Ipi 2155

HAYATI Journal of Biosciences June 2011
Vol. 18 No. 2, p 82-90

EISSN: 2086-4094
Available online at:

http://journal.ipb.ac.id/index.php/hayati
DOI: 10.4308/hjb.18.2.82
Influenza H5N1 Virus of Birds Surrounding H5N1 Human Cases

Have Specific Characteristics on the Matrix Protein
NI LUH PUTU INDI DHARMAYANTI1, FERA IBRAHIM2, DARMINTO1, AMIN SOEBANDRIO2,3
1
Indonesian Research Center for Veterinary Science, Jalan RE Martadinata 30, Bogor 16114, Indonesia
Microbiology Department, Medicine Faculty, Universitas Indonesia, Jalan Salemba Raya No. 6, Jakarta 10430, Indonesia
3
Kementerian Riset dan Teknologi, Jalan MH Thamrin No. 8, Jakarta 10340, Indonesia
Received October 20, 2010/Accepted June 9, 2011

The H5N1 influenza virus in Indonesia has caused more than 100 people died due to the virus infections.
Cases in humans were mostly due to the virus spread from the infected birds. This study characterized molecularly
the H5N1 virus from birds around the H5N1 infection cases in humans in Indonesia. Result from this study
revealed that in several cases, waterfowl species could become the source of H5N1 infections in human. We found
that the one of six viruses used in this study probably was a first antigenic shift virus in Indonesia. This study
shows that the AI viruses isolated from birds around humans infected by H5N1 virus has specific characteristics
namely the presence of several amino acid substitutions especially on the M1 and M2 proteins. The substitutions
are similar in most of H5N1 human cases in Indonesia.
Key words: molecular character, H5N1 virus, birds, surrounding, human H5N1 infection
___________________________________________________________________________
INTRODUCTION
The highly pathogenic avian influenza (HPAI) H5N1
virus was known for the first time in 1997, when the virus
was directly transmitted from birds to human in Hong Kong
and caused 18 respiratory disease cases including 6 people
died due to the disease (Claas et al. 1998; Suarez et al.
1998; Shortridge et al. 1998; Bender et al. 1999; Katz et al.
2000; Ungehusak et al. 2005). Then at the beginning of
2003, two H5N1 infection cases were identified in Hong
Kong (Peiris et al. 2004). Since the end of 2003, the HPAI
H5N1 has spread up to Central Asia, Europe and Africa,
causing endemic disease and death of domesticated birds
and wild birds. Until September 2006, approximately 40
laboratories have confirmed the human cases infected by
H5N1, and more than 50% of the infections in humans
were fatal (Centers for Disease Control and Prevention
2004; Hien et al. 2004).
Continuous exposure to the H5N1 virus on humans
will increase the possibility of the influenza pandemic in
humans. The avian virus can adapt more efficiently in
human through the reassortment with other influenza
strains in humans (Webster et al. 1992; Taubenbarger et
al. 2005). Several H5N1 infection cases were family
clusters; however, it can be stated that the virus
transmission from human to human is still very limited
(Ungehusak et al. 2005). The viral transmission inter human
has not yet been proven, thus most cases in humans
occurred due to virus spread from infected birds (World
Health Organization 2005a). Genetic analysis shows that
most of the H5N1 influenza viruses from birds and human
_________________
Corresponding author. Phone: +62-251-8331048,

Fax: +62-251-8336425, E-mail: nlpdharmayanti@yahoo.com
in Asia are the Z genotype, similar to the virus first

identified in South China (Guan et al. 2004; Li et al. 2004;
Puthavathana et al. 2005).
In Indonesia, since the first outbreak in the late 2003,
the H5N1 virus has rapidly became endemic (Smith et al.
2006; Sedyaningsih et al. 2007) and continued to cause
sporadic zoonotic transmission to human beginning in
July 2005 (Sedyaningsih et al. 2007). Avian influenza (AI)
is still a serious case requiring attentions considering the
number of victims died due to the infection till now. At
least, until 27 January 2009 there were 115 people died
due to this virus (World Health Organization 2009). The
data on the characters and genetic information related to
died victims caused by H5N1 virus infections of birds
origin in Indonesia are still limited. In this study, we
reported the molecular character of viruses isolated from
birds around the homes of human cases of H5N1.
MATERIALS AND METHODS
AI Viruses. During 2006-2007, six H5N1 viruses from
the avian species arounding human cases infected by AI
virus subtype H5N1 have been isolated (Table 1). Field
data of the viruses were identified to determine the avian
species origin of the viruses that can cause the H5N1
human infection.
The H5N1 viruses were propagated in 9-11 old day
embryo specific pathogen free (SPF) eggs (OIE 2000).
These six viruses were then further analyzed on four
segments namely hemagglutinin (HA), neuraminidase
(NA), matrix (M), and nonstructural (NS).
DNA Sequencing and Analysis of Influenza Virus
Genes (HA, NA, M, and NS). To identify the genetic
Vol. 18, 2011
H5N1 Virus on Birds Surrounding H5N1 Human Cases
characteristic of HA, NA, M, and NS of the H5N1 viruses,

