1. Influenza There are three types of influenza in humans: A,B, C
1. Flu A: Found in many animal species, in addition to humans
Closely related to Type B but not Type C
Main type responsible for human epidemics
Demonstrates the greatest antigenic variability (“antigenic drift”)
Reservoir in nature is waterfowl
2. Flus B and C: Found almost exclusively in humans
Flu C can also infect swine
Flu C is morphologically and antigenically distinct from A, B
3. Flu A strains designated by host from which isolated, where isolated, year of isolation, and type of HA and NA. An isolate (strain) number may also be included if there are multiple isolates.
Example: A/goose/Leipzig/137/79 (H7N2)

3. Influenza
Virus

4. Structure of Influenza Virus
Influenza A virus is an enveloped particle that when spherical is about 120 nm in diameter
Many particles are not spherical but filamentous in shape
There are two glycoproteins at the surface in surface “spikes”
HA (hemagglutinin) is present as homotrimers
NA (neuraminidase) is present as homotetramers
Protein M2 forms ion channels in the lipid bilayer
The matrix protein M1 lines the inner side of the lipid bilayer
The genome consists of 8 RNA segments present in helical nucleocapsids

5. Influenza Nucleocapsids
NP is the major nucleocapsid protein.
It has a major structural role
It is also required for the switch from mRNA synthesis to genome replication.
PA, PB1, and PB2 are minor components of the nucleocapsid and form the RNA synthesis machinery.
The function of PA is unknown but may be involved in the switch from mRNA synthesis to genome replication
PB1 is an endonuclease that process the mRNA primer; it also is a polymerase that catalyzes nucleotide addition
PB2 recognizes the cap of host cell mRNA required for priming mRNA synthesis
M is the matrix protein.
It is a peripheral membrane protein that underlies the viral membrane.
It interacts with the nucleocapsid and with the tails of HA, NA, and M2

6. Attachment & Entry
The HA spike is a homotrimer with a molecular weight of 110 kDa.
HA is synthesized as a 549 aa precursor called HA0 which is anchored in the membrane near the C-terminus.
HA0 is cleaved into HA1 (328 aa) and HA2 (221 aa)
At the N-terminus of HA is a 16 aa hydrophobic signal peptide for insertion into the ER.
A single Arg separates HA1 from HA2 and cleavage is by a cellular trypsin-like proteinase
HA1 and HA2 remain covalently associated after cleavage by a disulfide bridge
The C terminus of HA2 contains a 26 aa uncharged membrane-spanning domain followed by a 10 aa hydrophilic cytoplasmic domain
The HA polypeptide is glycosylated at specific asparagine residues

7. HA-mediated membrane fusion
The HA trimer is stabilized by a hydrophobic core formed between the three stalk regions. Attachment sites for the cellular receptors are located near the top of each large globular region, which also contains neutralization epitopes.
The exact glycoprotein(s) that serve as host cell surface receptors has not been identified, but it is known to contain sialic acid.
After binding of HA to the cell surface receptor(s) the virus is internalized by endocytosis.
The low pH of endosomes ( pH ) results in an irreversible conformational change in HA which results in the extrusion of the highly conserved hydrophobic amino terminus of HA2 from its position in the native protein.
This region, termed the ‘fusion peptide’, promotes membrane fusion.
The mechanism by which the ‘fusion peptide’ promotes membrane fusion is not completely understood.
The subsequent fusion of viral and endosomal membranes allows the release of the viral genome into the cellular cytoplasm

8. Activation of the HA Spike
After acid treatment
HA0 precursor
Cleaved spike
and proteolysis

9. Activation of Fusion Activity of Flu HA0 by Cleavage
View of One Monomeric Unit in the Spike

10. Change illustrated for one monomeric unit of the trimeric spike

11. Model for Fusion

12. Neuraminidase
NA spike consists of a tetramer. NA is a type 2 glycoprotein with the N terminus inside and the C terminus outside.
NA removes sialic acid from oligosaccharides on cell-surface proteins and glycolipids, thus destroying receptors for the virus.
Also removes sialic acid from HA so that progeny influenza virions cannot aggregate.
Separates virus particles from inhibitory mucopolysaccharides in the respiratory tract allowing efficient infection.

