4
4
7
4
0
0
38
25
Djibouti
0
0
0
0
0
0
1
0
0
0
0
0
0
0
0
0
1
0
Egypt
0
0
0
0
0
0
18
10
25
9
8
4
39
4
19
7
109 34
Indonesia
0
0
0
0
20
13
55
45
42
37
24
20
21
19
3
2
165 136
Iraq
0
0
0
0
0
0
3
2
0
0
0
0
0
0
0
0
3
2
Lao
People's
0
Democratic
Republi c
0
0
0
0
0
0
0
2
2
0
0
0
0
0
0
2
2
Myanmar
0
0
0
0
0
0
0
0
1
0
0
0
0
0
0
0
1
0
Nigeria
0
0
0
0
0
0
0
0
1
1
0
0
0
0
0
0
1
1
Pakistan
0
0
0
0
0
0
0
0
3
1
0
0
0
0
0
0
3
1
Thail and
0
0
17
12
5
2
3
3
0
0
0
0
0
0
0
0
25
17
Turke y
0
0
0
0
0
0
12
4
0
0
0
0
0
0
0
0
12
4
Viet Nam
3
3
29
20
61
19
0
0
8
5
6
5
5
5
7
2
119 59
Total
4
4
46
32
98
43
115 79
88
59
44
33
73
32
30
12
498 294
Total number of cases includes number of deaths.
WHO reports only laboratory-confirmed cases.
All dates refer to onset of illness.
Indonesia numbers indicate cumulative total of sporadic cases and deaths which occurred during 2009.
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Amantadine (Brand name Symmetrel: Treatment of influenza type A-2, but not type B). This drug
cannot treat the cytokine storm associated with avia n influenza
The pandemic H1N1 influenza
virus
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In 2009 a new H1N1 strain was passed from
pigs to humans
The virus rapidly dispersed worldwide in a
few months
Ultimately found to not be more virulent in
humans than the seasonal strains
No genetic markers for severe
disease detected
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HA - has only 1 arginine at the HA cleavage site - no
enhanced cleavability
PB1 - gene encodes a truncated, and most probably
non-functional PB1-F2 protein
PB2 lacks mutation in 627 and 701 that contribute to
better replication in mammalian cells
NS1 - has deletion of 11 carboxy-terminal encoding
the PDZ domain
H1N1 Pandemic
Threats
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Every year in the United States, on average:
 5% to 20% of the population gets the flu;
 more than 200,000 people are hospitalized from flu
complications
 about 36,000 people die from flu.
Research suggests that currently circulating strains of H5N1 viruses are
becoming more capable of causing disease (pathogenic) in animals than
were earlier H5N1 viruses.
Gambotto A, Barratt-Boyes SM, de Jong MD, Neumann G, Kawaoka
Y.Human infection with highly pathogenic H5N1 influenza virus. Lancet.
2008 Apr 26;371(9622):1464-75.
Blendon RJ, Koonin LM, Benson JM, Cetron MS, Pollard WE, Mitchell
EW, Weldon KJ, Herrmann MJ.Public response to community mitigation
measures for pandemic influenza. Emerg Infect Dis. 2008
May;14(5):778-86.
Song D, Kang B, Lee C, Jung K, Ha G, Kang D, Park S, Park B, Oh
J.Transmission of Avian Influenza Virus (H3N2) to Dogs. Emerg Infect
Dis. 2008 May;14(5):741-6.
Blumenshine P, Reingold A, Egerter S, Mockenhaupt R, Braveman P,
Marks J.Pandemic influenza planning in the United States from a health
disparities perspective. Emerg Infect Dis. 2008 May;14(5):709-15.
Take Home Message
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Influenza virus epidemics and pandemics have occurred
regularly since ancient times.
Influenza virus is an enveloped virus that has a SS, - RNA
genome of eight segments
Influenza virus epidemics and pandemics continue to occur
because of the genetic variability of the virus NA and HA due to
 Drift- genetic mutations
 Shift - genetic reassortment
 Recombination by template switching
Influenza infections are characterized by fever, headache, chills,
malaise and muscle aches.
 Secondary bacterial infections are common
Diagnosis is usually by immunological means
Treatment may target the HA or NA
New vaccines are needed every year

Live, attenuated cold adapted virus made by reassortment (FluMist)
Resources
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The Microbial Challenge, by Krasner; ASM Press; Washington DC; 2002.
Brock Biology of Microorganisms, by Madigan and Martinko, Pearson Prentice Hall,
Upper Saddle River, NJ, 11th ed, 2006.
Microbiology: An Introduction, by Tortora, Funke and Case; Pearson Prentice Hall;
9th ed, 2007.
Fundamentals of Molecular Virology, by Nicholas Acheson; Wiley and Sons; 2007
Suzanne L. Epstein, et al. 2002. DNA Vaccine Expressing Conserved Influenza Virus
Proteins Protective Against H5N1 Challenge Infection in Mice, Emerging Infectious
Diseases
http://www.brown.edu/Courses/Bio_160/Projects1999/flu/vaccines.html
http://www.hhmi.org/biointeractive/museum/exhibit99/4_6b.html
http://www.pandemicflu.gov/
http://www.who.int/csr/disease/avian_influenza/en/
http://www.cdc.gov/flu/avian/outbreaks/current.htm
Arias, Carlos F, et al. 2009. Molecular anatomy of 2009 influenza virus A (H1N1),
Archives of Medical research 40:643-654.
Thanh, Tran T., et al. (2008) Human H5N1 influenza: Current insight into
pathogenesis. The Int’l J. of Biochem. and Cell Biol 40:3671-2674.
Kuiken, Thijs and Taubenberger, Jeffery K. (2008) Pathology of Human Influenza
revisited. Vaccine 26S:D59-D66.
Basler, Christopher and Aguilar, Patricia V. (2008) Progress in identifying virulence
determinants of the 1918 H1N1 and the Southeast Asian H5N1 influenza A viruses.
Antiviral Research 79:166-178.