Genetic characterization of influenza A viruses (H5N1) isolated from 3rd wave of Thailand AI outbreaks

Page 1

S

A

fcrh©

K

pmaoui2iIrfsl

DHT

0d

Virus Research 122 (2006) 194–199

Short communication

Genetic characterization of influenza A viruses (H5N1) isolatedfrom 3rd wave of Thailand AI outbreaks

Alongkorn Amonsin a, Salin Chutinimitkul b, Nuananong Pariyothorn a, Thaweesak Songserm c,udarat Damrongwantanapokin d, Suphasawatt Puranaveja a, Rungroj Jam-on c, Namdee Sae-Heng c,Sunchai Payungporn b, Apiradee Theamboonlers b, Arunee Chaisingh d, Rachod Tantilertcharoen a,

Sanipa Suradhat a, Roongroje Thanawongnuwech a, Yong Poovorawan b,∗a Faculty of Veterinary Science, Chulalongkorn University, Pathumwan, Bangkok 10330, Thailand

b Faculty of Medicine, Chulalongkorn University, Pathumwan, Bangkok 10330, Thailandc Faculty of Veterinary Science, Kasetsart University, Kumphaengsaen Campus, Nakorn Pathom, Thailand

d Department of Livestock Development, National Institute of Animal Health, Bangkok 10900, Thailand

Received 22 March 2006; received in revised form 16 June 2006; accepted 28 June 2006Available online 28 August 2006

bstract

Three major outbreaks of avian influenza (AI) occurred in Thailand. During the third episode in October 2005, we have isolated H5N1 viruses

rom one human case and three poultry cases. The whole genomes of AI viruses from human, chickens and quail from the outbreaks wereharacterized. Sequence analysis of eight gene segments revealed that the 2005 H5N1 viruses isolated in October 2005 were closely related to thoseecovered from chicken, tiger(s) and human(s) in January and July 2004. In addition, the genetic changes of the AI isolates at the HA cleavage siteave been observed.

2006 Elsevier B.V. All rights reserved.

b5tci

rNfpt

eywords: H5N1; Characterization; Influenza A virus; Thailand

H5N1 influenza A virus causes avian influenza (AI), whichoses a serious threat to public health, as it can be directly trans-itted from poultry to humans. In Thailand, the AI outbreaks

ppeared to emerge in three major episodes. The first outbreakf H5N1 avian influenza was reported in Thailand in early Jan-ary 2004. It lasted until March, leading to 12 cases of humannfection with 8 fatalities. The second outbreak occurred in July004; it had run its course by the end of the year leaving ints wake five human cases with four fatalities (Tiensin, 2005).n October–December 2005, the third outbreak was reportedesulting in five human cases with two fatalities. One of the

atalities reported in October 2005 was a 48-year-old chickenlaughterer in Kanchanaburi province, western part of Thai-and. Moreover, his son was hospitalized and confirmed infected

∗ Correspondence to: Center of Excellence in Viral Hepatitis Research,epartment of Pediatrics, Faculty of Medicine, Chulalongkorn University andospital, Rama IV Road, Patumwan, Bangkok 10330, Thailand,el.: +66 2 256 4909; fax: +66 2 256 4929.

E-mail address: [email protected] (Y. Poovorawan).

pbtcobsoa

168-1702/$ – see front matter © 2006 Elsevier B.V. All rights reserved.oi:10.1016/j.virusres.2006.06.016

y H5N1. Another fatality, reported in December 2005, was a-year-old boy in the central province of Nakorn Nayok. In addi-ion, two infected individuals were reported as a worker in a localhicken farm in Nontaburi province and an 18-month-old boyn the suburban of Bangkok (Fig. 1).

