1. Influenza H1N1 A: A close insight Dr. Mustafa Ababneh Molecular Virologist

2. Outlines Virus structure, proteins and functions Influenza A replication Influenza A Reassortment Identification of reassortment Alignment of Current H1N1 influenza viruses, and between H1N1 and H5N1. Mutational effects

3. Virus charateristics Orthomyxoviridae Family Has a segmented negative-sense, single-stranded RNA viruses total genome length is 12000-15000 nt. 16 H antigens (H1 - H16) and 9 N antigens (N1 - N9), which can be found in all possible combinations. The nucleoprotein antigen (A, B, C) determines the virus type. The HA and NA antigens determine subtypes. Influenza virus A: both antigen shift and antigen drift noted. High antigenic variability in the surface glycoproteins HA and NA. Has 8 segments. Influenza virus B : Members infect only humans; associated with epidemics; antigenic drift noted. Has 8 segments Influenza virus C : human and swine, Has 7 segments.

7. Replication of orthomyxovirus Replication starts with the binding of HA protein to the sialic acid receptors Then penetration and uncoating Genome Replication takes place in the nucleus Cap-snatching NOT in the cytoplasm

10. Antigenic variation Antigenic shift: These are the major changes based on the reassortment of segments of the genome. In the reassortment entire segments of RNA can be exchanged between two or more viruses infecting the same host cell. Antigenic drifts: these are minor changes caused by point mutation in the genes encoding HA or NA glycoproteins

11. Antigenic shift will render the resulting virus the ability to cross species barriers. Antigenic drift will render the virus to transmit efficiently between the same species. RNA dependent RNA polymerase

13. 13 Influenza Reassortment

14. Reassortment in H1N1 influenza A SegmentSize in amino acidSpecies originGeographical origin PB2: polymerase basic subunit 2 760 a.aAvianNorth America PB1 :polymerase basic subunit 1 758 a.aHuman1993:H3N2 Human PA :polymerase acid717 a.aAvianNorth America HA : Heamagglutinin 567 a.aSwine (H1)North America NP: nucleoprotein499 a.aSwineNorth America NA: neurominidase470 a.aSwine (N1)Europe M : matrix (M1,M2)324 a.aSwineEuroasia NS : Non-structral proteins (NS1,NS2) 279 a.aswineNorth America

15. How we can Know the reassortments events

16. Multiplex RT-PCR identification of reassortment

17. Influenza A Current circulating strains are H1N1 and H3N2 Human subtypes include H1N1, H3N2, H1N2, and H2N2 Avian subtypes include H1 to H16 and N1 to N9 Bird  human H5N1, H9N2, H7N7, H7N2, H7N3

18. H1N1 sequence database

19. H1N1 genes sequences

20. PB1 (H1N1) PB2PAHANPNAMNS (H5N1) Jordan- 2006 95.9%84.5%96.1%9.7%93.6%87.6%10.1%77.4% Sequence similarity between current H1N1 and H5N1 (Jordan-2006) Alignment was done with ClustalW, lasergene DNA analysis Software.

21. Sequence similarity between current H1N1 isolates H1N1-CalPB2PB1PAHANPNAMNS Texas99.7%99.9% 99.8%100%99.9% DenmarkN.A 99.5%N.A99.9%100% AucklandN.A 99.6%100%N.A GermanyN.A 99.9%N.A

22. Mutational analysis Amino acid markers of host specificity,Does Influenza H1N1 A has the markers? Tamiflu resistant Mutation NS1 mutation- glutamic acid in position 92.

23. by compare the amino acids sequences of all influenza pandemic strains. If positions: 87 NS1, 103 NS1, 207 NS1 and 63 NS2 contains Human Amino acid: this means the virus will be cross to human from other species.

24. Tamiflu resistant H1N1 H275Y Histidine to tyrosine subsiotiution in the neuraminidase active site (H 275Y in N1 ) Detection by sequencing. The current H1N1 still Tamiflu sensitive

25. Tamiflu reseistant H1N1

26. Tamiflu resistant mutation in current H1N1

27. Tamiflu resistant mutation in Human H1N1 (Human strain)

28. NS1 H5N1 (D 92 E)

30. NS1 of H1N1 (D 92 E)

31. Refernces

33. Thank you