Tgp chi Cdngnghe Sinh hpc 11(1)

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Tgp chi Cdngnghe Sinh hpc 11(1): 77-82, 2013

D.AC D I E M N - G U C O S \ L H O A P R O T E I N N E U R A M I N I D A S E O Y i R U S H 5 N 1 L U U H.ANH T R E N G I A C A M T A I M I E N B A C M E T N A M

Ngujen Nam T h a n g ' , Dong Van Quy^n^, Dinh Duy K h a n g ^ Lirong Xuan H i l n ' 'Tnr&ngDai hoc Y Thdi Binh

^Vien Cdng nghe sinh hoc. Vien Hdn ldm Khoa hgc vd Cdng nghe Viet .\ain TOM TAT

Hemagglutinin (HA) va neuraminidase (NA) la cac glycoprotein co vai Iro quan trong doi vai su lay Iruyen trong quan the dgng vat, sy hoa mang ciing nhu sir giai phong cac virionciia virus ciim A. Hien tugng N- glycosyl boa protein HA va NA hen quan chat che den dac tinh nay ciia virus Trong nghien cim nay, chung toi da den hanh phan tich ve so luong, vi tri va tinh bao ton ciia cac vi tri N-glycosyl hoa protein NA cua 29 chung virus H5N1 lim hanh tren gia cam d mien Bac Viet Namgiai doan 2008-2009 RNA long so duoc tach chiet tir mau henh pham, sau do su dung lam khuon de khuech dai va tach dong loan bo cac phan doan NA cua cac chiing virus H5N1, Trinh tu nucleoUde ciia cac phan doan duac xac dmh bang phucmg phap Sanger, chuyen thanh trinh tu amino acid luong img va xac dinh cac vi iri co kha nang N-glycosyl hoa ban^ phan mem NetNGlyc 1.0. Ket qua nghien cim cho thay 3 vi tri tren protein NA (68, 126. 215) co linh bao ton cao a hau het cac chung virus, tuy nhien mot so dot bien lam ihay doi so luong va vi tri N-glycosyl hoa da duoc phat hien o mot so chung virus HSN I,

Tuklida: N-glycosyl hda, I ISN I, influc iraminida.se, dot hien DAT VAN DE

Virus cijin A la mgl Irong nhirng tac nhan gay benh quan trgng doi vai nhieu !oai dgng vat co vii, cac loai chim, gia cam va nguoi He gen cua virus gom 8 phan doan RNA ma hoa cho 11 protein, trong do neuraminidase (NA) la mgt trong hai khang nguyen b^ inal chu yeu eua virus va dong vai tro quan trong doi ven su ton tai cua virus, NA xiic lac phan img di dut lien ket glycosid giira sialic acid ctia td bao chti va glycoprotein tren be mat hat virus de virus c6 Ihe xam nhiem le bao va giiip cho cac hat virus mdl hinh thanh co the giai phong ra klidi be mSi te bao nhiem va xam nhiem sang cac te bao khae (Huang et al, 2008; Malrosovich el al, 2004). Trong so I 1 protein cua virus ciim A chi co hemagglutinin(HA) va NA la cac glycoprotein (Blake ei ai, 2009) Cac nghien cim cho thay dac di^m N-glycosyl hoa prolein HA va NA lien quan chat ehe den dSc linh khang nguyen. kha nang xam nhilm, bam gan \ a giai phong eua virus ciim ,\ thdng qua cac ca che true Hep (Abe cl al. 2004, Schuize et ai, 1997) va gian li^p (Deshpande et at. 1987, Gallagher et at, 1988; Klcnk et al, 2002. Maisuoka el al, 2009; Sun ci al. 2(112. Sun et ai. 2011, Wagner el al, 2000).

Qua trinh N-glyeosyl hoa dugc ihirc hien o he lh6ng lircn ngi ehal va phirc hap Golgi cua le bao

chii, Vi tri gan goc carbohydrate thuong la cac vi tri Asparagine cd motif N-X-S/T, trong do X la amino acid bat ky, trir Prolin (Baigent, McCauley, 2001), do do so luong va vi tri N-glycosyl hoa cua mot glycoprotein co the xac dinh bang cac chuang trinh phan mcm (Sun et at .2011, Thang etai. 2004)

Viet Nam la mot trong nhung niroc chui nhieu anh huong cua djch ciim gia cam Ke lir nam 2003 den nay. dich ciim gia cam hen tuc xay ra a hau hei cac lmh thanh tren loan quoe, hang irieu gia cam bi chei \a tieu huy O khu vyc Mien Bac Viel Nam.

