80% Influenza A — H3N2 (some H1N1) 20 % influenza B (orthomyoxiviridae – seals and humans only) Deaths above epidemic threshold – 50% hospitalizations ≥ 65 – 2x outpatient visits

95% Influenza A — H1N1 58.6% H3N? (1.6%), not typed (39.8%) 5 % influenza B (orthomyoxiviridae – seals and humans only)

Influenza Statistics 5-15% of World population each year (seasonal flu – not epidemic) 0.1 % or ~ ¼ → ½ million die 1918 H5N1 epidemic 2.5 % and 20 – 100 million deaths Flu, Gina Kolata (1999) Touchstone (Simon and Schuster) Past pandemics: 1918, 1957, 1968, 1971, H1N1 outbreak at Fort Dix – 13 severe cases & 1 death led to mass vaccination program but epidemic never materialized False correlations to Guillian Barré began fear of vaccinations

Influenza virus membrane proteins H1N1 Hemaglutinin (HA) 1-16 Neuraminidase (NA) infect humans 1 – 2 infect humans Cytokine Storm Cytokines are peptide signaling molecules generated as part of inflammation response. pulminary embolism common cause of death with H1N1 Cytokine deficient mice (influenza model) die at same rate?

Viral envelope Proteins Hemaglutinin (HA) Responsible for recognizing the type of cell to infect Binds to select host cell surface glycoproteins with external Sialic Acid Binding site accommodates 3 NeuNAc groups in ‘shallow’ wells - not conducive to anti-viral drugs Types 1-3 of 16 can infect human cells - a single aa mutation in avian H5N1 alters HA to allow human infection Sialic acids are N, or O, substituted derivatives of neuraminic acid

Viral envelope Proteins Neuraminidase (NA) Responsible for departure of mature virus from failing host - Mature virus particles tethered to host via NeuNAc glycoproteins. - Cleavage of the external NeuNAc group frees virus Binding site is very deep and amenable to small molecule inhibitors - oseltamivir (Tamiflu) and zanamivir (Relenza) NeuNAc Gal GalNAc Ser

Viral envelope Proteins Matrix Protein M2 (M2) Proton Channel Protein ↓pH in virus causes release of vRNP into host cell’s cytoplasm vRNP – viral Ribonucleoprotein (-) RNA (viral genome) Structural nucleoproteins Viral RNA Polymerase complex ― (-) RNA → (+) RNA - These contain signals that target them for transport from the host cell cytoplasm into the nucleus.

GP S S host target H --- V NN MM HA from virus binds to  -2,6 linked NeuNAc from host glycoprotein.

GP S S host target H --- V NN MM Host membrane engulfs virus particle forming endosome which enters host. H+H+ H+H+

host target V NN MM GP S S H HA changes conformation due to acidity of endosome. H2 exposed. H2 grabs endosome membrane bringing two membranes in contact. M2 opens to allow endosome protons to enter virus. vRNP released into host cell cytoplasm. --- GP S S host target H --- V NN MM H+H+ H+H+ H+H+ H+H+

GP S S host target H V NN MM (-) RNA → (+) RNA using viral RNA Pol complex of vRNP. Host RNA Pol II transcribes (+) RNA → (-) RNA. (?) RNA processed by host spliceosome while host mRNA processing inhibited. vRNPs form and acquire membrane from host cell membrane – then bud off.

GP S S host target H V NN MM GP S S host target --- H V NN MM However, virus tethered to host cell glycoproteins.

GP S S host target --- H V NN MM GP S S host target --- H V NN MM NA cleaves NeuNAc from host glycoprotein releasing virus.

GP S S host target H --- V NN MM New virus infects another host cell

Viral Life Cycle HA from virus binds to  -2,6 linked NeuNAc from host glycoprotein. Host membrane engulfs virus particle forming endosome which enters host. HA changes conformation due to acidity of endosome. H2 exposed. H2 grabs endosome membrane bringing two membranes in contact. M2 opens to allow endosome protons to enter virus. vRNP released into host cell cytoplasm. (-) RNA → (+) RNA using viral RNA Pol complex of vRNP. Host RNA Pol II transcribes (+) RNA → (-) RNA. (-) RNA processed by host spliceosome while host mRNA processing inhibited. vRNPs form and acquire membrane from host cell membrane – then bud off. NA cleaves NeuNAc from host glycoprotein releasing virus.

amantadine Inhibit M2 Don’t work for influenza B H --- V NN MM Binds to M2 and blocks H + entry H1N1 virus does not respond to amantadine or M2 inhibitors rimantadine Antiviral Drugs

oseltamivir (tamiflu) zanamivir (relenza) competitive inhibitors of viral NA prevents release from host at latter stages

GP S S host target --- H V NN MM GP S S host target --- H V NN MM competitive inhibitors of viral NA prevents release from host at latter stages X oseltamivir (tamiflu) zanamivir (relenza) Neuraminidase (NA)

Neuraminidase Inhibitor Resistance Testing Results on Samples Collected Since October 1, 2013 OseltamivirZanamivir Virus Samples tested (n) Resistant Viruses, Number (%) Virus Samples tested (n) Resistant Viruses, Number (%) Influenza A (H3N2)1090 (0.0)1090 (0.0) Influenza B320 (0.0)320 (0.0) 2009 H1N12,254*21 (0.9)8700 (0.0)