During the third outbreak, a great number of poultry waseported dead in the villages of Kanchanaburi, Nakorn Pathom,ontaburi and Nakorn Nayok provinces. Suspected samples

rom chickens and quail carcasses were collected and furtherrocessed for virus isolation by embryonated egg inoculation ashe standard procedure recommended by OIE. In addition, thelasma sample from human patient in Nakorn Nayok reportedy Chutinimitkul et al. (2006) was processed for virus isola-ion by using embryonated egg inoculation. The H5N1 virusould be isolated from plasma on the day 10 after patient devel-ped symptoms. This case showed the live virus in the patient’s

lood sample which could be raising a reminder of the neces-ity to carefully handle and transport serum or plasma samplesf patients suspected of H5N1 AI infection. Subsequently, wepplied hemagglutination (HA) and hemagglutination inhibition

Page 2

A. Amonsin et al. / Virus Research 122 (2006) 194–199 195

Kanc

(iMa

hf“dwC(dlPPpDTtEbCma(a

tT2(v(2(

TriCO(atsfifagNt(sr2

vvN2pbfr(wc(

Fig. 1. Map of Thailand. The provinces with reported H5N1 outbreaks (

HI) tests to identify influenza A virus and confirmed H5N1nfluenza A virus by multiplex RT-PCR aimed at detecting the

, HA and NA genes as previously described (Payungporn etl., 2004).

Four H5N1 viruses including one isolate derived fromuman, “A/Thailand/NK165/05” (NK-165), two isolates derivedrom chickens, “A/Chicken/Thailand/CK160/05” (CK-160),A/Chicken/Thailand/CK162/05” (CK-162) and one isolateerived from quail “A/Quail/Thailand/QA161/05” (QA-161)ere included in the present study. The chicken viruses (CK-160,K-162) were isolated from the same districts as the human case

Fig. 1). Whole genome sequencing was performed as previouslyescribed (Viseshakul et al., 2004). Briefly, viral RNA was iso-ated from allantoic fluid using the RNAeasy mini kit (Qiagen).CR products of each gene segment were generated using RT-CR with primers specific for each gene. The PCR products wereurified using the Perfectprep Gel Cleanup Kit (Eppendrof).irect PCR product sequencing was carried out using Big Dyeerminator V.3.0 Cycle Sequencing Ready Reaction (ABI, Fos-

er City, CA) and the ABI-Prism 310 Genetic Analyzer (Perkin-lmer, Norwalk, CT). The sequences were verified and assem-led using computer program; Bioedit 5.0.9 (Ibis Therapeutics,arlsbad, CA). The nucleotide sequences of eight gene seg-ents of 2005 H5N1 viruses were submitted to GenBank under

ccession numbers, NK-165 (DQ372591–DQ372598), CK-160DQ334757–DQ334762), QA-161 (DQ334765–DQ334772)nd CK-162 (DQ334773–DQ334780).

Phylogenetic and genetic analyses were carried out usinghe computer program MegAlign (DNASTAR, Madison, WI).he analyses were performed to compare all genes of the005 H5N1 viruses (human (NK-165), chicken (CK-160), quailQA-161) and chicken (CK-162)), with those of 2004 H5N1

iruses representing the 1st wave (chicken (CU-K2), humanSP-33) and tiger (Ti-1)) (Keawcharoen et al., 2004; Li et al.,004; Viseshakul et al., 2004) and 2nd wave of the outbreakschicken (CU-23) and tiger (CU-T3)) (Amonsin et al., 2005;

tsga

hanaburi, Nakorn Pathom, Nakorn Nayok and Nontaburi) are depicted.

hanawongnuwech et al., 2005). In addition, the H5N1 virusesepresenting the current outbreaks in late 2005 to early 2006n Qinghai, Western China (A/Goose/Qinghai/61/05(H5N1);hina-61) (Liu et al., 2005), Astrakhan, Russia (A/Cygnuslor/Astrakhan/Ast05-2-9/05(H5N1); Russia-2-9) and Africa

A/Chicken/Nigeria/641/06; Nigeria-641) were included in thenalyses. Phylogenetic analysis of the HA and NA genes showedhat H5N1 viruses from the 2005 AI outbreaks clustered in theame groups as H5N1 isolates from 2004, which had been identi-ed as genotype Z (Fig. 2). On the other hand, the H5N1 isolatesrom Western China, Russia and Nigeria were grouped into sep-rated cluster. In addition, phylogenetic analyses of the other sixene segments of the H5N1 isolates including PA, PB1, PB2,P, NS and M, also showed high degrees of genetic relatedness

o the human, chicken and tiger isolates from Thailand in 2004data not shown). Generally, phylogenetic analyses of all geneegments indicated that the 2005 H5N1 viruses were closelyelated and grouped with the H5N1 isolates responsible for thend wave of the AI outbreaks in Thailand.

