John R. LaMontagne Memorial Symposium on Pandemic Influenza Research

1. John R. LaMontagne Memorial Symposium on Pandemic Influenza Research Working Group 8 Virus Transmission: Understanding and Predicting Pandemic Risk

2. a2-3Gal Receptor specificity is a major factor a2-6Gal a2-3Gal a2-3Gal H5 a2-6Gal H9 a2-6Gal a2-6Gal a2-3Gal Molecular basis for interspecies transmission and pathogenesis. Influenza A virus host range is polygenic ? ? mixing vessel

3. What we know • Pandemic Human influenza A viruses contain genes from the avian reservoir • 1918 “Spanish” flu H1N1: avian? virus that killed >20,000,000 people. • 1957 Asian flu H2N2: reassortant between avian H2N2 and human H1N1 -> 3 avian genes: HA, NA, and PB1. • 1968 Hong Kong flu H3N2: reassortant between avian H3N? and human H2N2 -> 2 avian genes: HA and PB1. • Influenza viruses that circulate in terrestrial poultry have the potential to cross to humans (H5, H9). • Pigs could be potential intermediate hosts. • Evidence of H3N2 in pigs in or around 1968, with H3 having a2,3 receptor specificity (Kida et al. 1988)

4. What we know • Influenza viruses that have established permanent lineages in humans have altered receptor specificity • H1 viruses until 1957 have dual receptor specifity, contemporary H1 viruses bind a2,6 receptors preferentially (Rogers et al, 1989; Matrosovich et al, 2000; Gamblin et al, 2004; Stevens et al, 2004). • Glycosylation can affect the virus binding properties to receptors (Aytay & Schulze, 1991). • H3 viruses recognize a2,6 receptors almost exclusively (some of them do not grow in chicken eggs). • H5 and H9 viruses have altered receptor specificities (Matrosovich et al, 1999 & 2000).

5. Stevens et al, 2004 Ha et al, 2001 Gamblin et al, 2004

6. What we know • The internal genes limit the virus’ host range • Some internal gene constellations limit replication and transmission of influenza in humans (Snyder et al, 1987; Clements et al, 1992). • A single amino acid change in PB2 (627) is responsible for host range (Subbarao et al, 1993; Hatta et al, 2001). • Avian; Glutamic • Human; Lysine • Incompatibility between polymerase genes of viruses from human and duck origin (Hatta et al, 2002).

7. What we know • The internal genes limit the virus’ host range • Interactions between replication complex and host factors • Interaction of NS1 with… • Human U6 snRNA…disrupts binding of U6-U2 and U6-U4 during RNA splicing (Qi et al, 1995) • NS1-I, a human homolog of the porcine 17beta-estradiol dehydrogenase precursor protein (Wolff et al, 1996) • PABP1 and eIF4GI (Aragon et al, 2000) • 30 kDa subunit of CPSF, inhibits 3'end formation of cellular pre-mRNAs (Nemeroff et al, 1998; Chen et al, 1999; Noah et al, 2003) • Interferes with interferon pathways (Garcia Sastre, et al, 1998 a&b; Bergmann et al, 2000; Talon et al, 2000; Wang et al, 2000, Tumpey et al 2004)

8. What we know • The internal genes limit the virus’ host range • Interaction of the polymerase complex with… • PA and hCLE a potential transcription factor (Huarte et al, 2001) • Interaction of NS2 with… • Nuclear export pathway (O’Neill et al, 1998; Neumann et al, 2000) • M1 (Akarsu et al. 2001) • Interaction of NP with… • RAF-2p48/NPI-5/BAT1/UAP56 (Momose et al, 2001) • NPI-, NPI-3 (O’Neill & Palese, 1995; Wang et al, 1997) • Polymerase complex (Biswas et al, 1998) • Nuclear export pathway (Elton et al, 2001)

9. What we know • Some animal species may promote the emergence of novel influenza strains • Quail and other terrestrial birds as potential intermediate hosts (Makarova et al 200; Perez et al, 2003) • Changes on the globular heads of HA and NA, as well as changes in the internal genes modulate transmission and pathogenicity (Hulse et al 2004) • Presence of a2,3 and a2,6 receptors in terrestrial birds

10. What we do not know • Intermediate hosts that participate and the molecular features that render a virus potentially pandemic • The host that resulted in the emergence of the 1918, 1957, and 1968 pandemic viruses. • If pigs were really involved in the emergence of each one of these pandemics. • Circumstancial evidence of H1N1 in pigs in 1918 • No evidence of H2N2 in pigs prior, during, or after the 1957 pandemic

11. What we do not know • How the known interactions with host factors modulate virus replication in different species • If additional interactions exist with other cellular factors that may promote host restriction • The exact molecular mechanism for incompatibility among some replication complexes from different species • Whether different animal species favor certain molecular changes during adaptation

12. What we do not know • Whether alteration in receptor specificity is required to jump to other species, including humans • Whether any of the known interactions with the internal proteins are involved in host range

13. What needs to be done • Hypothesis: Pandemic influenza is the result of a complex process involving an expansion of the host range of one or more avian influenza viruses followed by a contraction with selection of one strain that transmits readily in humans. • Identify molecular changes associated with adaptation in different animal species • Classical virology and reverse genetics are instrumental to accomplish this goal.

14. What needs to be done • Changes associated with adaptation of influenza viruses to terrestrial birds and other potential intermediate hosts. • Are pathways of adaptation in different animal species ramdom? • Or is there a pattern of changes that are more favored than others?

15. What needs to be done • What further studies are needed to define the genetic loci important for virus transmission between species? • What studies are needed to determine whether changes in the viral RNA polymerase play a role in virus transmission? • What studies are needed to define environmental factors that contribute to virus transmission and how they interact with genetic factors? • What studies can be done to provide measures to predict intraspecies changes in virus transmission? • What studies are needed to identify what measures, if any, can alter intraspecies virus transmission and what the effects of these measures? • What studies are needed to determine whether transmission changes can be used to predict pandemic risk?