Tgp chi Cdng nghe Sinh hgc 9(1): 47-54,2011
PHAN TICH DAC DIEM CHUOI POLYPEPTIDE NA(N1) CUA M O T SO CHUNG VIRUS
CUM A/H5N1 G A Y BENH Cf GIA CAM THU THAP CAC NAM 2004 2009 TAI VIET
NAM
Nguyin Thi Bich Nga', Lirorng Thi Hong Van^ Le Thanh Hoa'
'Vien Cdng nghe sinh hgc
'Vien Khoa hgc su song, Dgi hgc Thdi Nguyen
T6M TAT
Protein khang nguyen neuraminidase (NA) cua cac chimg viras cum A/H5N1 cd dac tinh dot biln cao d
phan dau chudi polypeptide (diu -NH2), do ciing la mot trong nhiing dac diem quan trpng cua viras cum gia
cim vdi su thay ddi khang nguyen theo thdi gian. Toan bd chuli nucleotide (1350 bp) va amino acid (449 aa)
cua gen NA (Nl) d 11 chimg cum A/H5N1 phan lap phan lap tai cac viing/mien trong ca nude cac nam 2004 2009 dugc thu nhan va phin tich so sanh. Phan tich dac diem amino acid cua 11 chung nay vdi cac chiing dai
dien mpt sd dia phuang Viet Nam va the gidi, cho thay, do dai gen Nl tii nam 2003 den nay la 1350 bp, da co
sir CO ngan lai do bi xda di 60 nucleotide tuong iing 20 amino acid trong gen so vdi cac chung tir nam 2003 trd
ve trade. Thanh phan amino acid ciing da cd dot bien xay ra d mot sd chung nam 2008 va 2009. Mudi hai vi tri
sai khic amino acid giua cac chung so sanh da phan dinh 24 chimg thanh 3 nhdm chinh: nhom 2008 - 2009;
nhdm 2006 - 2007 va nhdm 2004 - 2005. Nhu vay, cd thi gen Nl da cd hien tugng dot bien quan trong dl tao
nen san phim gen Nl co chuc nang dac hieu cao nhat thich hgp vdi tinh gay benh tren ngudi va dong vat
(HPAI, highly pathogenic avian influenza) ngay cang gia tang d viras cum A/H5N1.
Tiir khoa: A/H5N1, dgt bien, Glycosyl hoa. Neuraminidase (NA), phd he, virus cum
DAT VAN DE
B?nh ciim gi (Avian Influenza - AI) li mdt benh
ti^yln nhilm cip tinh cua gia cim do virus ciim A
thudc hp Orthomyxoviridae gay ra. Protein be mat cd
vai frd quan frpng frong qua trinh xam nhap te bio
cua virus cum la hemagglutinin (HA) vi
neuraminidase (NA), frong do neuraminidase (NA),
Cling vdi HA, mang tinh khang nguyen vi la enzyme
chiu frach nhiem phan giii thy thi tl bio bang cich
cat lien kit glycosid cua phan tu sialic acid (Nacetylneuramic acid) giii phdng vfrus frong qui frinh
liy nhiem (Castrucci, Kawaoka, 1993; Beigent,
Cauley, 2001). Cic nghien cfru phan tii gen NA(N1)
cua vims ciim A/H5N1 cho thiy muc dp dot bien
chu ylu xiy ra d phin diu 5' lien quan din qua trinh
thich ling va gay b8nh fren nhieu ddi tugng vat chu
(Mafrosovich et al, 1999; Takao et al, 2002;
Keawcharroen et al, 2005). Qui tiinh tiln hda biln
ddi thanh phin gen NA(Nl) cua ciim A/H5N1 hii
qua nhilu dgt them vao roi bdt di nucleotide (gpi li
trugt-xod gen) dl tao nen NA hien nay, da dugc
nghien cuu kl ci d mdt sd chiing gay benh tai Viet
Nam (Chen et al, 2007; Le Thanh Hda et al,
2008a). Biln ddi phan tii d gen HA vi NA cd lien
quan din churc nang sinh hpc ma chung chiu frich
nhiem, cd the ciing lien quan tdi ddc luc ciia virus
(Castrucci, Kawaoka, 1993; Keawcharroen et al,
2005; Chen et al, 2007).