14. Synthesis of mRNAs and RNA Replication

15. Splicing to Produce Influenza A mRNAs
Since influenza RNA synthesis occurs in the nucleus, the cellular splicing machinery can be used
In Flu A two mRNAs are produced from both segments 7 and 8
One mRNA is unspliced, the second is spliced

16. Influenza C Has an Esterase
Flu C lacks NA and has only 7 segments
It has HEF that performs the functions of HA and NA in Flu AB
The receptor for Flu C is 9-O-acetyl-N-acetyl neuraminic acid
The Flu C esterase removes the 9-O-acetyl group to destroy the receptor
The HEF gene is also present in some coronaviruses, which must have obtained it by recombination with Flu C at some time in the past

17. M2 M2 tetramers form ion channels in viral and cellular membranes
Exposure to low pH is required to dissociate the nucleocapsid from the matrix protein, allowing the nucleocapsid to be transported to the nucleus
M2 also prevents premature activation of the fusion activity of HA
Amantadine interferes with the function of M2 and is an effective flu antiviral

18. Virus Assembly
Nucleocapsids assemble in the nucleus during genomic RNA synthesis
The encapsidation signal is at the end of the RNA and not present in mRNAs
Nucleocapsids are exported to the cytoplasm in a process that requires NS2 and M1
Glycoproteins are synthesized on the ER and transported to the plasma membrane
Nucleocapsids bud through the plasma membrane to form virions
More than 8 segments may be packaged: Ten segments randomly selected would result in ~3% of progeny virions having at least one each of the 8 segments
Random selection of segments would mean efficient reassortment during mixed infection, which is known to occur

19. Influenza - Some History
Oldest record of an epidemic probably caused by flu: Hippocrates, 412 BC.
Epidemics have occurred relatively frequently but at irregular intervals
Epidemics vary in severity but the very young and elderly are most at risk.
Epidemics appear to radiate from specific locations
Example: 1781 epidemic that spread across Russia from Asia.
Influenza has killed untold millions throughout the centuries
epidemic was particularly severe
million people died, more than died in World War I.
3. 80% of US WWI deaths were due to influenza
4. A significant factor in the German loss was influenza
First human influenza virus was isolated in 1933.
Different strains cause different epidemics, but human strains can recirculate

20. Antigenic Shift and Drift in Flu A
HA and NA are the major surface antigens of the virus
Antigenic drift describes the selection of variants by the immune system
Relatively slow
Resistance is only partial
Antigenic shift describes the results of recombination (reassortment)
There are 15 different subtypes of HA
There are 9 different subtypes of HN
Subtypes differ by 30% or more in amino acid sequence
A reassortant with a different HA and/or HN may cause a pandemic
Only a few of the subtypes have been isolated from humans

21. Influenza A in Birds The reservoir of influenza A in nature is birds
All 15 HA and 9 NA have been found in aquatic birds
In particular, migratory ducks are important in the maintenance and spread of influenza
Influenza infection of birds is usually asymtomatic
Influenza replicates in the respiratory tract and the intestinal tract of birds
It is excreted in the feces and high concentrations have been found in waters in which migratory ducks congregate
The virus appears to be in equilibrium in birds--little or no sequence drift has been found in bird viruses and disease seldom results from infection
In contrast, the virus drifts rapidly in humans and vaccines must be reformulated yearly, and serious illness is produced

22. Epidemic Influenza Strains
Year
Virus
Common Name
1889
H2N2
1900
H3N8
1918
H1N1
Spanish
1957
H2N2
Asian
1968
H3N2
Hong Kong
1977
H1N1
Russian
When a new strain appears the previous strain usually dies out
At present, H3N2 and H1N1 continue to cocirculate in humans
An H1N1 strains has circulated continuously in pigs in the U.S. since 1918

