PHAT HIEN VIRUS VIEM NAO NHAT BAN GENOTYP 1 VA MOT S6 VIRUS ARBO KHAC Tl> MUOI 6 TAY NGUYEN, 2006 - 2007
Bui Minh Trang, Nguyen Vi6t Hoang, Dfl Phuong Loan, Dang Tuan Dat*, Phan Thi Tuyet Nga*, Tong Thj Ha, Hoang Minh Due, Dang Thu Thao, Tran Nguyet Lan, Phan Thj Nga
Vien I e sinh dich te Trung trang. Hit Sdi
* I ien Ve sinh dich te Tay Nguyen
TOM TAT
Phan lap virus dupe thuc hien bang te bao Aedes albopictus dong C6/36, djnh loai virus bang ky thuat RT-PCR, ket qua cho thay, tu 285 mlu mudi gdm 13 982 ca thi thupc 11 loai mudi dupe thu thap 61 Tay Nguyen, 2006 - 2007, da phan lap va dinh danh dupe 24 chung virus gdm co: 09 chung virus thupc hp Flaviviridae. 05 chiing virus Nam Dinh, 10 chiing virus Banna. De phat hien virus VNNB genotyp 1, da giai ma toan bp trinh tu vung gen E ciia 09 chiing virus thudc hp Flaviviridae, xac dinh cd 05 chung la Culex Flavivirus, cdn 04 chiing la virus VNNB genotyp 1. Trinh tu toan bp viing gen E ciia 2 chiing virus VNNB genotyp 1 cd ky hieu: 07VN310 va 07VN311 da dupe dang ky trong ngan hang gen quoc te voi ma sd tuong irng la HM228922 va HM228923. Ngoai ra, cdn 12 mlu phan lap van chua djnh loai dupe, cho thly, Tay Nguyen la noi cd mot sd loai virus Arbo chua xac dinh danh dang luu hanh can dupe tiep tuc nghien ciru.
Tir khoa: RT-PCR, virus VNNB, Arbo, Tay Nguyen I. DAT VAN DE
Trong nhung nam gan day phat hien da phat hien mpt so virus moi do muoi truyen 6' Viet Nam nhu virus Sagiyanta thupc lip Al- phaviridae, virus Akabane. (ha... thupc hp
Bunyaviridae, virus Quang Binh - la mpt loai virus Ctdex Flavi thupc hp Flaviviridae, virus Banna thupc hp Reoviridae vit virus Nam Djnh
la Sidovirus thupc hp Ateriviridae o mpt so
\iing ciia Viet Nam [1], |2], [3], [4], [5].
Cac nghien ciiu ve virus hpc a Tay Nguyen trong nhiing nam 2004 - 2005, da xac djnh co sir luu hanh virus Nam Djnh. vi- rus VNNB tir muoi. virus Banna tir benh nhan hpi chiing nap cap [3], |4]. Nghien ciiu phat
hien virus VNNB genptyp 1 til 6 chiia virus (Ion) da dupe thuc hien trong nghien ciiu truoc day [2] nhirng chua cci nghien ciiu xac djnh genotyp ciia virus VNNB trong quan the muoi cung nhu mpt so loai virus arbo khac luu hanh a Tay Nguyen.
De gop phan cung cap thong tin cho viec xay dung ke hoach phong chong benh djch do muoi truyen, trong nghien ciru nay.
chung toi sir dung ky thuat phan lap virus bang te bao muoi Aedes albopictus, djnh loai virus bang ky thuat ELISA Sandwich, RT- PCR \a ky thuat Sequencing de phat hien su luu hanh cua virus VNNB genotype 1 mpt SP
\ irus arbo khac or Tay Nguyen, 2006 - 2007.
Tac gia: Bui Minh Trang
Dja chi: Vien Vc Sinh Djch Te Trung Uong. so 1 Yersin. Ha Npi Dien thoai: 04 3 9719721
Email: trangminhbui3007 d gmail.com
Tap chi Y hpc du phong. Tap XX. so 6(114) 147
II. PHI ()N(. PHAP NGHIEN CVfV 2.1. Vat lieu:
Mchi muoi: MuSi duoc thu thap tir 1 8 20 gid. trong cac thang 1, 3. 5, 6, 7, 8. 9, 11 va
12 6 Taj' Nguy en. nam 2006 - 2007 bao gom co 13.982 ca the muoi.