Trong bai bio niy chung tdi trinh bay ket qua
phin tich yl th^nh phin nucleotide va amino acid
ciia phaU' doan NA (Nl) d mdt sd chiing ciim
A/H5N1 do chiing tdi phin lap cac nam 2004 - 2009,
tren ca sd so sinh vdi mdt sd chung virus ciim
A/H5N1 tren gia cim tai Viet Nam vi thi gidi.
VAT LIEU VA PHUONG PHAP
Vat lieu
Cac mau benh pham chiia virus cudng ddc ciim
A/H5N1 thu nhin tai mdt sd dia phuang tir nim
2004 din 2009. Mau benh phim li huyen dich (chit
thim dich, exudate) cd chiia virus da dugc vd boat
dim bio an toin sinh hgc.
Cic chung vfrus ciim A/H5N1 cung cip chudi
gen Nl sir dimg trong nghien cuu dugc liet ke d bang
1. Bang 1 bao gdm cic chiing dugc phan thinh cic
nhdm theo thdi gian: i) Nhdm cac chung phin lap
cic nim 2008 - 2009 bao gdm 9 chung, trong dd cd 6
chiing do chiing tdi nghien cuu (ky hi$u *; dinh sd: 1
47
Nguyin Thi Bich Nga et al
- 6) va 3 chiing cua cic tic gia khic frong Ngan hing
gen (7 9); ii) Nhdm cic chung phin lap cic nam
2006 2007: gom 8 chung (frong do 2 chung ciia
chung tdi (dinh sd 10, 11) va 6 chung cd frong Ngan
hang gtn (12, 13, 14, 15, 16, 17) vi iii) Nhdm cac
chung phan lap cac nam 2004 - 2005: gdm 9 chung,
frong do cd 5 chiing cua chiing tdi (18, 19, 20, 23,
24) vi 4 chimg cd frong Ngan hing gen.
Tach chilt RNA tong s6, thuc hien RT-PCR va
thu nhan chuoi gen Nl
RNA tdng so trong do cd he gen ciia virus dugc
tach chiet true tilp tir miu benh phim, su dung bd
hda chit QIAamp vfral RNA kit (QL\GEN) theo
hudng dan cua nhi sin xuat.
Bd sinh phim RT-PCR (QIAGEN) dugc su
dung de nhan doan gen Nl. Phin iing RT-PCR dugc
sir dung vdi cap mdi:
N1F:5'- ATGAATCCAAATCAGAAGATAATAAC -3';
NIR: 5'- CTACTTGTCAATGGTGAATGGC -3'
Thuc hien phin iing RT-PCR, thu nhan toin bd
chudi gen Nl vdi chu trinh nhiet: 42''C frong 60 phut;
95''C frong 10 phiit; 35 chu k^ (94°C frong 1 phiit;
48°C trong 1 phiit; 72°C frong 2 phiit); 72°C frong 10
phiit. Dien di kiem fra sin pham RT-PCR fren thach
agarose 1%. Sin phim RT-PCR cua toan bd gen Nl
cd dp dai khoang 1,4 kb dugc tinh sach bing bd sinh
phim AccuPrepTw PCR Purication Kit ciia hang
Bioneer (Han Qudc) vi dugc gin vio vector tich
ddng pCR2.1 (Invifrogen), chuyin nap vio tl bio E.
coli chung DH5-a dl nhan ddng. Tach ddng, chpn Ipc
va tich DNA tai td hgp bing bd AccuPrep Plasmid
Exfraction Kit (Bioneer, Hin Qudc), cit DNA plasmid
kiem fra sin pham bang enzym gidi han ^coRI. San
phim DNA plasmid diing tieu chuin dugc giai trinh
tu fren may tu ddng ABI-3100 Avant Genetic
Analyzers (My) cd tai Vien Cdng nghe sinh hpc hoac
gui di giai tririh tu d nude ngoai.