we conducted sequencing for those genes. The sequences
of four genome segments of AI viruses (HA, NA, M, and
NS genes) of the six AI viruses of bird origin isolated
around cases in humans infected with AI in 2006-2007
were compared with the sequence data of human H5N1
virus available at GenBank (www.ncbi.nlm.nih.gov). The
information of the viruses can be seen in Table 1.
The strategy to amplify full length HA was using
primer Senne et al. (1996) to amplify HA1 region, and HA2
was modified using H5-155F (Lee et al. 2001) and NS890
primers published by Hoffman et al. (2001). Primers for
NA gene was from Komadina (2006, personal
communication), while the amplification of M and NS genes
used primer Hoffman et al. (2001). The PCR products were
separated in 1% agarose by electrophoresis and the
amplicon was excised and purified using QIAquick gel
purification kit (Qiagen). The sequencing method used
was direct sequencing using Cycle sequencing kit (BigDye
Terminator version 3.1; Applied Biosystem) on Genetyx
Analyzer 3130 (Applied Biosystems, USA).
The nucleotide sequencing data obtained in this study
were analyzed together with the genetic data available in
the avian influenza database (NCBI) based on each gene.
The production of multiple alignments each gene and the
residue analysis was carried out by using BioEdit version
7 (http://www.mbio.ncsu.edu/BioEdit). Phylogenetic trees
were generated by neighbor-joining bootstrap analysis
(1,000 replicates) by using the Tamura-Nei algorithm in
MEGA version 4 (Http: //www.megasoftware.net). All of
the viruses used in this study have been submitted to
GenBank (www.ncbi.nlm.nih.gov) with accession number:
83
A/Muscovyduck/Jakarta/Sum106/2006 (GU183453,
GU183472, GU183434, GU183414), A/Duck/Jakarta/
Slmt306/2006 (GU183454, GU183473, GU183435,
GU183415), A/Muscovyduck/West Java/Bks3/2007
(GU183455, GU183474, GU183436, GU183416),A/Ck/Pessel/
BPPVRII/2007 (GU183456, GU183475, GU183437,
GU183417), A/Ck/Inhu/BPPVRII/2007 (GU183457,
GU183476, GU183438, GU183418), A/Ck/Jakarta/DKINurs/2007 (GU183458, GU183477, GU183439,
GU183419).
RESULTS
The Avian Species Origin of the Viruses. All of the
victims in this study were infected by the A1 viruses after
direct contact with sick birds (Table 1). To collect the
viruses from the birds correlated with the human AI
infection in Indonesia relative difficult. One of the
problems is when AI human case accurs, the government
is directly to disinfect the location and stamping out the
birds before conducting investigation. During 2006-2007,
we just isolated six isolates and three of the viruses (A/
Muscovy Duck/Jakarta/Sum106/2006, A/Duck/Jakarta/
Slmt306/2006, A/Muscovy Duck/West Java/Bks3/2007)
were isolated from waterfowl (duck and muscovy duck).
In this case, muscovy duck is an infection source for the
AI infection in humans. Muscovy duck as the viral
reservoir did not show any clinical symptoms until the
presence of AI cases in human at that site (FJ and N,
Table 1) was known. From cloacal swab samples taken
from duck, we detected AI virus subtype H5N1 and then
Table 1. Samples taken from birds surrounding H5N1 cases in humans

Initiais Age Sex
Agreed
District Province Onset
occupation
Outcome
Date
death
Status
Human virus
code
Notes
Exposure
Urban
or no
Animal
virus
FJ
Student
South
Jakarta
DKI
Jakarta
7-9-06
Died
22-9-06
Confirmed CDC835
Poultry Urban A/Muscovy

deaths
Duck/
at
Jakarta/
Sum106/
home
2006
A/Duck/
Jakarta/
Slmt306/
2006
RI
14
Student
West
Jakarta
DKI
Jakarta
31-12-06 Died
10-1-07
Confirmed CDC887
**
Handled Urban A/Ck/

sick/
Jakarta/DKIdead
Nurs/2007
poultry
12
Student
Bekasi
Weat
Jakarta
12-1-07
No
No
Confirmed No
information information
information
***
Handled Rural
sick/
dead
poultry
A/Muscovy
Duck/West
Java/Bks3/
2007
14
Student
Pasir
Selatan
West
15-3-07
Sumatra
24-3-07
Died
Confirmed CDC1031
****
Slaughter Rural
sick/
dead
poultry
A/Ck/
Pessel/
BPPVRII/
2007
Yt
26
Paim
plantation
worker
Indragiri Riau
Hulu
12-6-07
Died
Confirmed No
information
*****
Slaughter Rural
sick/
dead
poultry
A/Ck/Inhu/
BPPVRII/
2007
3-6-07
*: The case had contact with sick chickens (his pets) in his household; **: Case had direct contact with bird/poultry (ducks on 24 Dec 06), since 1 month before
investigation bird/poultry in environment (owned by nelghbors) began dying and were disposed located near area were cases family caged their birds/poutry; ***: Case
had direct contact with sick birds belong to the neighborhood on 7 January 2007; ****: On 4 March 2007, chicken started dying in the neighborhood (brother,s house).
On 9 March 2007, case slaughtered and cleaned two sick chickens. The case lives in rural area 9 hours away from Padang; *****: Symtoms: cough, headache, fever.
84
DHARMAYANTI ET AL.
isolated it. This case showed that reared waterfowl could