Nguyen rat lieu sir dung cho qua trinh phim lap virus: Dung djch xu ly mau muoi la nude muoi sinh ly co khang sinh Penicillin 200 don vi/ ml, Streptomycine 100 don vj/ ml; Te bito mudi Aec/es albopictus dong C6/36; Moi truong Ml M de nuoi te bao co 10 % huyet thanh be bao thai.
Nguyen vdi lieu sir dimg de dinh loai virus: Su dung cac cap moi dac hieu virus VNNB. virus Nam Dinh. virus Banna va cac cap moi dac hieu virus nhom Alpha, Bunya va ITavi de phat hien vat lieu di truyen ciia virus trong cac mlu phan lap; Sinh pham va dung dich can thiet su dung cho ky thuat RT-PCR.
Sequencing cua hang Integrated DNA Tech- nologies (IDT); Cac nguyen vat lieu, trang thidt bi can thiet khac cho ky thuat phan lap va dinh loai \ irus trong nghien ciru nay.
2.2. Phuong phap
Ky thuat thu thap. dinh loai muoi: Muoi duoc thu thap theo phuong phap bay den.
muoi duoc chuyen vao hop luu giir muoi co du do am va thirc an trong qua trinh van chuyen muoi ve phong thi nghiem de djnh loai dua
vao dac diem hinh thai. Muoi sau khi djnh loai duoc hon lai theo tirng loai, khoang 20 - 50 con/mau, luu giir cac mau muoi o - 80"C cho den khi phan lap virus.
Ky thuat phim lap virus: Muoi duoc rira bang dung dich nude mudi sinh ly cd khang sinh 3 lan; nghien mudi trong 2 ml mdi trudng MEM cd 10 % huyet thanh be bao thai. Ty tam lav nude ndi lpc qua lpc 0.22 put. lay dich lpc giiy nhiem vao te bao mudi Aedes albopictus.
dc dich lpc hap phu \ ao tc bao trong khoang 90 phiit, bd sung mdi trudng MEM 2 % huyet thanh be bao thai. Theo ddi hien tupng benh ly cua tc bap sau gay nhilm bang kinh hien vi dao ngupc trpng khoang 7 ngay sau giiy nhilm. Nhung mau tc bao sau giiy nhiem benh pham va theo ddi. dupe luu gift d - 80°C dc dinh loai virus.
Kv thuat dinh loai virus being ky thuat RT-PCR: Phat hien vat lieu di truyen ciia cac virus thudc hp Alphaviridae, Bunyaviridae.
Flaviviridae. virus VNNB. virus Nam Dinh.
virus Banna theo quy trinh phat hien ARN spi don va ARN spi kep ciia virus [3]. |4],
Phuong phap xac dinh genotyp virus VNNB: Giai ma gen toan bp viing gen E ciia vi- rus VNNB bang ky thuat Sequencing, cay phat sinh loai dupe vc bang phuong phap Neibour- Joining de xac dinh genotyp ciia virus VNNB phan lap d Tiiy Nguyen, 2006 - 2007 |2|.
US Tap chi Y hpc du phdng, Tap XX. so 6 (114)
III. KET Q l A
Bang 1. Ket qua thu thap mu6i c> Tay Nguyen, 2006 - 2007
STT Ten loai muoi So mau mudi So ca
the muoi Ty le %
1 Anopheles vagus 12 419 3,00
2 Cx. fuscocephalus 16 613 4,39
3 Culex (Cx). gelidus 30 1449 10,36
4 Cx. pse'udovishnui 4 109 0,78
5 Cx. quinquefasciatus 16 864 6,18
6 Cx. tritaeniorhynchus 114 6396 45,74
7 Cx. vishnui 66 3339 23,88
8 Cx. vuscocephalus 1 36 0,26
9 Cx. whitei 2 66 0,47
10 Cx. whitmorei 10 337 2,41
11 Mansonia uniform 1 30 0,21
12 Hon hop muoi 13 324 2,32
Tong 285 13.982 100,00
Co 285 mau muoi gom co 13.982 ca the muoi duoc thu thap tai mot so dja diem ciia
Fay Nguyen trong hai nam 2006 — 2007. Theo ket qua djnh loai, cac mau muoi thu thap 6'Tay Nguyen dupe xac djnh co 1 1 loai muoi. Trong so I 1 loai muoi thu thap dupe tai thuc dja. loai
Cx. trilueniorhynehus chiem ty le cao nhat (45.74 %), tiep den la Cx. vishnui (23,88 %) va Cx. gelidus (10.36 %). so loai muoi con lai.