Xfr ly chuoi gen va phan tich s6 Ii§u
Toan bp trinh tu cac chudi gen Nl cua cac chiing
da dugc thu nhan va xu ly bing cic phin mIm ciia
may tinh Macintosh (bien tap bing chuang trinh
SeqEdl.03; so sanh bing AssemblyLIGN1.9c va xac
dinh thanh phin gen bing he chuang tiinh
MacVector8.2). Kit qui cac chudi frong nghien cuu
dugc so sanh vdi cic chuoi gen Nl cd frong Ngan
hing gen (GenBank) bing chuang trinh
GENEDOC2.5 (Nicholas, Nicholas, 1997). Thanh
phin nucleotide vi amino acid dugc thu nhan su
48
dung bd ma cua vi sinh vat bac thap (vi khuin,
bacterial code). Xay dimg cay phi he dugc thuc hien
bing chuong trinh MEGA4.0 (Tamura et al, 2007)
KET QUA VA THAO LUAN
Giai trinh tur va luru giii- chuSi gen Nl cac chung
virus cum gia cim A/H5N1 cac nam 2004 - 2009
Sau khi thuc hien phin ung RT-PCR tii cic miu
da dugc tich RNA tdng sd, tach ddng, giai trinh t^,
phan tich sd lieu, chiing tdi thu nhin dugc toin bp
chudi gen Nl cd dp dii li 1350 bp ma hda cho 449
amino acid ciia 11 chiing thu nhan qua cac nim 2004
2009 (Bang 1). Toan bd doan DNA cua gen Nl
(1350 bp) cua cic chung nghien cuu da dugc luu giir
trong plasmid, vdi muc dich ngoai viec su dimg giai
trinh tu, cdn phuc vu thu thap quy gen cum A/H5N1
tai Viet Nam. Mdt sd chudi da dugc sir dimg tray cap
Ngan hing gen de thu thap cic chudi gen Nl cua
mdt sd chung gay benh d gia cam theo cac nim da
duac ding ky de phan tich die tinh sinh hpc phan tit
(Bing 1).
Ket qua so sanh doi chiiu thanh phan amino acid
cua chuSi gen Nl
Ket qui so sinh ddi chieu chudi gen Nl cua 24
chimg (frong do 13 chung thuc hien d nghien cihi
niy) dugc frinh biy d hinh 1. Ket qua cho thiy,
ngoai nhiing sai khic cd tinh dan le, da cd su sai
khac vi khic biet rat ro ring, chii yeu tap trung d 80
amino acid dau tan ciing N cua protein Nl (hinh 1,
phan ddng khung ngang). Cic chimg phan lap nam
2006 2007, frong do cd mdt so chimg xic djnh
thudc phan ddng Phuc Kien cd thanh phin gen Nl d
phin dau 80 amino acid nay khic biet so vdi cac
chimg phan ddng Quing Ddng (phan lap nhiing nim
2004 - 2005 vi 2008 - 2009). Cac vi \n sai khic cua
24 chung gen Nl dugc het ke d bang 2.
So sinh cic chung cua nam 2008 - 2009 vdi cac
nam 2004 2007 cho thiy cic sai khac tap tiling d
80 amino acid diu N cua polypeptide Nl, cu the nhu
sau: P55L(Pro<^L); A63V(Ala-<-vVal); 2 chung
DkNA72(07) va DkNAl 14(07): A63I (Ala<->Ile)
day cung la 2 chung da dugc chiing tdi xac dinh
thudc phan ddng Phuc Kiln (Fujian sublineage,
clade 2.3.4) xuit hien tai Viet Nam nam 2007 (Le
Thanh Hda et al, 2008b). Ammo acid tai vi hi 64 la
K(Lys), vi tii 75 li N(Asp) d tit ci cac chung, frong
khi do d mdt sd chung: DkNA72(07);
DkNAl 14(07); CkVN42(07); DkHP208(06) vi
Tgp chi Cdng nghe Sinh hgc 9(1): 47-54, 2011
Mdl455(06) li T(Thr); vi tri 65 cic chung li L(Leu),
trii chung Dk0970(09) li F(Phe); DkDT9(07) vi
MdVN29(07) li I(Ile). Die biet khi xem xet 80
amino acid diu tien cua nhdm II (2006 2007) so
vdi cac chimg 2004, 2005, 2008, 2009, da sd cac
chung nhdm II da cd su thay ddi d cic vi tri
T17I(Thr<->Ile); V26I(Val^Ile); N39Q(Asp-M.Glu);
N75R(Asp.M.Arg);
V79I(Valf^Ile);
Y80H
(Tyr<->His)
Giiia nhdm I vi nhdm II, tai vi tri
K312T(Lys'.^Thr), hfr 2 chung CkDT382(09),
CkKG88(08) li R(Arg); vi fri 320 cd 22 chiing diu
la Ser(S) tifr 4 chung Dk0970(09) va CkVN42(07),
DkHP(06) vi Mdl455(06) la P(Pro) vi
DkNA72(07), DkNAl 14(07) li H(His); vi tri
G414S(GlyM.Ser) va DkCMl(06) la G(Gly); vi tri
S429N(Ser-*^Asp); vi tii G430S(Glyf^Ser); vi tii
434 da sd cac chiing la G(Gly) cdn mdt sd chung
nhdm II chung li S(Ser).