be a source of H5N1 infection in humans.
Molecular Characteristics of HA Protein.
Phylogenetic analysis of HA gene revealed that all of the
viruses used in this study belong to group 1 (Figure 1).
Based on the amino acid sequences on HA cleavage sites,
the six viruses used in this study have multiple basic amino
acids indicating that the viruses are highly pathogenic.
The four AI viruses used in this study had the substitution
on -6 of HA1 protein i.e. R S and one virus had
substitution RG, while the other one did not undergo a
substitution (Table 2). All viruses were analyzed in this
study had the preferential binding of sialic acid joined
with the sugar chain through 2,3 linkage as they had
glutamic residues at position 222 (at position 226 for H3
virus) and glysine residues at position 224 (at position
228 for H3 virus) that are avian receptors (Stevens et al.
2006). Seven potential glycosylation sites of HA1 at amino
acid positions 11, 29, 84, 154, 165, 193, and 286 were
maintained among human and bird isolates excluding
Pessel/BPPVRII/07. Mutation T/S156A at the Pessel/
BPPVRII/07 results in the loss of glycosylation sites (Li et
al. 2004; Chen et al. 2006). Glycosylation at the HA plays
a role in antigenic variation by masking and unmasking
antigenic site. In this study, Jakarta/DKI-Nurs/07 and West
Java/Bks3/07 viruses had no glycosylation site at position
165 and the Jakarta/DKI-Nurs/07 virus also had none at
position 193. Thus, Jakarta/DKI-Nurs/07 and West Java/
Bks3/07 viruses only have 4 and 6 glycosylation sites at
the HA1 respectively (supplemental data).
Molecular Characteristics of NA Protein. At the NA
gene level, the six viruses used in this study were closely
related to the H5N1 human origin virus and different from
virus Gs/Gd/96 or HK/483/97 (data not shown). Two
viruses from Sumatra, i.e. Riau and West Sumatra, (A/Ck/
Pessel/BPPVRII/07 and A/Inhu/BPPVII/2007) have genetic
relationship with H5N1 viruses from birds isolated from
Riau and West Sumatra such as the A/Ck/IDN/
PekenBaru161-11/06, A/Ck/Agam1631-2/06 and A/Ck/IDN/
Padang1631-1/06 viruses. Indonesian H5N1 viruses
including the six viruses in this study had 20 amino acid
deletions in the stalk region at positions 48-68 (data not
shown).
Molecular Characteristics of NS Protein. The PDZbinding motif of NS1 is a new virulence factor of influenza
A viruses (Jackson et al. 2008). The motif can bind to
cellular PDZ-containing proteins involved in host cellular
signaling pathways. Human influenza viruses contain
different such as RSKV or RSEV and ESEV or EPEV motif
belong to avian origin. From this study, four viruses
possessed the ESEV motif indicating that the viruses are
of avian origin. The two other viruses have another motif.
The Inhu/BPPVRII/07 virus showed KSEV motif and the
other hand Pessel/BPPVRII/07 virus possessed human
influenza motif i.e. RSEV motif.
Multiple alignment analysis on NS1 showed that the
deletions of amino acids at positions 80-84 in five isolates
(Table 2), excluding the Pessel/BPPVRII/07 virus. The five
amino acid deletions also characterize other H5N1 viruses
HAYATI J Biosci
of the Z+, Z, Y, A, B, and C genotypes and may contribute