Nucleotide identities between the genomes of the chickeniruses (CK-160) compared to those of the 2005–2006 H5N1iruses (QA-161, CK-162, NK-165, China-61, Russia-2-9 andigeria-641), the 2004 H5N1 viruses (CU-K2, Ti-1, SP-33, CU-3, CU-T3) and the 1996 H5N1 virus (GD-1), are shown asercentages in Table 1. Our results showed a striking similarityetween the H5N1 viruses from the 2005 outbreaks and thoserom the 2004 outbreaks with percentages of nucleotide identityanging from 97.8 to 100%. Conversely, the 2005 H5N1 isolateCK-160) displayed a lower percentage of nucleotide identityith the 1996 H5N1 isolate from China (GD-1) as well as the

urrent H5N1 isolates from Western China (China-61), RussiaRussia-2-9) and Nigeria (Nigeria-641), especially with regard

o the NS gene (96.4–96.6%). This result supported the conclu-ion that the 2005 H5N1 viruses circulating in Thailand wereenetically comparable with 2004 H5N1 isolates in Thailandnd Vietnam, but distinct from the H5N1 virus strains circulat-

Page 3

196 A. Amonsin et al. / Virus Research 122 (2006) 194–199

e HA

ia

bn(ao

tnlH

Fig. 2. Phylogenetic analysis of th

ng in Asia such as Hong Kong, China, Indonesia (1996–2005)nd Europe (2005) as well as in Africa (2006).

The 2005 H5N1 viruses harbor multiple insertions ofasic amino acids at the HA cleavage site, which by defi-

ition is characteristic for highly pathogenic avian influenzaHPAI) (Claas et al., 1998). Interestingly, the HA cleav-ge site of 2005 H5N1 viruses in this study containedne basic amino acid “SPQREKRRKKR” differing from

sEid

and NA genes of H5N1 viruses.

hat of 2004 H5N1 viruses in Thailand, Indonesia, Viet-am and Eastern China “SPQRERRRKKR” (arginine (R) toysine (K)). It is noted that HA cleavage site of the 20055N1 viruses from Vietnam (mallard/347/05) and Indone-

ia (duck/Parepare/BBVM/05) remain unchanged “SPQR-RRRKK”. On the other hand, the HA cleavage site variabil-

ty have been previously observed in some wild bird speciesuring earlier outbreaks in Thailand in 2004 (open-bill/CU-

Page 4

A.A

monsin

etal./VirusR

esearch122

(2006)194–199

197

Table 1Comparison of the gene segments of A/Chicken/Thailand/CK160/05 to those of H5N1 isolates from Thailand

Gene Region ofcomparison(bp)a

Nucleotide identity (%)

China Thailand: 1st wave of outbreaks Thailand: 2nd wave of outbreaks Thailand: 3rd wave of outbreaks China Russia Nigeria

GD-1b,1996(goose)

CU-K2c,Jan-04(chicken)

SP-33d,Jan-04(human)

Ti-1e,Jan-04(tiger)

CU-23,Jul-04(chicken)

CU-T3f,Oct-04(tiger)

QA-161,Oct-05(quail)

CK-162,Oct-05(chicken)

NK-165g,Dec-05(human)

China-61h,Oct-05(goose)

Russia-2-9i,Dec-05(CygnusOlor)

Nigeria-641j,Jan-06(chicken)