co so lupng ca the muoi thu thap dupe khong cao, dao dpng trong khoang 0,21 6,1 8 %.
Tap chi Y hoc du phong. Tap XX. so 6 (1 14) 149
Bang 2. Ket qua phan lap virus tir mudi 6> Tay Nguyen, 2006 - 2007
STT Ten loai muoi So mau phan lap
So mau duoc phat hien dirong tinh
bang PCR
S6 mlu chu'a djnh loai diroc,
co hien tirong benh ly tren te bao
1 Anopheles vagus 12 0 0
2 Cx. fuscocephalus 16 4 4
3 Cx. gelidus 30 2 1
4 Cx. pseudovishnui 4 0 1
5 Cx. quinquefasciatus 16 0 0
6 Cx. tritaeniorhynchus 114 12 3
7 Cx. vishnui 66 5 3
8 Cx. vuscocephalus 1 0 0
9 Cx. whilei 2 0 0
10 Cx. whitmorei 10 0 0
11 Mansonia uniform 1 0 0
12 Hon hop muoi 13 1 0
Tong 285 24 12
Su dung te bao muoi Aedes albopictus dong C6 36 dc phan lap virus tir 285 mau muoi thu thap a 'lay Nguyen, 2006 2007.
Quan sat hien tuong benh IV te bao sau khi gay nhiem benh pham. xae djnh co 36/285 mau giiy hien tupng benh ly tren tc bao. Trong so 36 mau gay hien tuong benh ly te bao, co 24 mau dupe xac djnh duong tinh bang ky
thuat RT-PCR vdi cac cap mdi sir dung trong nghien ciru nay. cdn 12 mau chua djnh loai dupe, ty Ic phan lap virus tir mudi la 12.63
% (36/285). Trong sd 1 1 loai muoi thu thap a Tay Nguyen, virus dupe phan lap tu 5 loai mudi. dd la cac loai mudi: Cx. fuscocephalus.
( v. gelidus. Cx. pseudovishnui. ('.v. iritaenio- rhynchus va Cx. vishnui.
150 Tap chi Y hoc du phdng. Tap XX. so 6(114)
Bang 3. Ket qua djnh loai mot s6 virus Arbo tir cac mlu phan lap bang ky thuat RT-PCR o Tay Nguyen, 2006 - 2007
(+) vai (+) vui STT Ten loai muSi cap mdi cap moi
Alpha Flavi
1 Cx. fuscocephalus 0 2 0 2 4 2 Cx. Gelidus 0 0 1 11
3 Cx. pseudovishnui 0 0 0 0 1 4 Cx. tritaeniorhynchus 0 5 3 4 3 5 Cx vishnui 0 2 1 2 3
6 H6n hop muSi 0 0 0 1 0 Tong 0 9 5 10 12
(+) voi
(+) voi Virus cap moi . ,. , .. ,
cap moi vi- chua dinh virus Nam
rus Banna loai duoc Binh
Su dung cac cap moi dac hieu voi nhung virus thuoc hp .•Uphcivirrichre. Bunyaviridae.
Flaviviridae. virus Nam Dinh. virus Banna de djnh loai mau phan lap bang ky thuat RT-PCR.
ket qua xac djnh co 9 mau duong tinh voi cap moi dac hieu virus thupc ho Flaviviridae. 5 mau duong tinh voi cap moi dac hieu virus Nam Djnh. 10 mau duong tinh voi cap moi
virus Banna, con 12 man phan lap chua djnh loai dupe. Kiem tra so bp sir nhan len cua vi- rus trong te bao voi 2 mau phan lap bang ky thuat lat cat mong. quan sat bang kinh hien vi dien tu. xac djnh co virus nhan len trong tc bao. cho thay day co the la nhirng virus moi chua duoc dinh loai.