to the increased virulence (Long et al. 2008).
Using pair wise alignments the Pessel/BPPVRII/07 virus
showed that nucleotide identities between Pessel/
BPPVRII/07 or Hong Kong virus and five viruses used in
this study were about 86-88% and 84-88% for amino acid,
respectively, even though the identity of NS1 among the
five viruses used in this study was 97-99% for nucleotide
and 95-99% for amino acids sequence (data not shown)
and located in same group with HK/497/97 and HK/498/97
and China virus group (Figure 1b). This is very interesting
because the virus isolated from indigenous chicken
outbreak around the human H5N1 case in Riau Province,
Sumatra Island is in one cluster with Indonesian H5N1
virus on the HA, NA, and M gene, but slightly different
on NS1 gene, because the virus is similar to Hong Kong
viruses especially H3N2 viruses namely HK/497/97 and
HK/498/97.
Pessel/BPPVRII/07 and H3N2 Hong Kong viruses have
8 similar amino acids at the positions 22, 70, 81, 114, 127,
207, 215, and 227 (supplemental data). This result
demonstrates that Pessel/BPPVRII/07 virus has HA, NA,
and M genes belonging to Indonesian H5N1 viruses
originated from avian but for NS1 gene belongs to Hong
Kong viruses which is a human influenza virus; hence
Pessel/BPPVRII/07 virus is probably a reassortment virus.
Molecular Characteristics of M Protein. The six
viruses used in this study demonstrated M2 mutation of
V27A indicating their resistance to amantadine (data not
shown). The AI viruses from birds isolated from around
H5N1 human cases possessed specific amino acids similar
to amino acid sequence of H5N1 human viruses at the M
protein level. The amino acid on the M1 protein namely
Ala/A, Lys/K, Ala/A, and His/H at positions 37, 95, 137,
and 249 respectively were identified from human-origin of
H5N1 virus or from birds around H5N1 human cases at
the location. All of the viruses had four specific amino
acid excluded Jakarta/DKI-Nurs/07 virus, the virus had R
at position 249 replacing H. On the other hand, most of
the H5N1 viruses from bird origin had the amino acids
composition of Thr/T, Arg/R, Thr/T, and Gln/Q at the above
mentioned positions.
On the M2 protein, though it is not as specific as in
the M1 protein, several amino acids were possessed
exclusively by the bird origin virus surrounding AI cases
in humans, which are Tyr/Y, Lys/K, Ile/I, Ala/A, and Phe/
F (at positions 8, 18, 19, 27, and 50 respectively) except for
the DKI-Nurs/07 virus that had only 2 amino acids from
the amino acid motifs above (A and F amino acids) (Table
3).
DISCUSSION
The molecular character of the avian viruses isolated
from around H5N1 human cases on the cleavage site
region of HA protein showed that the geographical
variation in cleavage site motif may occur and it is not
related to the virulent changes and infections in humans
(Writing Committee of Second World Organization
Vol. 18, 2011

