1. presented by Jan Haas
Institute for Immunology

2. Influenzaviruses: Orthomyxoviridae
16 Haemagglutinin
9 Neuraminidase
- types
3 Polymerase Subunits
Nucleoprotein
Nuclear Export Protein (NEP)
Matrix Protein (M1)
Ion Channel Protein (M2)
Interferon Antagonist (NS1)
PB1-F2 Protein
Virions are decorated with two surface glycoproteins, HA and NA. The NA protein facilitates virus release from infected cells by removing sialic acid from cellular and viral HA proteins.
The genome is composed of eight segments of single-stranded RNA that interact with the nucleoprotein and the components of polymerase complex (PB2, PB1 and PA). The 3 polymerase subunits and the nucleoprotein carry out the replication and transcription. Viral ribonucleoprotein complexes are exported from the nucleus to the cytoplasm by the nuclear export protein (NEP) and the matrix protein.
M2 = release of vRNP into the cytoplasm, PB1-F2 = apoptosis by interaction with mitochondrial proteins

3. The viral life cycle (-) RNA virus enveloped
Influenza A responsible for pandemic outbreaks
spread via aerosols and droplets
Infection occurs by binding of the viral HA to sialic acid receptors on the target cell surface and subsequent fusion of the viral envelope with the host cell membrane and receptor-mediated endocytosis. The low pH inside the endosomes (pH 5-6) maintained by proton pumps within the endosomal membrane triggers the fusion reaction between the viral envelope and the endosomal membrane. This is a key step in viral infection. It is through this fusion process that the viral RNA gains access to the cytosol of the host cell. The RNA then enters the nucleus of the cells where it is replicated. Viral proteins are being synthesized in the cytosol ultimately resulting in the production of many new viral particles. Early viral proteins – those required for replication and transcription are transported back into the nucleus. During the production of progeny virus, the host cell’s own protein synthesis is effectively shutdown. Late in the infection cycle, the M1 and NEP = NS2 proteins facilitate the nuclear export of the newly synthesized vRNPs. PB1-F2 associates with mitochondria. Finally after assembly and budding of progeny virions - many thousands of new viral particles - the cell lyses and dies as a result of the infection.

4. Historical overview
Anitgenic-shift and antigenic-drift combined with reassortment events of the 8 different gene segments of the virus are made responsible for the regular outbreak of influenza pandemics. Especially in swine reassortment of gene segments can occur.

5. ‘Spanish’ influenza (H1N1, 1918-1919) ‘Russian’ influenza (H1N1, 1977)
Genetic Relationships among Human and Relevant Swine Influenza Viruses, 1918–2009.
Yellow arrows reflect exportation of one or more genes from the avian influenza A virus gene pool. The dashed red arrow indicates a period without circulation. Solid red arrows indicate the evolutionary paths of human influenza virus lineages; solid blue arrows, of swine influenza virus lineages; and the blue-to-red arrow, of a swine-origin human influenza virus. All influenza A viruses contain eight genes that encode the following proteins (shown from top to bottom within each virus): polymerase PB2, polymerase PB1, polymerase PA, hemagglutinin (HA), nuclear protein (NP), neuraminidase (NA), matrix proteins (M), and nonstructural proteins (NS). The genes of the 1918 human and swine H1N1 and the 1979 H1N1 influenza A viruses were all recently descended from avian influenza A genes, and some have been “donated” to the pandemic human H1N1 strain.
‘Spanish’ influenza (H1N1, )
mortality pattern unusual: young adults
restricted to the respiratory tract, lack of systemic infection
most patients died of bacterial pneumonia, some as well of viral pneumonia
aberrant innate immune responses contributing to its virulence
‘Russian’ influenza (H1N1, 1977)
the re-emerging H1N1 virus did not replace the H3N2 viruses circulating at the time
both subtypes are co-circulating in humans to this day
reassortment between viruses of these subtypes resulted in the emergence of H1N2 viruses in human populations in 2001
H1N2 viruses have since disappeared.
‘Asian’ influenza (H2N2, )
was caused by a human/avian reassortant that introduced avian virus H2 HA and N2 NA genes into human populations
furthermore, the Asian influenza virus also possessed a PB1 gene of avian virus origin
‘H5N1’ influenza ( )
Hong Kong: highly pathogenic avian virus
six fatalities, marked the first reported fatal infections of humans with avian influenza viruses
after a period of local and sporadic outbreaks, a new outbreak started in 2003
sustained human-to-human infection has not occurred
H5N1 viruses are characterized by a high mortality rate but inefficient spread among humans
 in contrast, S-OIVs seem to spread efficiently among humans but have caused a limited number of fatal infections
‘Hongkong’ influenza (H3N2, )
viruses of the H2N2 subtype were replaced by another human/avian reassortant that possessed an H3 HA gene of avian virus origin
again, the PB1 gene of the pandemic virus was derived from an avian virus
‘S-OIV H1N1’ influenza (2009)
S-OIVs probably resulted from the reassortment of recent North American H3N2 and H1N2 swine viruses (that is, avian/human/swine ‘triple’ reassortant viruses) with Eurasian avian-like swine viruses
S-OIVs possess: PB2 and PA genes of North American avian virus origin, a PB1 gene of human H3N2 virus origin, HA (H1), NP, and NS genes of classical swine virus origin, and NA (N1) and M genes of Eurasian avian-like swine virus origin (hence their original description as ‘quadruple’ reassortants)