Bang 4. Ket qua djnh loai genotype virus VNNB trong so cac mau dirdc xac djnh dircrng tinh voi cap mdi dac hieu vdi virus thuoc hp Flaviviridae
Ket qua Sequencing So mau Sd mau (+) xac djnh virus STT Ten loai mudi phan lap
(+) vdi cap moi
Flavi So chung virus VNNB
So chung Culex Flavivi-
rus
1 Cx. fuscocephalus 6 2 0 2
2 Cx. tritaeniorhynchus 9 5 2 3
3 Cx vishnui 5 2 2 0
Tdng sd 2C 9 4 5
Tap chi V hpc du phdng. Tap XX. so 6 (I 14) 151
Trong so 11 loai muoi thu thap d Tay Nguyen. 2006 2007. nhung man phan lap (+) vdi cap mdi v irus thuoc ho Flaviviridae
la nhung mau phan lap tir 3 loai mudi dd la Cx, fuscocephalus. Cx. tritaeniorhynchus va Cx. vishnui. Bang ky thuat Sequencing giai ma viing vung gen 12 xac djnh cd 4 chiing virus VNNB genotyp 1 va 5 chiing la Culex Flavivirus. Cac chung virus VNNB duoc phan lap tir Cx. tritaeniorhynchus thu thap o Kon
"Turn thang 6 2007 (chiing virus cd ky hieu la 07VN310. 07VN31C) vii tir Cx. vishnui thu thap d Kon Turn thang 7 2007 (chung virus ed ky hieu la 07VN479 va 07VN486). trong sd 4 chiing virus V NNB genotyp I. da dang ky trinh tu vung gen C ciia hai cluing v irus cd ky hieu la 07VN3 10 \a 07VN3 11 vao ngan hang gen qudc te v di mii so tuong irng la HM228922 va I IM228923.
IV. HAN LUAN
Tinh th ich irng cua cac loai virus tren tc bao rat khac nhau. cho den nay chua cd mot dong tc bao nao thich irng vdi tin ca cac loai virus, qua thuc nghiem cho thay. cac virus do mudi iruycn rat thich irng tren cac ddng te bao mudi thudng true, dac biet la tc bito \ciles albopictus ddng C6 36 duoc xac djnh la rat nhay cam va sir dung rdng rai d nhieu phdng thi nghiem khap noi Iren the gidi tic phan lap v irus |3 |. [4], [5|. Do vay. da sir dung ddng te brio niiy de phat hien v irus VNNB genotyp 1 va mot so virus Arbo khac lii mudi o l ay Nguy en trong cac nam 2006 - 2007. Sir nhan len ciia virus tren te bao Aedes albopictus ddng C6 36 cd the phiit hien duoc qua hien tuong benh ly tc bao, tuy nhien ciing cd trudng hop. virus nhan len trong te bao nhung khdng gay hien tuong benh ly tc bao.
nen phai kiem tra till ca c<ic man nudi cay tc bao de phiit hien v irus [6|.
Dc djnh loai virus phan lap. cd nhieu ky thuat thudng duoc su dung nhu ky thuat 152
nhupm am ban dd phat hien vii xac djnh nhanh virus, ky thuat huynh quang. ky thuiit EIT- SA Sandwich ai phat hien virus hoac khang nguyen virus, ky thuat trung hda de djnh loai virus, ky thuat RT- PCR de phat hien viu lieu di truyen cua virus...[6]. |7]. Nhirng ky thuat thudng dupe irng dung cho muc dich cu the.
nhu ddi vdi ky thuat nhupm am ban co the phai hien nhanh dupe hinh thai v irus. nhung chi thuc hien khi hieu gia virus trong mau dat tren 10'', neu dudi ngud'ng hieu gia nay se khdng phat hien dupe: Con ddi vdi ky thuat huynh quang hoac ELISA Sandwich cd the djnh typ dupe virus, nhung khdng the xiic djnh dupe genotyp ciia virus |6J. Do vay. de phat hien sit luu hanh cua v irus VNNB genotyp 1 va mpt sd loai virus Arbo khac. vice lira chpn ky thuat RT-PCR vii Sequencing la phii hop.