6 7 A/ Ind o nes ia/ TL L0 10 /2 00 6
A/ Ind o nes ia/ 54 2H/ 20 06
A/In do nes ia/ CDC6 10 /2 00 6
A/I nd on es ia/C DC8 35 /20 06
A/M u sco vyd uc k/Ja ka rta /DKI -Su m1 06 /2 00 6* * *
78
A/ Chic ken /In do ne sia /Ba nd un g1 63 14 9/2 00 6
A/M us co vy D uck /Ja kar ta /HABW IN/2 00 6
95
A/In do ne sia /CDC 73 9/2 00 6
A/In do ne sia /CDC6 69 /2 00 6
92
A/In do ne sia /CDC7 59 /2 00 6
A/ Chic ken /In d on esia /G ar ut1 63 15 1/ 20 06
A/ Qu ail/J ak ar ta/J U1 /20 06
A/C hic ken /W est Jav a/T ASIKSO L/ 20 06
83
A/I nd on esi a/CD C6 99 /20 06
A/In do nes ia/ CDC1 03 1/ 20 07
72
A/M us cov ydu ck/W es t J ava /Bks3 /2 00 7* * *
76
A/I nd on esia /CD C88 7/ 20 06
99
97 A/I nd on esia /CD C10 47 /2 00 7
A/I nd on esi a/CD C10 46 /2 00 7
82
A/In do ne sia /T LL 01 2/2 00 6
98
94 A/In do ne sia /CDC1 03 2/ 20 07
A/ Ind on es ia/C DC6 44 /2 00 6
A/In do nes ia/ 56 7H/ 20 06
84
A/ Chic ken /In d on esia /L am pu ng 16 31 23 /2 00 6
A/Ch ick en/ In do nes ia/ Sem er an g1 63 16 2/ 20 07
79
A/Swa n/ Ind on es ia/M a lan g1 63 16 1/ 20 07
90
9 9 A/ Ind on es ia/T L L0 09 /20 06
A/ Ind on es ia/C DC3 70 /20 06
A/ Ch ci ke n/W est Ja va/ SMI ENDRI 1/2 00 6
68
A/ Pige on /In do ne sia /Ro khit 16 31 6/2 00 6
6 6 A/ Chic ken /In d on esia /Pa da ng 16 31 1/2 00 6
85
A/ Chic ken /In d on esia /Sia k16 31 2/ 20 06
96
A/ Chic ken /In d on esia /Ag am 16 31 3/ 20 06
76
A/Ch icke n/ Ind on es ia/ Peke nb ar u1 63 11 1/ 20 06
99
A/I nd on es ia/T L L0 08 /20 06
65
A/M us co vy Du ck/ Ind on es ia/ Ked ri1 63 12 4/2 00
98
A/ Ch ci ke n/W est Ja va/ SMI PAT/ 20 06
65 A/ Ch ci ke n/W est Ja va/ PW T WIJ /20 06
A/C k/J aka rta /DKI -Nu rs /20 0 7* **
99
A/c hick en /We st Jav a/SM ICS LKEB/2 00 6
68 A/C hick en /We st J ava /SM IC SL KEC/ 20 06
A/ Ind on es ia/C DC2 92 N/2 00 5
A/ Ind on es ia/2 45 H/ 20 05
79
A/Ch ick en/ In do nes ia/ Belitu ng T im or 16 31 18
60
A/ch ci ke n/W es t Ja va/ TASI KSOB/2 0 06
A/Ch icke n/W es t Ja va/ T ASIK2/ 20 06
A/fe line /In do ne sia /CDC 1/2 00 6
66
A/In d on esia /2 83H /2 00 6
64
79
A/In do nes ia/ 5/2 00 5
A/Ch icke n/ Paku n Bar u/BPPVI I/2 00 5
94 A/Ch icke n/ Mu ra o J am b i/BBPVII/ 20 05
A/ Ind on es ia/1 75 H/2 00 5
A/Duc k/I ndr a ma yu /BBPW1 09 /20 06
99 A/In do ne sia /CDC 18 4/2 00 5
99
A/In do ne sia /16 0H /20 05
A/In do ne sia /CDC 7/2 00 5
77
A/In do ne sia /T LL 00 2/2 0 06
99
A/ Ind on es ia/T L L0 01 /20 06
67
A/In do ne sia /7/ 20 05
A/D uck /Ja kar ta /DKI- Slm t30 6/ 20 06 ** *
A/Ck /Pes sel/ BPPVRII/ 20 07 ** *
A/Ck /In hu /BPPVRII /2 00 7* * *
63
A/C hic ken /Pa pu a/T B1 /20 06
99
A/C hic ken /Pa pu a/T B1 5/2 00 6
A/C hic ken /Pa pu a/T A5 /20 06
A/ Ch ci ken /In d on esia /W ate s1/ 20 05
A/Ch ick en/ In do nes ia/W at es1 30 /2 00 5
A/Ch icke n/I nd on esi a/W ate s1 26 /20 05
81 A/c hick en /In do ne sia/ CDC 24/ 20 05
A/c hick en /In do ne sia/ CDC 25/ 20 05
A/Ch icke n/W ay Kan an /BBPVIII /20 0 6
97
A/Ch icke n/B and ar L am pu ng /BBPVI II/2 00 6
99 A/Ch icke n/ Ind on es ia/B ang ka Sel eta n1 63 12 1
A/Ch icke n/ Ind on es ia/B ang ka Sel eta n1 63 12 0
A/Ch ick en/ Sem b awa /BPPVII I/2 00 5
A/C hick en /Pa lem ba ng /BPPVII I/2 00 5
65
6 9 A/I nd on es ia/6 /20 0 5
A/Ch icke n/ Ma diu n/B BVW 14 20 /2 00 5
A/C hic ken /In do ne sia /M ag ela ng 16 31 57 /20 07
66
A/ Chic ken /In d on esia /Kulo n 16 31 47 /20 06
98
99
A/ Chic ken /G un un g Kidu l/BBVW/ 200 6
A/Q ua il/Ce ntr al Ja va/S MRG /2 00 6
A/ Chic ken /In do ne sia /Sop pe n g1 63 17 1/2 00 7
99
A/Ch icke n/W es t Ja va /GAR UT MAY/ 20 06
64
A/Ch ick en/ In do nes ia/W at es8 0/ 20 05
A/Du ck/ T aba na n/ BPPV1/ 20 05
99
A/Du ck/P ali/BBVW 13 58 /20 05
A/D uck /Buf ele ng /BPPV1 /20 05
A/C hick en /We st J ava /HAM D/ 20 06
99
A/C hic ken /In do ne sia /Wa tes 77 /20 05
A/Ch ci ke n/I nd on esia /W ate s83 /2 00 5
A/Ch icke n/ Deli S er da ng/ BPPV1/ 20 05
80
A/c hic ken /M ed an /BPPV1 53 4/2 00 5
A/Ch icke n/Pid ie/ BPPV1/2 00 5
62
A/Ch icke n/M e da n/BBP V15 71 /20 05
A/Ch icke n/ Kar o/BBPVI I/2 00 6
A/C hick en /De li De rd an g/ BBPVI/2 00 5
A/C hick en /M ed an /BPPV1 49 8/2 00 5
A/C hic ken /M ed an /BBPV1 57 6/2 00 5
95
A/C hick en /T ap ut/ BBPV15 76 /20 05
99 A/In do nes ia/ CDC5 97 /2 00 6
99
91
95
A/ T urk ey /La ng ka t/BBPVI/ 20 05
A/Ch icke n/ La ng kat /BBPV15 76 /2 00 5
A/C hick en /Aga m /BBPVI/2 00 5
A/C hick en /Pul au Ram p an g/BBP VI I/2 00 6
91
A/C hick en /Pad an g /BBPVII/ 20 06
A/Ch icke n/Sia k/BPPVI I/2 00 5
A/Ch icke n/R oka n Hilli/BPPVII /20 05
A/C hick en /Du m a/BBPVII /20 0 5
Group 1 (Indonesian human