6. Genesis of swine-origin H1N1 influenza viruses
‘mixing vessel’
The S-OIV probably resulted form the reassortment of recent North American H3N2 and H1N2 swine viruses (an avian/swine/human ‘triple’ reassortant) with Eurasian avian-like swine viruses.

7. Electron microscopic picture: H1N1
Two different reports about the shape and size of H1N1 virions. TEM from this paper and negatively stained virions from the CDC which are spherical and not of filamentous shape.

8. Role of HA in viral pathogenicity
Receptor distribution on host cells:
human influenza preferentially bind to sialic acid that is linked to galactose by an a2,6-linkage (SAa2,6Gal)
this preference is matched by SAa2,6Gal on epithelial cells in the human trachea
in contrast, avian influenza viruses preferentially recognize SAa2,3Gal that is matched by SAa2,3Gal on epithelial cells in the intestinal tract of waterfowl (the main replication site of avian influenza viruses)
surprisingly H5N1 binds preferentially to SAa2,3Gal
studies showed avian-type receptors (SAa2,3Gal) on human epithelial cells that line the respiratory bronchiole and the alveolar walls, but human-type receptors (SAa2,6Gal) on human epithelial cells in nasal mucosa, paranasal sinuses, pharynx, trachea and bronchi
HA receptor specificity: amino acid residues in the HA receptor binding pocket determine binding to human/avian type receptors
HA cleavage: HA cleavability determined by the amino acid sequence at the cleavage site
Low pathogenic viruses possess a Arg residue at the cleavage site
highly pathogenic H5 and H7 viruses possess several basic amino acids at the HA cleavage site
 pathogenicity correlates with acquisition of multibasic HA cleavage sites

9. Role of PB2, NS1 and PB1-F2 in pathogeniciy and host specificity
PB2: belongs to the viral replication complex
PB1-F2: is expressed from the +1 reading frame, induces apoptosis by interaction with two mitochondrial proteins
NS1: the NS1 protein is an interferon antagonist that blocks the activation of transcription factors and IFN-b-stimulated gene products, and binds to double-stranded RNA (dsRNA) to prevent the dsRNA-dependent activation of 2’-5’ oligo(A) synthetase, and the subsequent activation of RNase L; can block RIG-I, MDA5 and TLR-3,7 & 8
RNAse L = innate immune response
RIG-I = retinoic acid inducible gene-I
MDA5 = melanoma differentiation antigen 5
NS1 blocks antiviral interferon and is associated with high levels of pro-inflammatory cytokines  cytokine imbalance and high mortality
NS1 amino acid changes affect virulence in a strain specific manner, unlike HA cleavage affects universal pathogenicity

10. Prevention and control
Antiviral drugs:
M1: Adamantanes
NA: Oseltamivir (Tamiflu) and Zanamivir, Peramivir
CS-8958 (NA)  Phase II
T-705 (Nucleoside analogue)  Phase III
mAb against HA
Vaccines: produced in allantoid fluid of embryonated chicken/cell culture
Pandemrix (GSK) (dead vaccine)
Focetria (Novartis) (dead vaccine)
Celvapan (Baxter) (cell culture)
Celtura (Behring) (cell culture)
Live attenuated viruses yield higher humoral and cellular immune responses
Most circulating human H1N1 and H3N2 viruses, some H5N1 viruses, and most European porcine H1N1, H1N2 and H3N2 viruses, are resistant to adamantanes. The S-OIVs are also resistant to ion channel inhibitors.
Neuraminidase inhibitors interfere with the enzymatic activity of the NA protein, which is critical for the efficient release of newly synthesized viruses from infected cells. Oseltamivir-resistant human H1N1 viruses may have emerged in immunocompromised patients in which prolonged replication79–81 may have resulted in the selection of mutations that increase the fitness of oseltamivir-resistant viruses. The S-OIVs are sensitive to neuraminidase inhibitors when tested in vitro in enzymatic assays.

11. Thank you for your attention!

12. “History” of the virus
Two mechanisms that are not mutually exclusive, reassortment and interspecies transmission, result in the introduction of viruses with new HA subtypes into human population.