Rieng ddi vdi chung Dk0970(09) li mdt chung
mdi phan lap nam 2009 khi so sanh vdi cic chimg
phan lap frong ciing nhdm ciing cd su sai khac tai
mdt sd vi tri amino acid: F65L (Phe<->Leu); P71L
(Pro<^Leu); S202N (Ser<^Asp); Y217S (Tyr-f^'Ser).
Trong toan bd cac chudi gen Nl, glycosyl hda
chi xiy ra d hai vi tri amino acid 68-70 (NSS) (I) vi
vi tii 126-128 (NGI) (H) (Hmh 1).
Bing 1. Danh sach cac chung cum A/H5N1 thu thap qua cac nam 2004 - 2009 da giii trinh tu va cac chiing the gidi cung
cap chuoi gen Nl sd dpng de so sanh thinh phan amino acid.
TT
Ky hi^u
chung
Danh phap quoc te
Vat
chu
Oja danh phan lap
1
Dk0970*
A/Di<A/N/0970/2009(H5N1)
Vjt
Sdc Trang
2
CkDT382*
A/CkA/N/DT382/2008(H5N1)
Ga
3
CkDT381*
A/CkA/N/DT381/2008(H5N1)
Ga
Nam phan So dang ky trong
Ngan hang gen
lap
2009
Dang dang ky
Ddng Thip
2008
Dang dang Icy
Ddng Thip
2008
Dang dang ky
GS
Tra Vinh
2008
Dang dang icy
4
CkTV98*
A/Ck/VN/TV98/2008(H5N1)
5
CkKG88*
/VCI<A/N/KG88/2008(H5N1)
Gi
Kien Giang
2008
Dang dang Icy
6
DI<U\11A
A/DI</VN/11 A-1JV2008(H5N1)
Vit
Long An
2008
FJ812010
7
DkST41
A/DI</VN/ST-41/2008(H5N1)
Vit
Sdc Trang
2008
FJ812006
8
DI<TV7B
A/DI</VN/7B-TV/2008(H5N 1)
Vit
TrS Vinh
2008
FJ812012
Vit
Hung Nguydn - Ngh? An
2007
Dang dang Ic;^
Vjt
Quynh Luu - Ngh# An
2007
Dang dang k;^
Vi#t Nam
2007
CY030517
Dong Thip
A/DI</VN/NA72/2007(H5N 1)
9
DkNA72*
10
DkNA114* A/Di</VN/NA114/2007(H5N1)
11 CI<VN42
A/CI</VN/NCVD-42/2007(H5N1)
GS
12
DI<DT9
A/DI<A/N/DT-9/2007(H5N1)
Vjt
2007
FJ811999
13
IVIdVN29
A/iVldA/N/NCVD-29/2007(H5N1)
Ngan Vi$t Nam
2007
CY030440
14
Di<CM1
A/Di<A/N/CM-1 /2006(H5N 1)
Vit
Vi$t Nam
2006
FJ811997
15
DI<HP208
A/Di</VN/HP208/2006(H5N1)
Vit
Hai Phong
2006
GU052504
16
IVId1455
A/iVldA/N/l 455/2006(H5N 1)
Ngan Viet Nam
2006
CY029537
17
DkAG*
A/Dk/VN/AG/2005(H5N 1)
Vjt
An Giang
2005
EF057804
Ca IVIau
2005
Dang dang ky
18
CkCaM*
A/CkA/N/ Cai\/I/2005(H5N1)
Ga
19
CI<VN11
A/CkA/N/NCVD 11 /2005(H5N 1)
Gi
Vi#t Nam
2005
CY036721
2005
CY031629
2004
EF057807
20
Md02
A/iVldA/N/NCVD02/2005(H5N1)
Ngan Vidt Nam
21
CkHD1*
A/CI<A/N/HD1/2004(H5N1)
Ga
A/MdA/N/GU2004(H5N 1)
Ngan Gia Lim - Ha Npi
2004
EF057805
CY033171
CY036705
22
23
24
MdGL*
Hoai Due - Ha Npi
CI<VN830
A/CI<A/N/830/2004(H5N1)
Gi
Vi$t Nam
2004
CI<VN925
A/CkA/N/925/2004(H5N 1)
Gi
Vi$t Nam
2004
Ghi chii: *Cic chung do chiing tdi phin lip.