H5N1 viruses 2006-2007)
68
98
64
92
61
76
90
0.0 05
Figure 1a. Phylogenetic trees of the H5N1 viruses.
Group 2 (avian H5N1 viruses)
85
86
DHARMAYANTI ET AL.
HAYATI J Biosci
A/chicken/Pangkalpinang/BPPV3/2004
89
A/turkey/Kedaton/BPPV3/2004
A/chicken/Jembrana/BPPV6/2004
A/chicken/Ngawi/BPPV4/2004
A/chicken/Wonosobo/BPPV4/2003
A/Ck/Indonesia/BL/2003
A/Ck/Indonesia/5/2004
A/chicken/Bangli Bali/BBPV6-1/2004
A/chicken/Bantul/BBVet-I/2005
A/chicken/Mangarai-NTT/BPPV6/2004
A/chicken/Kulon Progo/BBVW/2005
A/quail/Boyolali/BPPV4/2004
A/Ck/Indonesia/2A/2003
64 A/Dk/Indonesia/MS/2004
A/chicken/Pekalongan/BPPV4/2003
A/chicken/Sragen/BPPV4/2003
A/Ck/Indonesia/PA/2003
A/quail/Tasikmalaya/BPPV4/2004
A/chicken/Kulon Progo/BBVet-XII-2/2004
A/chicken/Yogjakarta/BBVet-IX/2004
76
A/quail/Yogjakarta/BBVet-IX/2004
A/chicken/Purworejo/BBVW/2005
A/chicken/Indonesia/CDC25/2005
A/chicken/Gunung Kidal/BBVW/2005
A/Indonesia/CDC184/2005
A/duck/Parepare/BBVM/2005
66
A/chicken/Wajo/BBVM/2005
A/chicken/Magetan/BBVW/2005
82
63 A/Indonesia/341H/2006
A/Indonesia/298H/2006
A/Indonesia/CDC370E/2006
A/Indonesia/5/2005
66 A/Indonesia/CDC194P/2005
98 A/chicken/Malang/BBVet-IV/2004
A/chicken/Purwakarta/BBVet-IV/2004
A/chicken/Kupang-1-NTT/BPPV6/2004
A/Vietnam/CL01/2004
68
A/Vietnam/CL119/2005
61
A/duck/Vietnam/258/2004
87
A/chicken/Vietnam/5/2003
A/Thailand/1(KAN-1)/2004
A/mallard/Vietnam/347/2005
62
A/chicken/Salatiga/BBVet-I/2005
98 A/Indonesia/CDC594/2006
61
A/chicken/Tebing Tinggi/BPPVI/2005
67
A/chicken/Dairi/BPPVI/2005
79 A/chicken/Tarutung/BPPVI/2005
A/chicken/Deli Serdang/BPPVI/2005
A/Muscovyduck/West Java/Bks3/2007***
A/Ck/Pessel/BPPVRII/2007***
A/Ck/Inhu/BPPVRII/2007***
94
A/Muscovyduck/Jakarta/DKI-Sum106/2006***
A/Duck/Jakarta/DKI-Slmt306/2006***
A/Ck/Jakarta/DKI-Nurs/2007***
0.005
Figure 1b. Phylogenetic trees of the M1 gene.
The H5N1
viruses
2003-2005
Group of
Indonesian
human
H5N1
viruses
2006-2007
Vol. 18, 2011
A/Muscovyduck/West Java/Bks3/2007***
61
61
87 A/Indonesia/CDC1032N/2007
A/Indonesia/CDC287E/2005
A/feline/Indonesia/CDC1/2006
A/Duck/Jakarta/DKI-Slmt306/2006***
A/Muscovyduck/Jakarta/DKI-Sum106/2006***
A/Ck/Jakarta/DKI-Nurs/2007***
65 A/Indonesia/CDC292T/2005
83 A/chicken/Indonesia/CDC24/2005
60 A/chicken/Indonesia/CDC25/2005
A/Ck/Inhu/BPPVRII/2007***
64
A/Indonesia/CDC194P/2005
A/chicken/Wajo/BBVM/2005
A/Indonesia/7/2005
A/Indonesia/5/2005
A/Ck/Indonesia/PA/2003
A/chicken/Salatiga/BBVet-I/2005
A/Ck/Indonesia/2A/2003
A/Dk/Indonesia/MS/2004
A/chicken/Bantul/BBVet-I/2005
A/Ck/Indonesia/BL/2003
A/chicken/Kulon Progo/BBVet-XII-1/2004
65
61 A/Indonesia/6/2005
A/chicken/Wonosobo/BPPV4/2003
A/chicken/Yogjakarta/BBVet-IX/2004
A/duck/Shanghai/13/2001
A/duck/Fujian/17/2001
10064 A/duck/Guangxi/35/2001
A/duck/Guangxi/50/2001
A/duck/Guangxi/13/2004
A/duck/Fujian/13/2002
A/Hong Kong/483/97
A/Hong Kong/514/97
A/duck/Vietnam/1/2005
A/duck/Vietnam/8/05
A/Hong Kong/532/97
95
A/Hong Kong/156/97
A/Hong Kong/542/97
A/Ck/Pessel/BPPVRII/2007***
74
84 A/Hong Kong/497/97
A/Hong Kong/498/97
73
79
100
87
Indonesian H5N1 viruses
A/Ck/Pessel/BPPVRII/2007
A/duck/Guangdong/12/2000
A/duck/Shanghai/08/2001
A/Goose/Guangdong/1/96
A/duck/Guangdong/07/2000
A/duck/Zhejiang/11/2000
0.1
Figure 1c. Phylogenetic tree of NS1 gene. Phylogenetic trees of the H5N1 viruses. a) Phylogenetic tree of the hemagglutinin (HA), b)
Matrix 1 (M1) and c) Non Structural (NS1) gene of the H5N1 viruses isolated from birds surrounding AI human cases in
Indonesia. The stars sign showed the viruses used in this study. Nt 49-1680 of HA gene, nt 1-1157 of The NA gene, nt 1-756
of M1 gene and nt 25-690 of NS1 gene were used for the analysis. The construction of phylogenetic using MEGA version
4. A neighbor-joining bootstrap analysis (1,000 replicates) using the KimuraNei model is shown.
88
DHARMAYANTI ET AL.
HAYATI J Biosci
Table 2. Genetic characters of H5N1 viruses of this study compared with H5N1 human closest isolates from Indonesia in GenBank
Viruses
CDC938/2006
CDC835/2006
CDC887/2006
CDC1031/2007
Bks3/2007
Pessel/BPPVRII/2007
Inhu/BPPVRII/2007
DKI-Nurs/2007
Sum106/2006
Slmt306/2006
222
Q
Q
Q
Q
Q
Q
Q
Q
Q
Q
HA sequence
224
G
G
G
G
G
G
G
G
G
G
at aa
Cleavage site
PQRESRRKKR
PQRESRRKKR
PQRESRRKKR
PQRESRRKKR
PQRESRRKKR
PQRERRRKKR
PQREGRRKKR
PQRESRRKKR
PQRESRRKKR
PQRESRRKKR
NS sequence
Deletion of aa 80 to 84
PDZ-binding ligand
YES
ESEV
YES
ESEV
ESEV
YES
ESEV
YES
YES
ESEV
NO
RSEV
YES
KSEV
ESEV
YES
YES
ESEV
YES
ESEV
M 2 sequence at aa
27
31
A
S
A
S
A
S
S
A
S
A
A
S
A
S
A
S
S
A
A
S
Table 3. Amino acid prediction at the M gene from H5N1 viruses of avian species from this study correlated with H5N1 human cases
Viruses
Sum106/2006
Slmt306/2006
DKI-Nurs/2007
Bks3/2007
Pessel/BPPVRII/2007
Inhu/BPPVRII/2007
Indonesian H5N1 viruses origin from avian species
Most Indonesian human H5N1 viruses 2006-2007
Amino
37
A
A
A
A
A
A
T
A
Consultation, 2008). The presence of glycosylation sites