PB2 82–2220 93.9 99.2 98.9 99.3 99.0 98.8 99.7 99.8 99.4 97.2 96.8 NAPB1 63–2226 93.2 99.2 99.2 99.4 99.2 99.1 99.6 99.8 99.4 97.4 NA NAPA 28–2119 93.2 99.4 99.2 99.5 99.3 99.2 99.8 100 99.3 97.7 NA NAHA 46–1623 96.5 97.9 99.0 99.2 99.2 99.0 99.5 99.9 99.5 96.5 96.1 96.1NP 58–1427 92.4 99.4 99.4 99.4 99.2 99.2 99.9 99.9 99.6 97.7 97.4 NANA 28–1296 91.2 98.9 98.9 99.0 98.6 98.5 99.6 99.7 99.4 96.8 96.8 NAM 1–952 96.5 99.9 99.8 99.8 99.4 99.5 99.7 99.6 99.7 98.2 97.7 NANS 36–815 69.3 99.1 99.4 99.5 99.5 99.1 99.7 99.6 99.9 96.6 96.4 NA

a Position of nucleotides is based on A/Chicken/Thailand/CK160/05(H5N1).b A/Goose/Guangdong/1/96(H5N1).c A/Chicken/Nakorn Pathom/Thailand/CU-K2/04(H5N1) (Viseshakul et al., 2004).d A/Thailand/2(SP-33)/04(H5N1) (Li et al., 2004).e A/Tiger/Suphanburi/Thailand/Ti-1/04(H5N1) (Keawcharoen et al., 2004).f A/Chicken/Ayutthaya/Thailand/CU-23/04(H5N1) and A/Tiger/Thailand/CU-T3/04(H5N1) (Amonsin et al., 2005).g A/Chicken/Thailand/Kanchanaburi/CK-160/05(H5N1), A/Quail/Thailand/Nakorn Pathom/QA-161/05(H5N1), A/Chicken/Thailand/Nontaburi/CK-162/05(H5N1), A/Thailand/NK165/05(H5N1) (this study).h A/Goose/Qinghai/61/05(H5N1) (Liu et al., 2005).i A/Cygnus Olor/Astrakhan/Ast05-2-9/05(H5N1).j A/Chicken/Nigeria/641/06(H5N1).

Page 5

198 A. Amonsin et al. / Virus Research 122 (2006) 194–199

F se ofN basic(

2p2aiui“Fagcfia1

(lnTaAcNTaboo(

o

Hapt2Hri(ut

(2g(ctwpne6geHaa

ig. 3. Alignment of HA1 amino acid sequences of 2005 AI viruses with thoigeria from 2005 to 2006. The hemagglutinin cleavage site contained multiple

SPQREKRRKKR).

/04 “SPQREKRRKKR”, white peafowl/CU-16/04 and Kalijheasant/CU-18/04 “SPQRERKRKKR”) (Keawcharoen et al.,005). This finding showed evidence of amino acid substitutiont the pathogenic site of the HA gene of H5N1 viruses circulatingn Thailand. In addition, the genetic change at one amino acidpstream of the HA cleavage site of 2005–2006 H5N1 virusesn Western China, Russia and Africa have also been observedSPQGERRRKKR” (arginine (R) to glycine (G)) as shown inig. 3. The 2005 H5N1 viruses also contained the same aminocids (Q222 and G224) at the receptor binding site of the HAene (positions 222–224) as has been previously reported withhicken, human and tiger isolates (Amonsin et al., 2005). Thisnding demonstrated that the 2005 H5N1 isolates still possessvian-specific receptor binding properties (Matrosovich et al.,999).

In this study, a 20-amino acid deletion at the NA stalk regionpositions 49–68) was discovered in all 2005–2006 H5N1 iso-ates as well as 2004 H5N1 isolates from Thailand, yet couldot be found in the 1996 H5N1 isolates from China (GD-1).his shortening of the NA stalk region presumably representsn adaptation of the H5N1 viruses (Matrosovich et al., 1999).mino acids conveying oseltamivir resistance were not dis-

ernible among the conserved residues (E119V, H274Y, R292K,294S) at the NA active site of any 2005 H5N1 isolates fromhailand as well as the isolates from Western China, Europend Africa, whereas oseltamivir resistant H5N1 isolates hadeen reported in Vietnam (Le et al., 2005). In our investigationf more than 100 H5N1 isolates, we have never encountered

seltamivir resistant amino acids in those particular residuesdata not shown).