Irong giai doan 2008-2009, dich ciim gia cam H5N1 da xay ra tai nhieu dia phuang Do do.nghien ciru nay duoc Ihuc hien nham phan tich ve so luong, vi tri va tinh bao Ion ciia cae vi tri N-glycosyl hoa prolem NA ciia 29 chung virus H5NI luu hanh tren gia cam tai Mien Bac Viei Nam giai doan 2008- 2009

PHUONG PHAP XGHIEN CU'U Doi tirgnng nghien eiru

Nghien ciru su dung 29 mau benh pham thu ihap Iren gia cam dm hoac ehet trong cac \u dich ciiin H5N1 Iren gia cam 6 khu vuc Mien Bac Vicl Nam giai doan 2008-2009

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Nguyen Nam Thang el ai Phutmg phap nghien ciiu

Tdch chiit RNA

M^u benh pham dugc sir dung de tach RNA long so bang bo sinh pham RNeasy mini Kit (Qiagen, Hilden, Germany) theo hudng dan ciia nha san xuat Khoang 30 mg mau mo dugc cho vao ong ly tam, bo sung 600 pi dung dich dem RET, lac ky bang vortex trong 30 giay de ly giai va thuan nhat Ly tam mau 14000 vong trong 3 phiit, thu dich noi sang mgt ong ly Iam khac, bo sung 550 pi ethanol 70% va Iron deu bang pipette Chuyen mau sang egt tinh sach, nia cot mgt lan voi 700 pi dem R W l , hai lan vdi 500 |il dem RPE. Chuyen cot sang ong 1,5ml, RNA sau do dugc day ra khoi egt bang 50 pi nudc sieu sach da loai bo RNase va bao quan d -20°C.

Tdng hop cDNA (complementary DNA) Nam pi RNA tach chiet Ui mau benh pham dugc su dung lam khuon de thyc hien phan ung tong hgp cDNA vdi bg sinh pham Revert A id'^'^ H Minus First Strand cDNA Synthesis Kit (Fennentas, Vilnius, Liihuama) theo hudng dan cua nha san xuat. Phan irng sao ma ngugc dugc thyc hien vdi moi dae hieu Unil2' 5 ' - A G C A A A ' A G C A G G - 3 ' (Hoffmann el al. 2001)

Khuech dai, tdch dong va xdc djnh Irinh lu nucleotide phdn dogn f^A

Ky thual khuech dai, tach dong va xac dinh Irinh tu nucleotide phan doan NA ciia cac chung virus H5NI dugc thyc hien theo quy trinh da dugc cong bo Irong mot nghien ciru trudc day (Nguyen Nam T h i n g el al, 2011) Sau do, trinh ty nucleotide ciia cac phan doan NA dugc ndi lai vdi nhau bang chuang trinh Seqman cua phan mem DNAStar (http //wvvw.dnastar.com) tao Ihanh cac phan doan gen hoan chinh va dang ky tren GenBank.

Phdn lich dgc diem N-glycosyl hoa

Nghien cim nay img dung chuong trinh NetNGlyc I 0 (hllp wwwcbs dlu.dk/sen-ices/NeiNGhi) de phan tich dac diem N-glycosyl hda protein NA ciia cac virus phal hien trong nghien ciru va so sanh vdi mdt sd chung H5NI da dang ky tren GenBank Trinh tu ammo acid protein NA ctia cac chung virus duoc xac dinh dya tren trinh ty nucleotide bang phan mem MEGA5 (blip,//www megasoftware net, Tamura et at. 2011). NelNGKc I 0 su dung 9 he thdng mang

de phan tfch trinh tu ammo acid va danh gia kha nang glycosyi hda ciia timg motif N-X-S/T, Kha nang glycosyi hda ciia moi motif N-X-S/T co gia tri trong khoang tir 0 - 1 va la gia tri trung binh cua 9 he thdng mang.