49
Nguyen Thi Bich Nga et al
Dk0970(09)
C%DT381(08
C3cDT382(08
C%KG88 (08)
C3CTV98 (08)
DkL%llA(08
DkST41(08)
DkTV7B(08)
D k i a 7 2 (07)
DkIIA114(07
cncVN42 (07)
DkDT9(07)F
IIdVN29(07)
DkCMl(06)F
DkHP208(06
Hdl455(06)
DkAG (05)
CkCaM(05)
CatVHll (05)
Hd02(05)CY
HdGL(04)
CkHDl(04)
Ck-™83 0(04
cncVl(92 5 ( 0 4
Dk0970(09)
ClcDTaSKOS
CkDT382(08
CkKG88(08)
CkTV98(08)
DkLAllA(08
DkST41(08)
DkTV7B(0e)
DkHA72(07)
DkIIA114(07
CkVH42(07)
DkDT9(07)F
KdVN29(07)
DkCHl(0e)F
DkHP208(06
Hai455(06)
DkAG(05)
C3cCaM(0S)
CkVNlKOS)
Hd02(05)CY
HdGL(04)
CkHDl(04)
CkVN83 0 ( 0 4
CkVH925(04
Dk0970(09)
C%DT381(08
CkDT3e2(0S
CkKG88(08)
CkTV98(08)
DkLAllA(08
DkST41(08)
DkTV7B(08)
DkNA72(07)
DkNA114(07
CkVN42(07)
DkDT9(07)F
HdVH29(07)
DkCHl(Oe)F
DkBF208(06
I(dl455(06)
DkAG(05)
cncCaM(05)
CfcVHlKOS)
Md02(05)cy
HdGL(04)
CkaDl(04)
C3[VN830(04
C%VN925(04
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Tgp chi Cdng nghe Sinh hgc 9(1): 47-54,2011
Dk0970(09)
OcDT381(08
CncDT382(08
CkKG88(08)
(3cTV98(08)
DkLAllA(Oe
DkST41(08)
DkTV7B(08)
DkNA72(07)
DkNA114(07
CkVH42(07)
D)cDT9(07)F
MdVN29(07)
DkCMl(06)F
DkBP208(06
Hdl455(0e)
DkAG(05)
CkCaH(05)
CkVNll(05)
Hd02(05)Cnr
Hd6L(04)
CkHDl(04)
CkVN830(04
CkVN925(04
Dk0970(09)
CkDT381(08
(3cDT382(a8
CkKG88(08)
CkTV98(08)
DlcLAllA(08
DkST41(08)
DkTV7B(08)
DkNA72(07)
DkNA114(07
CkVN42(07)
DkDT9(07)F
HdVN29(07)
DkCHl(06)F
DkBP208(0e
Hdl455(06)
DkAO(05)
CkCaH(05)
CntVNll(05)
Hd02(05)C:Y
HdGL(04)
CkBDl(04)
CkVN830(04
CkVN925(04
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Hinh 1. So sinh doi chieu trinh tu amino acid gen N1 cic chung vims cum gia cam A/H5N1 thu thip qua cac nam 2004 2009. Ghi chu: diu (.) the hien sg giong nhau vdi amino acid cCia chung Dk0970 (nam 2009). Sg sai khic the hien bang cic
chij cii k^ hidu cQa chiing. Nhung sai khac idn giua cic chCing dugc dong khung dpc. Vi tri gllycosyl hda dugc danh s6 La
Ma (I v i i l ) , chi dan bang mui ten va gach ben dudi. Sai khac ro rang giua cac chung chii yeu t i p trung d 80 amino acid
phin diu N cua protein N1 (phan dong khung ngang).
Phan tich moi quan h§ cua cac chung virus cum
A/H5N1 tfr nam 2004 2009 vdi cac chiing cua
Vif t Nam da dang ky trong Ngan hang gen
Bing chuomg trinh MEGA4.i sfr dung thanh
phin amino acid cua Nl chung tdi xay dung cay pha
he cua cic chung nphien ciiu vdi cic chung cd frong
Ngan hing gen. Ket qui dugc thi hien fren hinh 2.