Trong nhung nam gini day xuiit hien nhieu tac nhan virus mdi giiy benh. tru virus IIIV va v icm gan B. cdn phan Idn nhung virus mdi xuat hien la do mudi Iruycn. va chau A dupe cho lii noi tiem an ciia nhieu loai virus do mudi truyen nhung chua dupe phat hien [7].
Dc phiit hien cac virus Arbo do mudi truyen o Tiiy Nguyen, cd 285 mau mudi cua 11 loai mudi gdm cd 13.982 ca the mudi da dupe thu thap. Trong sd 11 loai mudi thu thap dupe, ty le thu thap dupe Cx. tritaeniorhynchus. la cao nhat (45.74 %). tiep den lii CA. vishnui (23.88
%) va Cx. gelidus (10.36 %). day la nhiing loai mudi da dupe xiic djnh la vec-to truyen virus VNNB trong nhung nghien ciru trudc day [2], 14 |.
Mac du cd I I loai mudi Ihu thap dupe.
nhung virus mdi dupe phai hien d 5 loai mudi dd lii Cx. tritaeniorhynchus. Cx. vishnui, Cx.
gelidus. Cx. fuscocephalus va Cx. pseudovish- nui. Ket qua djnh loai da xac djnh cd mot so loai muc i cd the la v ec-to tu nhien cua VNNB.
virus Nam Djnh. virus Banna. Culex Flavivi- rus nhu CY: tritaeniorhynchus. Cx. vishnui.
Cx gelidus. va Cx. fuscocephalus.
Tap chi Y hpc dir phdng. Tap XX. so 6(114)
Hang ket qua RT-PCR va Sequencing da xac djnh 4 chung virus VNNB phan lap trong nghien cuu nay la virus VNNB genotyp I. mot lan nua khang djnh sir luu hanh rpng rai cua virus VNNB genotyp 1 a Viet Nam khong nhung o 6 chiia virus ma con a mpt so loai muoi la vec-to truy en virus d Tay Nguyen |2].
Theo ket qua phan lap va djnh loai, co 24 mau phan lap da djnh loai dupe virus, con 12 mau phim lap chua djnh loai dupe voi nhung cap moi su dung trong nghien ciru nay, cho thay can co nhirng nghien ciru tiep theo de djnh danh nhirng mau virus phan lap nay, lam co sO cho vice djnh huong du phOng benh dp muoi truy en co hieu qua.
V. KET EUAN
Prong nghien ciru nay. bang ky thuat phan lap virus tren te bao Aedes albopictus dong C6/36. djnh loai virus bang ky thuat RT-PCR va Sequencing, da phan lap va djnh loai dupe 24 cluing virus tir 285 mau muoi thu thap o l ay Nguyen. 2006 2007. Cac chiing virus dupe phat hien va djnh loai gom CP: 05 cluing virus Nam Djnh. 10 chiing \ irus Banna.
05 cluing Culex I'/avivirus va 04 cluing vi- rus VNNB genotyp 1 (trinh tir toan bp viing gen I ciia 2 cluing virus VNNB cp ky hieu 07VN3I0 va 07VN31I dupe diing ky trong ngan hang gen quoc te voi ma so tuong irng la I1M228922 vii HM228923). ngoai 24 chiing virus da dupe djnh danh, con 12 chiing virus chua djnh loai duoc can co nghien ciru tiep de xiic djnh.
Pol cant on: Cac tdc gia xin chdn thanh cam on sir hd tro mot phan kinh phi tir l ien Qudc gia cua benh truyen nhiem Tokyo Slid!
Ban cho nghien ciru phiit hien virus "Culex I'Tavivirus " o \ let Xam
TAI LIFT THAM KHAO
1. Bryant J. P.. Crabtrce M. B.. Nam V. S., Yen N. L. Due H. M.. Miller B. R. (2005).
Isolation of arboviruses from mosquitoes collected in northern Vietnam. The Ameri- can Society of Tropical Medicine and Hy- giene. 73(2). 470- 473.