at 154-156 from six viruses in this study showed that Z
strains of H5N1 isolated since late 2002 in Hong Kong,
Indonesia, Thailand, Vietnam, and Yunnan Province, China
in late 2003 and 2004 had acquired a potential N-linked
glycosylation site at the position. Glycosylation at this
site, adjacent to the receptor-binding and antigenic sites
at the globular tip of the H5 influenza HA molecules, is
capable of altering the receptor-binding and may help the
virus to evade the host antibody response (Li et al. 2004).
Gain or loss of glycosylation sites in HA clearly plays a
role in determining morphological characteristics of the
virus antigenicity and influences the selection of potential
vaccine strains, but the role these sites play in disease is
unknown (Bean et al. 1985).
Glycosylation sites in the stalk of the neuraminidase
play a role in maintaining the tetrameric structure of the
protein (Luo et al. 1993). All of the isolates from this study
have no glycosylation at the stalk of the NA due to the
deletion in the stalk region. Deletion in the stalk of the NA
is thought to increase the retention of virions at the plasma
membrane (Matrosovich et al. 1999) to balance weaker
binding of sialic acid receptor by the HA with newly
acquired N154 glycosylation (World Health Organization
2005b). The isolates in this study have conserved three
out of the four glycosylation sites present in the NA at
the amino acid positions 88, 145, and 235.
For the NS gene, a large-scale sequencing analysis of
avian influenza viruses indicates that the C-termini of avian
influenza virus NS1 proteins have the consensus
sequence of a PDZ domain ligand (Obenauer et al. 2006).
They specifically recognize and bind to short C-terminal
peptide motifs of 4-5 amino acids (X-S/T-X-V type) at the
227-230 positions of NS1 (Obenauer et al. 2006). PDZ ligand
binding motifs with ESEV or EPEV sequence were found
acid position of M1
95
137
249
K
H
A
K
H
A
K
R
A
K
H
A
K
H
A
K
H
A
R
Q
T
K
H
A
8
Y
Y
C
Y
Y
Y
C
Y
Amino acid
18
K
K
R
K
K
K
R
K
position of M2
19
27
50
I
A
F
I
A
F
S
A
F
I
A
F
I
A
F
I
A
F
S
V
C
I
A
F
in the NS1 from HPAI H5N1, H9N2, and H7N7 viruses

