This document summarizes the evolution and pathobiological features of avian influenza viruses, with a focus on H5N1. It discusses how H5N1 viruses have evolved increased pathogenicity in chickens through mutations including additions of basic amino acids in the HA cleavage site. It also describes the emergence of novel H5N8 and H5N2 viruses in Taiwan and their spread through poultry farms and wild birds. The document emphasizes the importance of transboundary cooperation to control the spread of avian influenza viruses throughout the Asian Pacific region.

1. Management and Control of Important
Transboundary Animal Diseases in the Asian Pacific
Region
Evolution and Patho-biological
Features of
Avian Influenza Viruses
Ching-Ho WANG
National Taiwan University
July 26, 2016, 1:30 – 2:15
at Bay Leaf Hotel, Manila, Philippines
(Food and Fertilizer Technology Center
for the Asian and Pacific Region, FFTC) 1

2. Outline
• Avian influenza virus
• Evolution of H5N1 AIV
• Virus persistence and pathogenicity
• The appearance of H5N8
• Avian influenza outbreaks in Taiwan
• Transboundary cooperation in the Asian
Pacific region
2

3. Influenza virus: Structure and Gene
Type A
Subtypes
HA: H1 to H18
NA: N1 to N11
3

4. Highly pathogenic avian influenza
• OIE definition (Office International des
Epizotics)
– Intravenous pathogenicity index (IVPI) greater
than 1.2
– at least 75 percent mortality
• or
– multiple basic amino acids at the cleavage
site of the haemagglutinin molecule (HA0)
[OIE code] 4

5. 5
Cleavage of HA0 is necessary for virus Entry:
HPAIV has multiple basic amino acid at the HA
cleavage site but LPAIV doesn’t
LPAIV HA1 Trypsin HA2
Respiratory and enteric tracts
Local infection
HPAIV protease
Systemic infection
Fusion peptide
RRKRR
R

6. 6
The HA cleavage is hindered by
carbohydrate molecules
Glycosylation
at 11th amino acid
is close to
HA cleavage site

7. N-glycosylation at 11st of HA protects
cleavage site
• MERIVIAFAIISIVTG- signal peptide
• 11 12 13 NXT, NXS
• DRICIGYHANNSTKQVDTIMEKNVTVTHA
QDILEKEHNGRLCSLKGVKPLILKD-
• 327HA2
• -NVPQKKKR/GLFG
7

8. Disappearance of N-glycosylation loses
cleavage site protection
• MERIVIAFAIISIVTG- signal peptide
• 11 12 13
• DRICIGYHANNSKKQVDTIMEKNVTVTHA
QDILEKEHNGRLCSLKGVKPLILKD-
• HA2
• -NVPQKKKR/ GLFG
8

9. 9
The number of basic amino acids at the cleavage
site of HA affects pathogenicity
Strain A. a. Pathogenicity
Most avirulent RETR/GLF Avirulent
USA avirulent RKTR/GLF Avirulent (BBXR)
A/Penn/1/83 (H5N2) KKKR/GLF Avirulent
Penn/1370/83 (H5N2) KKKR/GLF Virulent
Scotland/59 (H5N1) RKKR/GLF Virulent
Italy/00 (H7)
Texas/04 (H5N2)
KKKKR/GLF Virulent
KRRRR/GLF
QRKKR/GLF HPAI (USDA)
LPAI (Texas)Clin Microbiol Rev 14:129
13
12
11-CHO
10

10. Outline
• Avian influenza virus
• Evolution of H5N1 AIV
• Risk factors for virus persistence and
pathogenicity
• The appearance of H5N8
• Avian influenza outbreaks in Taiwan
• Transboundary cooperation in the Asian
Pacific region
10

11. Strain Gs/Gd is the ancestor of recent H5N1
11

12. H5N1 evolution in China
12
http://www.ncbi.nlm.nih.gov/
A/goose/Guangdong/1/1996
A/Henan/09/2015

13. H5N1 persistence in Asia
13
[WHO, 2014]

14. AIVs transmitted by migrating birds,
wild bird trade, and poultry
14

15. Outline
• Avian influenza virus
• Evolution of H5N1 AIV
• Virus persistence and pathogenicity
• The appearance of H5N8
• Avian influenza outbreaks in Taiwan
• Transboundary cooperation in the Asian
Pacific region
15

16. AIV-circulation can be significantly reduced
by closing the live poultry market
• Temporary LPM closure
• Periodic rest days
• Market depopulation
overnight
• Improved hygiene and
disinfection.
16
[Offeddu, 2016][Mario Ruckh]

17. Impact of incoming poultry prevalence on
the effectiveness of rest days in LPMs.
17
[Offeddu, 2016:55]

18. Pahtologic changes from H5N2 LPAIV to
HPAIV in chicken farms
PQREKR
PQRKKR
PQRRKR/)
18

19. Date County Poultry virus HA0 cleavage/IVPI
2003.12 Changh Layer 0312
-REKR-
IVPI=0.0
2012.1.4 Changh Layer 12010
–RKKR-
IVPI=2.01
2012.2.9 Tainan Breeder A1997
-RRKR-
IVPI=2.53
2012.5.8 Yunlin Native 1205
-RRKR-,
IVPI=2.91
Pathogenicity of H5N2/TW viruses
19