23. Sialic Acid (N-Acetyl Neuraminic Acid)
Terminal NANA is attached to galactose by a2,3 or a2,6 linkages
Different HAs prefer one or the other linkage
Avian intestine contains predominantly a2,3 linkages
Human trachea contains predominately a2,6 linkages
Pig trachea contains both linkages and serves as an efficient intermediate host in which reassortment can take place--pigs are often referred to as mixing chambers
Other components also contribute to host specificity, best studied for NP

24. U.S. Life Expectancy

25. 1918 Influenza Deaths

26. The 1918 Flu in America

27. Influenza affects 10-20% of U. S
Influenza affects 10-20% of U.S. population each year, causing up to 70,000 deaths. Average death rate in people over 65 is 1/2200 but in it was 1/300.

28. Illness Induced by Influenza Virus
Influenza virus infects superficial cells throughout the respiratory tract.
There is little or no spread to other organs.
High temperatures often accompany the infection, C, that last 3-6 days.
Cough and weakness can last 1-2 weeks longer.
Extensive destruction of epithelial cells of the LRT can result in primary viral pneumonia.
Influenza infection can result in secondary bacterial infection of the LRT resulting in bacterial pneumonia.
Death following influenza infection is usually due to pneumonia, whether viral or bacterial or combined.
Immunity following influenza infection is incomplete and appears to fade in time.
It has been suggested that the high death in young adults in the 1918 pandemic could have resulted from a more active immune response to the virus.

29. Bird Influenza
An epidemic of influenza in chickens occurred in Hong Kong in 1997
The virus was highly virulent, killing % of infected chickens
Bird viruses are not normally transmitted to humans but the 1997 Hong Kong virus resulted in 18 humans becoming infected
This virus was highly virulent in humans--6 of 18 infected people died
The virus was H5N1 and did not spread in humans--no person to person transmission occurred
To eradicate the virus and to prevent new reassortants from arising that might give rise to epidemic virus by direct person to person transmission, 1.6 million chickens were slaughtered

30. Defenses against Influenza
Antivirals
Amantadine and Rimantadine licensed for use and ameliorate symptoms
Inhibitors of NA being developed
Vaccines
Inactivated vaccines are in widespread use
These vaccines must be reformulated every year because of shift and drift
They are 60-80% effective
Attempts being made to develop attenuated virus vaccines that could be reformulated yearly by reassortment
An emergency response to swine flu in 1976 demonstrates the difficulties in preparedness decisions

31. Bunyaviridae
Sin Nombre Virus
La Crosse Virus

38. Arenaviridae
Budding Machupo Virion
Lassa Virions
Tacaribe Virion

40. Representative Arenaviruses
Rodent Host
Disease
Where Found
Old World
Lymphocytic
Mus musculus
Meningitis
Worldwide
choriomeningitis
Lassa
Mastomys sp. HF
West Africa
Mobala
Praomys sp. ?
CAR
New World
Tamiami
Sigmodon hispidus
None?
Florida
Whitewater Arroyo
Neotoma albigula
3 fatal ARDS
Western U.S.
Pichinde
Oryzomys albigularis
None?
Colombia
Guanarito
Zygodontomys brevicauda
Venezuelan HF
Venezuela
Junin
Calomys musculinus
Argentine HF
Argentina
Machupo
Calomys callosus
Bolivian HF
Bolivia
Sabia ?
3 severe cases
Brazil
Tacaribe ? ?
Trinidad

41. Arenaviruses in the New World

42. Some Viruses
Causing
Hemorrhagic fever

43. Representative Viruses Causing Encephalitis
Flaviviridae
Alphaviruses
St. Louis encephalitis
Eastern equine enceph.
Japanese encephalitis
Western equine enceph.
West Nile
Venezuelan equine enceph.
Murray Valley enceph.
Herpesviridae
Tick-borne enceph.
Herpes simplex
Bunyaviridae
Paramyxoviridae
La Crosse
Mumps
California enceph.
Measles