Ket qui hmh 2 cho thiy, frong sd 11 chiing chiing tdi
nghiSn cuu hiu hit cic chung (9 chung li
CkDT381(08);
CkKG88(08);
CkTV98(08);
Dk0970(09);
CkDT382(08);
CkCaM(05);
DkAG(05); CkHDl(04); MdGL(04)) tiiugc clade 1,
kl ci cac chiing mdi phin lap 2008-2009; khdng cd
chung nio thudc clade 2.3.2; nhung cd 2 chimg
tiiupc phan ddng Phiic Kiln (DkNA72(07) vi
DkNAl 14(07)) nim frong cung nhdm vdi chung
CkVN42(07) thudc clade 2.3.4 (Nguyen et al, 2008)
(Hmh 2).
Dilu niy cho thiy cac chung thupc clade 1 van
cdn tdn tai kl tu khi xam nhip vio Viet Nam vi chu
51
Nguyen Thi Bich Nga et al
yeu gay benh ciim gia cim d vung ddng bang sdng
Cfru Long.
Nhu vay cic chung ciim A/H5N1 tii khi xuat
hien tai Viet Nam din nay khdng cd dot biln ldm
khic biet, khdng gidng nhu Nl dot biln "trugrt-xda"
(slippage mutation) gen ciia cic chiing d giai doan
1996 1997 va 1997 2003 (Le Thanh Hda et al,
2008a), ma cac dot biln chii yeu li dot biln "diem"
(point-mutation) van tap tnmg vio mdt ddng gen Nl
da tiln hda. Gen Nl do mang dac tinh dot biln qua
cac thdi ky frong do die tinh ndi bat do la dot biln
truat-xoa gen theo thdi gian de tao nen cac ddng gen
rieng biet, cac ket qua phan tich frong bai bio nay
cung hoin toin phii hgp vdi nhan xet cua cic tic gia
da cdng bd trudc day (Chen et al, 2007; Le Thanh
Hoaetal, 2008a).
Bing 2. Liet ke cic vi tri amino acid cd sai khac idn cua polypeptide Nl glOa c i c chung cac nam 2004 - 2009.
Nhdm
NH6M
Cac vj tri aminolacid sai khac
Nam
phan
lap
55
63
64
75
79
80
312
320
414
429
430
434
1.Dk0970*
2009
P
A
K
N
V
T
K
P
G
S
G
G
2. CkDT382*
2008
P
A
K
N
V
T
R
S
G
S
G
G
Ten chung
3. CkDT388*
2008
P
A
K
N
V
T
K
S
G
S
G
G
1
4. CkTV98*
2008
P
A
K
N
V
T
K
S
G
8
G
G
(20082009)
5. CkKG88*
2008
P
A
K
N
V
T
R
G
8
G
G
6. DkLAIIA
2008
P
A
K
N
V
T
K
G
8
G
G
7. DkST41
2008
P
A
K
N
V
T
K
G
S
G
G
G
8
G
G
8. DkTWB
2008
P
A
K
N
V
T
K
s
s
s
s
9. DkNA72*
2007
L
1
T
R
1
H
T
H
s
N
S
S
10. DkNA114*
2007
L
1
T
R
1
H
T
H
N
8
8
11.CkVN42
2007
L
V
T
R
1
H
T
P
s
s
N
S
8
12. DkDT9
2007
L
V
K
N
V
T
T
S
N
8
G
13. i\/ldVN29
2007
L
V
K
N
V
T
T
s
s
S
N
8
G
14. DkCMI
2006
L
V
K
N
V
T
T
s
S
N
S
G
NH6M
II
(20062007)
NH6M
III
(20042005)
52
15. DkHP208
2006
L
V
T
R
1
H
T
P
S
N
S
S
16. i\/ld1455
2006
L
V
T
R
1
H
T
P
S
N
8
8
17. DkAG*
2005
L
V
K
N
V
T
T
s
S
N
8
G
18. CkCaM*
2005
L
V
K
N
V
T
T
s
S
N
8
G
19. CkVNII
2005
L
V
K
N
V
T
T
s
S
N
S
G
20. i\/ld02
2005
L
V
K
N
V
T
T
s
S
N
8
G
21. CkHOr
2004
L
V
K
N
V
T
T
s
S
N
8
G
22. MdGL*
2004
L
V
K
N
V
T
T
s
S
N
8
G
23. CkVN830
2004
L
V
K
N
V
T
T
s
S
N
8
G
24. CkVN925
2004
L
V
K
N
V
T
T
8
S
N
8
G