20. H5N2 AIV increases pathogenicity
after embryo passages
Passage ICPI IVPI IVPI HA
cleavage
sequence
0 0.19 0.0 REKR
5 0.71 0.01 REKR
10 1.64 0.45 REKR
20 1.64 NT RRKKR
30 1.61 NT RRKKR
40 1.61 1.45 REKR
50 1.88 1.92 REKR
[Cheng, Wang, 2011]20

21. Plaque forms and increases in size
by H5N2 after embryo passages
Passage Plaque size with
trypsin
Plaque size
without trypsin
0 1.6 0
5 1.0 0
10 1.4 1.0
20 2.0 1.0
30 2.0 1.0
40 2.0 1.2
50 2.0 1.2
[Cheng, Wang, 2011]21

22. Vaccines might push higher H5N1
mutation
Country Date Vaccine
used
Genetic
clade
Evolutio
nary rate
Selected
sites
Egypt 2006-10 Yes 2.2.1 5.36 9
Egypt A 2007-10 Yes 2.2.1 4.07 4
Egypt B 2007-10 Yes 2.2.1 8.87 5
Indonesia 2003-07 Yes 2.1 6.13 6
Nigeria 2006-08 No 2.2 5.20 2
Turkey 2005-08 No 2.2 4.04 0
Thailand 2004-08 No 1 2.52 3
[Cattoli, 2011:9368]
Evolutionary rate: 10*-3 sub/site/year. P = 0.17
22

23. Outline
• Avian influenza virus
• Evolution of H5N1 AIV
• Virus persistence and pathogenicity
• The appearance of H5N8
• Avian influenza outbreaks in Taiwan
• Transboundary cooperation in the Asian
Pacific region
23

24. The appearance of H5N8: a novel H5 HPAIV
HA clade 2.3.4.4
24
[de Vries, 2015:842]
HA reassorts with different NA
Antigenicity difference
R-7 vaccine protects only 60%
High mortality in geese

25. H5N5 virus (A/duck/Guangdong/wy24/2008) is the
first detected reassortant subtype within this clade
25[de Varis, 2015]

26. Clade 2.3.4.4 is widely spread in the world
Verhagen, 2015 26

27. Outline
• Avian influenza virus
• Evolution of H5N1 AIV
• Virus persistence and pathogenicity
• The appearance of H5N8
• Avian influenza outbreaks in Taiwan
• Transboundary cooperation in the Asian
Pacific region
27

28. AI outbreaks in poultry farms in
Taiwan
• 1. H6N1 since 1972
• 2. H5N2 LPAI outbreaks in chicken farms
in Dec. 2003 - Mar. 2004. H5N2 HPAI
appeared in chicken farms
• 3. The appearance of novel H5 HPAI in
Taiwan in 2015
28

29. Phylogenetic tree of H6N1 in
Taiwan
29[Wang, exp]

30. PB2 E627K is critical for virus growth
and plaque size in MDCK cells
• E627K: Mutation of the 627 of PB2 from E to K
30[Wang, exp]

31. Discovery Studio shows the HA
structure of Taiwan H6N1
31

32. Check the location of amino acid
change
32

33. 33

34. 34
HA1 changes more than HA2
[Wang, 2011]

35. H5N2 avian influenza
outbreaks - in Dec. 2003
to Mar. 2004
•Low pathogenic avian
influenza (LPAI) H5N2 virus
caused outbreaks in 21 chicken
farms in December 2003 to
March 2004.
•No relationship with wild bird
H5N2.
•Sequence is similar to Mexican
vaccine strains
35

36. Epi – curve of H5N2 LPAI in 2004-2012
2004 2008 2010 2011 2012
Year
No. of cases
36

37. 37

38. 38
(American lineage)
(Eurasian
lineage)
2012/12/13
Phylogenetic tree
of HA gene
CK/TW/2004
CK/Tw/2010-2012
CK/Tw/2008-2012
CK/TW
DK/TW/H5N2
Smuggled bird/H5N1
WB/Tw/H5
38

39. Genetic analysis of the eight segments of
Tw/H5N2 AIVs
American lineage
strain(s)
-Mexican-like viruses
Eurasian lineage
strain(s)
-H6N1/TW-like
viruses
Taiwan H5N2 AIV
 Tw/H5N2 AIV was a
reassortant whose HA
and NA gene segments
belonged to the
American lineage and
the internal genes to the
Eurasian lineage.
39

40. Novel H5 outbreaks in Taiwan in
2015
40

41. 41
HA phylogenetic tree of AIVs in 2015
Two clades
Novel H5
Classical
H5

42. Transportation of geese spread the virus
H5 viruses from
different counties
show the same sequence
H5 spreads from one
county to another via
transportation
42