2. Nguyen Viet Hoang, Do Phuong Loan. Biii Minh Trang, Le Thj Hien Thu va Phan Thj Nga (2008). Phat hien su xuat hien virus Viem nao Nhat ban Genotype 1 a mien Trung. Mien nam, Tay nguyen. Viet Nam.
Tap chi Y hpc du phong tap XVIII. so 5 (97): 38-45.
3. Phan Thj Nga. Nguyen Thanh Thuy, Biii Minh Trang. Nguyen Thj Tuan. Dang Tuan Dat, Nguyen Tran Hien (2007). Phat hien mpt thanh vien vi rut thupc hp Reoviridae gay hpi chiing nao cap 6'tinh Gia lai. 2005.
Tap chi y hoc du phong. tap XVII. so 2 (87): 5 - 9.
4. Phan Thj Nga, Pham Thj Minh ITang, Doan Hai Yen, Pham Do Quyen, Nguyen Anh Vuong. va Dang Tuan Dat (2005). Phan lap virus viem nao Nhat Ban. virus Nam Djnh 6' Gia Lai, 2004. Tap chi Y hpc du phpng, tap XV, s6 2 i 3 (74): 15-19.
5. Crabtree M. B.. Nga P. T. and Miller B. R.
(2009). Isolation and characterization of a new mosquito flavivirus. Quang Binh virus from Vietnam. Arch Virol. 154 (5): 857 - 860.
6. Dang Due Anh, Nguyen Thj Hong Hanh.
Phan Thj Nga (2010). Virus Y hpc. Nha xuat ban Y hpc: 7 - 30.
7. Kenta Okamoto, Yushirou F.ndo, Shingo Inoue. Takeshi Nabeshima. Phan Thi Nga.
Posadas LI. Guillermo. I uxun Yu, Do Phuong Loan, Bui Minh Trang. Filipinas F.
Natividad. Futoshi Hasebe, Kouichi Morita (2010). Development of a rapid and com- prehensive protcomics-based arboviruses detection system. Journal of Virological Methods. 167: 31 -36.
Tap chi Y hoc du phong. Tap XX. so 6 (114) 153
DETECTION OF JAPANESE ENCEPHALITIS (JE) VIRUS GENOTYPE 1 AND OTHER ARBOVIRUSES FROM MOSQUITOES IN TAY NGUYEN, 2006 - 2007
Bui Minh Trang, Nguyen Viet Hoang, Do Phuong Loan, Dang Tuan Dat*.
Phan Thi Tuyet Nga*, Tong Thi Ha, Hoang Minh Due, Dang Thu Thao, I ran Nguyct Lan, Phan Thi Nga
Xatiomil Institute of Hygien and Epidemiology, Ha Noi
^Institute of Hygien and Epidemiology, Tay Nguyen The isolation of viruses were conducted
by Aedes ulbopieius clone C6/36 cell line, typ- ing virus by RT-PCR. the results showed that from 285 mosquito pools, including 13,982 mosquitoes belonging 1 I mosquito species, which were collected in Tay Nguyen during 2006 2007. there are 25 virus strains to be detected and identified which including: 09 virus strains belong to Flaviviridae. 05 Nam Dinh virus strains and 10 Banna virus strains.
I or the detection of JL genotype I. sequenc- ing whole R gene region of 09 virus strains, which belong to Flaviviridae family, confirm-
ing 05 virus strains to be Culex Flavivirus. 04 virus strains to be JE virus genotype I. The nucleotide sequence of E gene of two JE vi- rus strains code: 07 VN 310 and 07VN 311 were submitted in GenBank with the acces- sion number: IIM228922 and HM228923. re- spectively.
Besides, there are 12 mosquito isolates have not identified yet, show ing Tay Nguyen possible a place where several unknown Arbo- viruses are circulating need for further study.
Key word: RT-PCR. JE. virus. Arbo, lay- Nguyen
154 Tap chi Y hoc du phong, Tap XX. so' 6(114)