Tgp chi Cong nghe Sinh hgc 9(1): 47-54,2011
ioo|CkDT381(08).^
1001 CkKG88(08)
100
CkTV98(08) -^
^
Dk0970(09) CkDT382(08) - 4 — DkST41(08)FJ812006 -^
100 DkLA11A(08)FJ812010
100 ^
DkTV7B(08)FJ812012
'-^"DkCM1(06)FJ811997
IOC
99
99
100
Clade 1.0
DkDT9(07)FJ811999
< MdVN29(07)CY030440
CkVN830(04)CY003171
Md02(05)CY031629
'°«S0DkAG(05)^
^«*5^CkCaM(05)
CkVNII (05)CY036721
100
CkVN925(04)CY036705
-MdGL(04) - ^
—
CkHD1(04) - ^
_ j DkHP208(06)GU052504
Clade 2.3.2
~^ Md1455(06)CY029537
CkVN42(07)CY030517
100
Clade 2.3.4
DkNA72(07) ^
~| 100
• DkNAll4(07)-^
Hinh 2. Ciy phi h$ dua tren thanh phan amino acid NA (Nl) cua cic chiing phin lip trong cic nam 2004 - 2009 vdi mpt so
Chung trong Ngan hang gen, bang chuang trinh i\/IEGA4.0, h$ sd tin ciy 1000 bootstrap. Cic chung nghien cdu cua chiing
tdi (mui tdn chi din d cuoi chudi), t i p tmng d clade 1.0 va clade 2.3.4.
KETLUAN
Chudi gen Nl ciia 11 chiing ciim A/H5N1 phin
lap qua cac nim 2004-2009, ciing nhu cac chiing
khac cd frong Ngan hang gen, cd dg dii 1350 bp, ma
hda cho 449 amino acid, phu hgp vdi tiln hda Nl tii
2003 din nay. Phin diu tan cung N cua protein.
NA(N1) cd biln ddi Idn ban rit nhieu so vm cic
phan cdn lai, cd xu hucmg phin chia thinh timg
nhdm theo thdi gian (2004 - 2005; 2006 - 2007; 2008
- 2009). Tuy nhien, tii khi xuit hien tai Viet Nam
din nay khdng cd dot biln Icm khac bipt cic ddng Nl
nhu d giai doan trudc 2003, mi chii ylu vin tap
trung vio mdt ddng gen Nl da tien hda.
PGS TS Le Thanh Hoa vd ThS (NCS) Dodn Thi
Thanh Huang ldm chu nhiem vd Phong Thi nghiem
trgng diem cdng nghe gen, Vien Cdng nghe sinh hgc
hd trg trang thiet bi thuc hien cdng trinh ndy.
TAI LIEU THAM
KHAO
Baigent SJ, McCauley JW (2001) Glycosylation of
haemagglutinin and stalk-length of neuraminidase combine
to regulate the growth of avian influenza viruses in tissue
culture. Virus Res 19(\-2): 177-185.
Castmcci MR, Kawaoka Y (1993) Biologic importance of
neuraminidase stalk length in influenza A vims. J Virol 67:
759-764.
Chen JM, Ma HC, Chen JW, Sun YX, Li JM, Wang ZL
(2007) A preliminary panorama of the diversity of Nl
Ldi cim OTI: Chirng tdi cdm an Bd Khoa hgc vd subtype influenza vimses. Virus Genes 35(1): 33-40.
Cong ngh4 cdp kinh phi cho Nhiem vu Nghi dinh thu
Vi^t Nam - Thdi Lan ve nghien ciru sinh hgc phdn tu Nguyen TD, Nguyen TV, Vijaykrishna D, Webster RG,
cum A/H5N1 tgi Viet Nam giai dogn 2008 - 2010, do Guan Y, Peiris MJS, Smith GJD (2008) Multiple
53
Nguyin Thi Bich Nga et al
Sublineages of Influenza A Vuus (H5N1), Vietaam, 20052007. Emerg Infect Dis 14(4): 632-636.
