1. Orthomyxoviridae
Molecular Virology

2. Orthomyxoviridae Myxo means mucus
Myxovirus (old name): particularly affinity to mucopolysaccharide and glycoprotein, in particular, for sialic acid-containing receptor on cell surface
Sialic acid: neuraminic acid
Myxovirus divided into Orthomyxoviride and paramyxoviridae
In term of Genetic organization
Enveloped glycoprotein
Replication strategy

3. Classification
On the basis of antigenic cross-reactivity of RNP between members of each type, but not between types
Orthomyxoviridae has 5 genera
The influenza virus A, B and C
Thogotovirus (ticks)
Isavirus (infectious salmon anemia virus)
Virus is named
influenza A/chicken/Hong Kong/220/97(H5N1)
(species origin omitted if human)
Hemagglutinin (HA): H1-H16 for type A
Neuraminidase (NA): NA1-NA9 for type A
Hemagglutinin-esterase-fusion (HEF): type C

4. Virion structure Influenza B: HA, NA, NB, and BM2
Influenza C: has hexagonal reticular structure
on the particle surface.
No NA, but has HEF

5. Internal structure of influenza virus under EM

6. Genome structure and organization
Types A and B have 8 segments of (-)ssRNA, encoded 11 viral protein
Types C has 7 segments of (-)ssRNA, NA is missed, but has hemaglutinin-esterase-fusion (HEF) protein

7. Genome structure and protein function of type A

8. Genome structure and protein function of type B

9. Genome structure and protein function of type C

10. Virus replication cycle

11. Attachment
HA of virus has specificity forward sialic acid with different linkages
Mutation of HA amino acid may make virus adapt to different species

12. Entry and uncoating Mediated by clathrin-endocytosis for virus entry
Required a low pH to initiate membrane fusion of endolysosome and envelope
Low pH induces conformational change of HA (HA1 and HA2) and fusion peptide of HA2 was exposed. Can be blocked with weakly bases (NH4Cl, chloroquine) or ionophores (monensin).
HA sequences at HA cleavage site determine pathogenicity
Also, M2-mediated release of the RNA results in appearance of free RNA complexes in cytoplasm
M2 allows the influx of H+ ions from endosome into the virus particle and disrupts protein-protein interaction and results in the release RNP from M1 protein. Can be blocked by amantadine or rimantadine which block the ion channel activity of M2 protein (inhibited uncoating).
BM2 and CM2 have similar functions to M2 (type A)

13. Hemaglutinin (HA)

14. Hemaglutinin (HA)

15. Hemaglutinin (HA)

16. Hemaglutinin (HA)

17. M2 protein Is a tetrameric type III integral membrane protein
Has ion channel activeity
Involves in capturing the RNPs at the assembly site and be a role in virus assembly and budding

18. Nuclear import of ribonucleoproteins
One unusual characteristics of influenza virus in its replication cycle is its dependence on nuclear functions
Four proteins (NP, PB2, PB1, and PA) associated with vRNA to form ribonucleoprotein complexes (RNA) and create a panhandle structure. These proteins possess nuclear localization signals (NLS) that mediated their interaction with nuclear import machinery.

19. Viral RNA synthesis
mRNA is made by a primer-dependent mechanism, is capped and polyadenylated
Replication of vRNA occurs via a two-step process. A full length, (+) copy of vRNA(cRNA) is first made and used as the template to make more (-)vRNA
They use the same viral polymerase complex

20. Viral RNA promoter

21. mRNA synthesis of viral RNA

22. mRNA synthesis of viral RNA
Needs cellular mRNA producing enzyme(RNA polymerase II)
Actinomycin D inhibits virus replication
Cellular DNA destroyed by UV
α-amanitin inhibits RNA poly II
Nucleus removing
PB2 recognizes cap structure of cellular mRNA
PB1 also has endonuclease, resulting in cap-snatching phenomenon to supply capped primer for mRNA synthesis
Polyadenylation by stutter mechanism
Viral splicing is using the cellular splicing machinery

23. Switch from mRNA to cRNA synthesis
Be structurally different in the replication-competent pol from the transscription-competent pol. Due to different domains of pB1 are involved in binding vRNA or cRNA
Involves in newly synthesized cRNA and vRNA by soluble NP protein
Cap structure may have an effect on RNA synthesis

24. Regulation of viral gene expression
Early expression genes: NP, NS1 mRNA and protein
Late expression in genes: HA, NA, M1 and protein
NP is required for replication
NS1 is a factor in combating the host immune response
M1 inhibits viral replication, involves in RNP export from nucleus, only occurs once replication is complete.

25. RNP export from nucleus
Proteins M1 and NS2 involve in directing the nuclear export of RNP.
M1 binds RNA (vRNA and NP), also binds to nucleosome, suggesting that M1 interactions cause the dissociation of RNP from nuclear matrix.
NS2 interacts with the cellular export machinery

26. Virus assembly and release
Assemble and bud from the apical plasma membrane of polarized cells, is an asymmetric procession
Important in viral pathogenesis and tissue tropism Basolateral procession tends to cause systemic disease. HA or NA transmembrane domain is replaced to contain a basoleteral targeting signal and virions still bud from apical plasma membrane
Matrix (M1) protein is absolutely required for assembly of virus particles
M2 captures RNP at the assembly site

27. Budding of influenza virus

28. Packaging of eight RNA segments
Mechanism is not well known
Random incorporation model
Selective incorporation model

29. Release
Neuraminidase (NA) involves virus release by removing the sialic acid
NA activity is also required to remove sialic acid from the carbohydrates present the viral glycoproteins themselves so that the individual virus particles do not aggregate on cell surface

30. Reservoir of virus

31. Reassortment of virus

32. Reverse genetics

33. Antigenic drift Antigenic shift
Induces by point or accumulative point mutation of HA gene, resulting in less antigenic change, no new type formed
Antigenic shift
Induces by gene reassortment of HA gene, resulting in a significant antigenic change, new types of virus formed
Defective interference(DI) particle (von Magnus phenomenon)

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