(Hale et al. 2008). The Inhu/BPPVRII/07 virus showed
KSEV motif as well as the PDZ-binding motif of 1918 virus
and surprisingly, the motif did not belong to avian-like
PDZ-binding motif. The KSEV motif was rarely found in
nature, and in 2005 it was recorded that two of the H5N1
Indonesian viruses have that motif i.e. A/chicken/
Indonesia/CDC24/2005 (Acc. number of GenBank
CY014196) and A/chicken/Indonesia/CDC25/2005 (Acc.
number CY014189) and Arabian H5N1 viruses in 2007
(Monne et al. 2008). On the other hand Pessel/BPPVRII/
07 virus possessed human influenza motif i.e. RSEV motif.
It is not known whether the motif in Inhu/BPPVRII/07 and
Pessel/BPPVRII/07 viruses affects the virulence or
adaptation of the virus in human. In vitro and in vivo
studies are required to answer this question.
Several studies reported that NS1 protein is also
associated with the virulence and host range of influenza
viruses in different animal models (Seo et al. 2002;
Quinlivan et al. 2005; Solorzano et al. 2005; Li et al. 2006).
The Pessel/BPPVRII/07 virus is the virus without deletion
at NS1 and like other Indonesian H5N1 viruses did not
undergo mutation at position 92; they have aspartic acid
instead of glutamic acid at position 92 of the NS1 molecule.
The glutamic acid at position 92 of the NS1 protein of the
H5N1 influenza virus transmitted to human in 1997
becomes critical in conferring virulence and resistance to
antiviral cytokines in pigs (Seo et al. 2002). However,
H5N1 virus with this amino residue is no longer circulating
in nature and glutamic acid is not found in the NS1 proteins
of other influenza A virus subtypes. In addition, it was
recently reported that a deletion of amino acids at positions
80-84 in NS1 enhances the virulence of H5N1 viruses
(Long et al. 2008).
Vol. 18, 2011
This study shows that two viruses of chicken origin

(Pessel/BPPVRII/07 and Inhu/BPPVRII/07 viruses)
isolated surrounding H5N1 human cases have NS1 unique
genetic character which may correlate with the adaptation
of the viruses in human. We recommend to conducting
the integral surveillance in Riau due to unique viral
characteristic from viruses isolated in 2007 and continue
to compare between human and animal viruses genetic
information and animal experiment for understanding the
pathogenicity and virulence of the viruses to human.
From the Matrix protein analysis, most of the virus
from human-origin have the above five amino acid motifs,
and had at least two of the amino acids and only a few had
other motifs. These findings are in agreement with the
previous result suggesting that at the M gene level, the
human and animal origin viruses differ in the presences of
substitutions in M1 and M2 (Chen et al. 2006). From the
results obtained in this study, it was assumed that there
were visible differences between virus from birds that may
or may not infect humans. Unlike in M1 and M2 proteins,
a specific difference between the viruses that could or
could not infect humans was not found in the surface
protein genes of H5N1 viruses. However, the motif in M
protein was specifically found only in the viruses from
Indonesia, not in the other country. This result is in line
with Benders et al. (1981) in his study that stated that the
analysis of the surface protein genes of the H5N1 viruses
from Hong Kong 1997 outbreak did not provide immediate
answers regarding the molecular basis for virulence. Even
though this study indicates that M1 and M2 proteins may
have important roles in predicting whether or not an AI
virus can infect humans, in vitro and in vivo studies are
still required to answer the question about the role of M
protein as a marker for predicting whether or not a virus is
capable of infecting human.
ACKNOWLEDGEMENT
This study was supported by research grants by the
2007-2008 the Department of Agriculture State Budget,
AARD, Ministry of Agriculture. The authors would like
to thank the Animal and Fish Health Institute Jakarta,
Livestock Services of Bekasi and Yuli Miswati for their
help during this field study and to Nana Suryana for his
technical assistance. Thanks to Gina Samaan to sharing
data information from the human aspect.
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HAYATI Journal of Biosciences June 2011

Vol. 18 No. 2, p 82-90

Available online at:

Influenza H5N1 Virus of Birds Surrounding H5N1 Human Cases

Received October 20, 2010/Accepted June 9, 2011

Corresponding author. Phone: +62-251-8331048,

in Asia are the Z genotype, similar to the virus first

Vol. 18, 2011

H5N1 Virus on Birds Surrounding H5N1 Human Cases

characteristic of HA, NA, M, and NS of the H5N1 viruses,

Table 1. Samples taken from birds surrounding H5N1 cases in humans

Poultry Urban A/Muscovy

Handled Urban A/Ck/

isolated it. This case showed that reared waterfowl could

of the Z+, Z, Y, A, B, and C genotypes and may contribute

Vol. 18, 2011

H5N1 Virus on Birds Surrounding H5N1 Human Cases

Group 1 (Indonesian human

Figure 1a. Phylogenetic trees of the H5N1 viruses.

Group 2 (avian H5N1 viruses)

Figure 1b. Phylogenetic trees of the M1 gene.

Vol. 18, 2011

H5N1 Virus on Birds Surrounding H5N1 Human Cases

Indonesian H5N1 viruses

Consultation, 2008). The presence of glycosylation sites

in the NS1 from HPAI H5N1, H9N2, and H7N7 viruses

Vol. 18, 2011

H5N1 Virus on Birds Surrounding H5N1 Human Cases

This study shows that two viruses of chicken origin

Monne I, Fusaro A, Al-Bodowi MH, Ismail MM, Khan OA, Dauphin

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