Keawcharoen J, Amonsin A, Oraveerakul K, Wattanodom
S, Papravasit T, Kamda S, Lekakul K, Pattanarangsan R,
Noppompanth S, Fouchier RA, Osterhaus AD,
Payungpom S, Theamboonlers A, Poovorawan Y (2005)
Characterization of the hemagglutinin and neuraminidase
genes of recent influenza virus isolates from different
avian species in Thailand. Acta Virol 49(4): 277-280.
Le Thanh Hda, Nguyin Thi Bich Nga, Le Trin Bmh
(2008a) So sanh va phan tich dac tinh dot biln tnrgt xoa
gen NA(N1) theo thdi gian tiln hda cua vims cum
A/H5N1 d cac chiing cua Vi?t Nam va thi gidi. Tgp chi
Cong nghe Sinh hoc 6(2): 161 -167
LS Thanh Hda, Nguyin Thi Bich Nga, Tran Quang
Vui, Nguyen Manh Kien, Nguyen Xuan Vu, Le Tran
Binh (2008b) Phat hien biln chung virus cum A/H5N1
ddng Phuc Kien gay benh tren gia cim/ngudi tai Viet
Nam (pp 699-702). Hoi nghi khoa hoc todn quoc Idn
thu tu: Hoa sinh vd Sinh hgc phdn tu phuc vu nong,
sinh-y hoc vd cong nghiep thuc phdm (Hd Ngi, 1617/10/2008). Nha xuit ban Khoa hpc v i ky thuat, Ha
Npi (924 trang).
MaUosovich M, Zhou N, Kawaoka Y, Webster R (1999)
The surface glycoproteins of H5 influenza vimses isolated
from humans, chickens, and wild aquatic birds have
distmguishable properties. J Virol 73(2): 1146-1155.
Tamura K, Dudley J, Nei M, Kumar S (2007) MEGA4.0:
Molecular Evolutionary Genetics Analysis (MEGA)
software version 4.0. Moi Biol Evol 24: 1596-1599.
Nicholas KB, Nicholas HB (1997) Genedoc: a tool for
editing and annotating multiple sequence alignments.
Distributed by the author.
Takao S, Shimazu Y, Fukuda S, Kuwayana M, Miyazaki K
(2002) Neuraminidase Subtyping of Human Influenza A
vimsres by RT-PCR and Its Application to Clinical
Isolates. Jpn J Infect Dis 55: 204-205.
C H A R A C T E R I Z A T I O N O F T H E N E U R A M I N I D A S E ( N A ) P O L Y P E P T I D E O F THE
A V I A N I N F L U E N Z A V I R U S A / H 5 N 1 S T R A I N S I N P O U L T R Y C O L L E C T E D DURING
2004 - 2009 I N V I E T N A M
Nguyen Thi Bich NgaS Luong Thi Hong Van^ Le Thanh Hoa'''
Institute of Biotechnology
College of Sciences, Thai Nguyen
University
SUMMARY
Antigenic polypeptide neuraminidase (NA) of the avian influenza vims A/H5N1 strains possess a highly
variable property at the -NH2 terminal, and this is one of the antigenic variation during evolution. The entire
nucleotide sequence (1350 bp) of the neuraminidase (NA) coding for 449 deduced amino acids from 11
A/H5N1 strains isolated in different regions of Vietnam were obtained for comparative analysis. The obtained
results revealed that the NA nucleotide sequence for 11 A/H5N1 strains in this study is 1350 bp in length
remaining unchanged since 2003; this was resulted by deletion of 60 nucleotides (20 amino acids) from the
fragment of strains isolated prior 2003 (1410 bp NA (Ml) gene translated for a peptide of 469 amino acids).
There were some drift mutations observed in amino acids sequence of NA derived from the strains collected in
2008 and 2009. Based on twelve major variation sites in NA sequences, 24 strains were clarified to three
groups: group I: 2008 - 2009; group 11: 2006-2007 and group HI: 2004 - 2005. Possibly the NA has evolution
time by time to possess several important variations that involved in its pathogenicity in order to easily invade,
exist and replicate in humans and animals (HPAI, highly pathogenic avian influenza).
JTey words: A/H5N1, Avian influenza virus, Glycosylation, Neuraminidase (NA), Mutation, Phytogeny.
' Author for correspondence: Tel: 84-4-37567297; E-mail: imibtvn(d),smail com
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