K E T Q U A

Xac dinh trinh ttr nucleotide gen NA

Vdi phuong phap trinh bay d tren, trinh tir gen NA cua 29 chung virus H5N1 luu hanh tren gia cam d mien Bac Viet Nam giai doan 2008-2009 da duoc xac dinh. Tat ca cac trinh tu gen deu duoe dang ky tren GenBank vdi cac ma so dang ky:CY095681;

CY095684, CY095687, CY095690; CY095693;

CY095696, CY 103800;

CY 103808;

CY103816;

CY 103824;

CY103832;

CY095699, CY 103802 CY 103810;

CY103818:

CY 103826;

CY095702;

CY 103804;

CY103812;

CY 103 820, CY 103828,

CY095705;

CY 103806;

CY 103814;

CY 103822;

CY 103830, CY103834, CY103836, CY103838 Ket qua trong bang 1 cho thay sd lugng, vi tri va trinh ty amino acid tai cac motif N-glycosyl hoa tren protein NA eua cac chung virus H5N1 phal hien trong nghien cuu cd sy tuong ddng cao. Hau nhu cac chiing virus phat hien trong nghien ciru va cac chiing virus tham chieu deu cd 3 vi tri N- glycosyl hda vdi cac motif khong ddi la 68-NSSL, 126-NGTV va 215-NGSC. Tuy nhien chung toi phat hien cd 4 chung virus mang dot bien tai cac v|

tri glycosyi hda Chiing virus TMU029, mac dii van cd 3 VI tri glycosyi hda nhung thyc te da cd sy thay ddi ve vi tri va motif N-glycosyl hda, Chiing virus nay mang 2 dot bien: dot bien thay the L71P lam motif 68-NSSL chuyan thanh 68-NSSP va mil kha nang glycosyi hda tai vi tri nay nhung dot bien I86T lai lam xuat hien motif 84-NNTR cd kha nang glycosyi hda Chiing virus TMU012 va TMU0I3 ciing mang dot bien S70T tuy nhien dot bien nay khdng lam thay doi kha nang glycosyi hda tai vi tri 68. Chung virus T M U 0 I 2 con cd them dot bien I203T lam xuit hien motif 201-NNTL nhung motif nay duac xac dinh la khdng cd kha nang glycosyi hoa Chimg virus TMU004 chi cd 2 vi tri glycosyi hda (68-NSSL va 126-NGTV) vi d chung virus nay xual hien ddi bien N 2 I 5 D , mat vi tri glycosyi hda 215,

Dac diem N-glycosyl hoa protein \ A

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Tap chi Cong nghe Smh hpc\\(\y. 77-82. 2013 Bang 1. D$C di^m N-glycosyl hoa prolein NA

Chiing virus

Anhui/1/05, Dk/Laos/3295/06

Japaness white eye/HK/1038/06

Env/Guizhou/7/09

Clade

2 3 4

2 3 4 1 28 NMIS

NWIS NITS {+)

Vi tri motif N-X-S/T tren protein NA 68- 84 126- 201 NSSL

NSSL

NSSL NSIR

NSIR

NSIR NGTV

NGTV

NGTV NNIL

NNIL

NNIL 215*

NGSC

NGSC Tong

3

4 Hunan/2/09,

Dk/VNrTMU017/09;

DkA/N/TMU019/09, Dk/VN/TMU021/09, DkA'N/TMU023/09;

Ck/VN/TMUO 25/09;

DkA'N/TMUO 27/09;

DkA/N/TMU018/09;

OkA/N/TMU020/09;

DkA/N/TMU022/09;

DkA/N/TMU024/09;

DkA/N/TMU026/09;

Dk/VN/TMU028/09

NSSL NGTV NGSC NSIR NNIL +++) (++) {++)

Ok/VN/TMU029/09 NTTP

(")

^NNTR(++)

NGTV

VN/HN31432M/08, Env/Guizhou/2/09, DkA/N/TMU002/08: MdkA/N/TMU003/08, 07CkA/N/TMU007/08; Dk/VN/TMU008/08, Ck/VN/TMU011/08; MdkA/N/TMU014/08 Mdk/VN/TMU012/08

Ck/VN/TMU013/08

VN/UT31413ll/08,Avian/HK/1993/07, VN/UT31203A/07,

MdkA/N/TMU001/08; DkA/N/TMUOOS/Oa, CkA/N/TMU0O6/0e;quailA/N/TMUO10/08, CkA/N/TMU015/08; CkA/N/TMU016/08;

CkAfN/TMU004/08

CkA/N/TMUO 09/08

common-magpie/HK/5052/07, Hubei/1/10, hllle egrel/HK/8863/07, common buzzardHK/9213/07 MdkA/N/1455/06

VN/1194/04

2 3 4 2

2 3 4 3

2 3 2 1

2 3 2

1 NIIS

NIIS

NMIS

NMVS

NMIS

NMIS NSSL

NSTL

NSSL

NSSL (+* + ) NSSL

NSSL

(*)**

NSIR

NSIR NGTV

NGTV

NGTV NNIL

NNTL

(-)