43. HPAI outbreak
in Taiwan in 2015
Totally confirmed 965 farms
novel H5N8 210
Novel H5N3 19
Novel H5N2 413
nH5N8 & nH5N2 66
nH5N2 & nH5N3 2
Slaughterhouse 22
nH5N2, nH5N3, H5N8 131
cH5N2 land fowl 57
nH5N2, cH5N2 land fowl 1
H5 water fowl 25
H5 land fowl 21
43

44. 44 44
Multi-basic amino acid in cleavage sites of
Novel H5 HPAI viruses
Cleavage site Critical basic aa Phenotype
A/Goose/Taiwan/0103 (H5N8) PLRERRRKR/GLF 5 HP
A/Goose/Taiwan/0104 (H5N2) PLRERRRKR/GLF 5 HP
A/Goose/Taiwan/01042 (H5N3) PLRERRRKR/GLF 5 HP

45. Clinical signs in different poultry in
novel H5 HPAI in Taiwan 2015
45

46. Geese
46

47. 47
Ducks

48. H5N8 HPAIV is poorly chicken
adapted
• high chicken infectious dose50 = 105.7
EID50 vs 102.4 EID50 of A/hHong
Kong/486/1997 (H5N1)
• A limit blood vessel endothelial cell
replication
• Poorly field transmissible (less than
transmission cutoff 104.7 EID50)
48
[Bertran, 2016:190]

49. 49
Chickens

50. Outline
• Avian influenza virus
• Evolution of H5N1 AIV
• Risk factors for virus persistence and
pathogenicity
• The appearance of H5N8
• Avian influenza outbreaks in Taiwan
• Transboundary cooperation in the Asian
Pacific region
50

51. Wild birds in duck and chicken farms
51

52. Chinese bulbuls get AIV H5N3
infection in Taiwan in Jan 2015
52

53. Night heron gets AIV H5N2
infection in Taiwan in Jan 2015
53

54. Black spoon bill gets AIV nH5 infection
in Taiwan
54

55. 55
Sampling by Taipei Wild Bird Society
Bird fecal sample collections
since 1998 till now

56. 56
Active surveillance in wild birds in Taiwan
Year Ducks Shore
birds
Gulls Egrettas Others Total
1998 1,457 0 0 30 0 1,487
1999 1,418 75 0 0 0 1,493
2000 1,825 44 0 0 0 1,869
2001 2,516 45 3 0 0 2,564
2002 2,060 652 72 0 98 2,882
2003 1,831 553 59 0 0 2,443
2004 1,902 1,149 190 4 106 3,351
2005 2,806 1,357 112 38 194 4,507
2006 2,352 1,413 161 436 179 4,541
2007
2008
2009
2010
2011
2,645
2,768
2,755
2,791
2,301
1,147
920
835
754
1,068
20
80
57
79
40
317
389
417
504
326
21
108
91
18
26
4,150
4,265
4,155
4,246
3,761
Sum 31,427 10,034 873 2,461 841 45,714
isolates 340 14 2 6 1 363
Prevalence
(%) 1.08 0.14 0.24 0.23 0.24 0.80
Detected subtypes
H1N1, H1N2, H1N3,
H2N3, H2N7, H2N9,
H3N2, H3N6, H3N7, H3N8, H3N9,
H4N2, H4N3, H4N5,H4N6, H4N7, H4N8,
H5N2, H5N6,
H6N1, H6N2, H6N5, H6N9,
H7N1, H7N2, H7N3, H7N5, H7N6, H7N7, H7N9,
H8N3, H8N4,
H9N6, H9N9,
H10N1, H10N2, H10N3, H10N4, H10N6, H10N7,
H10N8, H10N9,
H11N3, H11N9,
H12N2,
H14N7
All isolates are LPAIV
Subtype Cleavage site of HA
protein
H7 PEIPKGR*GLF
H5 PQRETR*GLF

57. Five million birds use the East Asia-
Australasian Flyway (blue)
[U.S. Fish and Wildlife Service/Alaska/Wikimedia]
[OneOcean.org] 57

58. Some migrating birds appear in NTU
campus only in winter each year
58
a heron

59. Extensive surveillance for HPAIV in wild birds is
essential through transboundary cooperation in
the Asian Pacific region countries
59
http://www.oneocean.org/

60. The distribution of Chinese Crested-
tern (Sterna bernsteini)
http://www.birdlife-asia.org/eng/chinese_crested_tern/

61. Conclusion
• The unprecedented epidemic of HPAI is not
yet under control
• The novel H5 AIV has different antigenicity
and different pathogenicity
• The novel H5 HA is very aggressive to re-assort
with other genes, forms H5N8, H5N3 and
H5N2
• Migrating birds are to blame for spreading
• Cooperation among countries
61

62. Thanks for your attention
62