NNIL

SNtL

NNIL NGSC

NGSC

DGSC NGSC

NGSC

NGSC 3

3

2

3

3 Chii t h i c h K^t qua xac dinh cac vi Iri Aspargine cb kha nang glycosyi hoa dua tren tnnh tu' ammo acid protein NA cua cac virus phat hi0n Irong nghien c i i u dong thoi su' dung Irinh t u ammo acid protein NA cua mot so chung I-I5N1 da dang ky tren GenBank ah so sanh Clade cua cac chiing virus H5N1 duoc xac dmh Iheo danh phap cua WHO/OIE/FAO H5N1 Evolution Working Group (WHO/OIE/FAO, 2012) V| tn cua cac molif glycosyi hoa duoc danh so dua tren tnnh lu' ammo acid prolein NA cua Chung vims A/Anhui/1/2005 (accession number ABU94738) Tai cac vi tn glycosyi hoa. cac Innh tu ammo acid khflng t h u ^ motif N-X-S/T v§n dug-c tnnh bay trong bang dfi so sanh Ten cac virus trong nghien cuu su dung phdng chu d j m • cSc v| Iri c6 kha nang glycosyi hda voi molif h i n g dmh a virus H5N1 Cac vi iri glycosyi hoa mang ddt bien duoc dSnh d i u bang n^n sSm mau V i ^ l t a i Ck chicken, Dk duck, Env environment HK Hong Kong, Mdk muscovy duck VN Vi$l Nam

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Nguyen Nam Thang et al BAN LUAN

Sau khi xuat hien va gay benh tren ngdng d Qtiang Dong nam 1996, virus H5N1 tiep tuc luu hanh Iren gia cam va ngudi, dong thdi cung xuat hien nhieu bien doi trong he gen Mdt trong nhiJng thay ddi co ban la hien tugng mai 20 amino acid tren vung than NA d hau het cac chiing virus H5NI phan lap tit 2003 den nay, Hai muoi chin chung virus phat hien trong nghien eiru deu mang dot bien nay (ket qua khong duac trinh bay), do do dac diem N- glycosyl hda cua cac virus cting cd su khac biet so vdi chiing gdc Quang Dong.

O chiing virus Gs/Gd/I/96. proiein NA cd 7 vi tri N-glycosyl hda, 4 vi tri thudc viing than N A (50, 58, 63, 68) va 3 vi tri thudc vimg dau NA (88, 146, 235 - tugng img vdi cac vi tri 68, 126. 215 trong nghien ciru nay). Ket quajighien cuu eiia chiing tdi cho thay hau het cac virus trong nghien cuu ciing nhu cac chiing virus tham chieu deu chi cd 3 vi tri N-glycosyl hda a viing dau NA ma khong vi tri glycosyi hoa nao Iren viing than NA (rieng chiing virus tham chieu A/en Vl ron ment/Guizhou/7/2009 cd 4 vi tri glycosyi hda, trong dd cd vi tri 28 thudc viing than NA) Cac chung virus ciim ngudi H l N l luu hanh tir 1918 den nay deu cd mdt sd vi tri N-glycosyl hda tren viing than NA (Sun et at, 2011), tuy nhien cac chiing virus H5N1 luu hanh trong thdi gian gan day deu khdng cd vi tri glycosyi hda nao tren viing than NA Su loai bo cac vi tri glycosyi hda tren viing than N.A cd the la mdt dac diem thich nghi cua virus H5N1 Irong qua trinh iuu hanh tren gia cam.

Ket qua nghien ciru ctia Sun va cdng sy (2011) ciing cho thay vi tri glycosyi hda 146 d virus cum 111N1 (tuong ung vi tri 126 d virus HSN I) dugc duy In d hau het cac chiing H l N l . Trong nghien cuu nay, VI tri N-glycosyl hda 126 tren protein NA xuat hien d lat ca cac chung virus H5N1 phat hien trong nghien cdu ciing nhu cac virus tham chieu. Nhu vay, glycosyi hda tai vi tri nay cd vai trd quan trpng ddi vdi chuc nang ciia NA \ a cd the hen quan den kha nang tdn lai cua virus H5NI ciing nhu virus H l N l

Thay ddi vi tri glycosyi hda tren viing dau NA da dugc xac dinh la ca che de virus ciim ngudi H l N l tranh khdi tac ddng mien dieh bao ve ciia vat chii (Sun et at, 2011). Tuy nhien ket qua trong bang 1 cho ihay 3 vi tri glycosyi hda thugc viing dau NA khdng thay ddi d hau het cac chdng virus ciim gia cam HSN 1 Trong sd 29 chung virus phat hien trong nghicn cim chi cd 2 chung (TMU004, TMU029) mang dot bien lam thay ddi sd luong hoac vi tri

glycosyi hda tren protein NA Hai chiing TMU012, TMU013 mac dii mang dot bien tai cac motif N- glycosyl hoa nhung cac dot bien do khdng lam thay ddi sd lugng hay vi tri N-glycosyl hda

Sy khac biet ve dac diem glycosyi hda viing dau NA giUa virus HSNl va virus H l N l cd le lien quan den ap lyc chgn Ipc ciia he mien dich vat chii Virus ciim H l N l thudng gay benh nhe d ngudi va sau khi nhiem virus ngudi benh se cd mien dich vdi virus H l N l . Trong nhiing lan nhiem tiep theo, virus HIN!

se chiu ap lyc chgn lgc eua he mien dich vat chii, chi nhirng chung virus mang cae dot bien phii hpp mdi cd the tdn tai. Su thay ddi vi tri glycosyi hda tren viing dau NA d virus H l N l la mdt ca che dot bien nham trdn thoat khdi he mien dich ciia vat chu Ngugc lai. virus ciim gia cam HSNl ed dgc lyc cao ddi vdi gia cam nen hau het gia cam bi nhiem virus deu bl chet, sd cdn lai thi bi neu huy de dap dich. Do dd virus HSNl hau nhu khdng chiu ap lyc chgn lgc ciia he mien dich vat chu va hien tugng thay ddi vi ui glycosyi hda tren viing dau N A khdng xay ra,

KET LUAN

Ket qua nghien ciiu cho thay cac vj tri N- glycosyl hda tren protein NA (68, 126, 215) ciia virus HSNl cd tinh bao tdn cao, tuy nhien d mgt sd chung virus da xuat hien dot bien lam thay ddi ve sd lugng va vi tri N-glycosyl hda.

Ldi cam on: Nghien cim dupc ihuc hien vdi su ho Ira kinh phi cua de ldi hap ldc theo Nghi dinh ihu giira Viel Nam vd Thdi Lan "Hap ldc nghien cuu dgc diem lull hdnh vd su bien ddi gen cua virus ciim gia cdm H5NI a khu vuc phia Bdc Viet Nam " do Tnrang Dgi hoc Y Thai Binh chu Iri

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Nguyen Nam Thang el al

N - G L Y C O S Y L A T I O N P A T T E R N S O N T H E N E U R A M I N I D A S E P R O T E I N O F H 5 . \ l V I R U S C I R C U L A T I N G IN P O U L T R Y IN N O R T H E R N V I E T N A M

Nguyen Nam Thang'", Dong \ an Quyen', Dinh Duy Khang', Luong Xuan Hien ' Thaibinh Medical University

'Inslilule of Bioiechnolog}-, Vtelnam Academy of Science and Technology SUMMARY

Hemagglutinin (HA) and neuraminidase (NA) are glycoproteins Ihat play crucial roles in Iransfaunalion, membrane fusion and Ihe release of progeny virions of influenza A virus Previous reports showed that N- glycosylation of HA and NA proteins are closely related to these properties of Ihe virus In this study, we analyzed the number, position and conservaiion of N-glycosylation sites on NA of 29 H5Ni virus strains circulanng in poultry in northern Viet Nam during Ihe penod from 2008 to 2009 Total RNA of each clinical sample was exd-acted then used as template for lull-length ampHficalion and cloning of NA segments of H5NI virus The nucleoUde sequences of segments were determined by Sanger's method, translated into amino acid sequences and Ihen determined potential N-glycosyi at ion sites of the sequences by using NelNGlyc LO software The result showed that though 3 potential N-giycosylauon sites (68, 126, and 215) in NA are conserved m almost all of H5NI virus strains, mutations that altered the number and position of potential N- glycosylalion sites were found in some HSN I virus strains

Keywords: \-glycosylalion. muiation, H.>.\'l influenza A uriis, neuraminidase.

.•spondence Tel -84-912188101: E-mail: nnlhans tmuid.vahoo.com.vn

Tgp chi Cdngnghe Sinh hpc 11(1)

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