Alterations of the amino acid at position 31 (serine (S))f the M2 protein can result in amantadine resistance. All

tc

g

H5N1 isolates from 1996 to 2004 and those of Western China, Europe andamino acids of previous outbreaks (SPQRERRRKKR) and current outbreaks

5N1 viruses isolated in 2004–2005 contained asparagine (N)t residue 31 of the M2 protein and therefore, the viruses wereresumably resistant to the amantadines. Five amino acid dele-ions at positions 79–83 of the NS gene were observed in all004–2005 H5N1 viruses in Thailand, but were not found in the5N1 virus from China (GD-1). The purpose of this deletion

emains as yet unknown. In addition, another virulence markern NS gene, the mutation of aspartic acid (D) to glutamic acidE) at position 92 of the NS1 protein, associated with high vir-lence of the virus in mammalian species, was not observed inhis study.

We further analyzed the amino acids of the polymerasePA, PB1, PB2) and nucleoprotein (NP) genes. In none of the004/2005 H5N1 isolates from poultry did we discover any sin-le amino acid substitutions at position 627 of the PB2 proteinglutamic acid (E) to lysine (K)). In contrast, we observed ahange of the amino acid at position 627 from glutamic acid (E)o lysine (K) in human H5N1 isolate in this study (NK-165) asell as H5N1 isolates from tigers and human in Thailand fromrevious analyses (Amonsin et al., 2005). It is interesting toote that all avian isolates from the current outbreaks in West-rn China, Russia and Nigeria contain Lysine (K) at position27 of the PB2 protein. Lysine (K) at position 627 of the PB2ene is deemed responsible for an increase in virus replicationfficiency especially in mammals (Shinya et al., 2004). Since5N1 viruses circulating in Thailand, particularly the human

nd tiger isolates, harbor both lysine (K) and glutamic acid (E)t PB2 627 (Amonsin et al., 2005; Li et al., 2004), it is important

o comprehensively monitor the PB2 627 position, especially inhicken isolates.

To summarize, this study highlights the significance ofenetic characterization presenting as examples of 4 H5N1

Page 6

Resea

vlsbavAHstst

A

RSUSS

R

A

C

K

K

L

L

L

M

P

C

S

T

T

A. Amonsin et al. / Virus

iruses isolated from human, chickens and quail which iso-ated from Thailand in 2005. Sequence analysis of eight geneegments revealed that the 2005 H5N1 viruses isolated in Octo-er 2005 were related to those recovered from chicken, tiger(s)nd human(s) in January and July 2004 but not related to theiruses from the recent outbreaks in Western China, Eurasia andfrica. Interestingly, amino acid substitution especially at theA cleavage site has been observed. However, the amino acid

ubstitution still basic protein which can cause highly pathogenico host. It shows that H5N1 viruses have continued to evolveince early 2004–2005 of Thailand with minor change, alterna-ively unchanged in pathogenicity.

cknowledgements

This study was supported by a grant from the Nationalesearch Council of Thailand, the Thailand Research Fund,enior Research Scholar and the funding from Chulalongkornniversity. The authors would like to thank Dr. Surangratrisuratanon, Department of Pediatrics, Faculty of Medicine,rinakharinwirot University, Nakorn Nayok, for her assistance.

eferences

monsin, A., Payungporn, S., Theamboonlers, A., Thanawongnuwech, R.,Suradhat, S., Pariyothorn, N., Tantilertcharoen, R., Damrongwantanapokin,S., Buranathai, C., Chaisingh, A., Songserm, T., Poovorawan, Y., 2005.Genetic characterization of H5N1 influenza A viruses isolated from zootigers in Thailand. Virology 344, 480–491.

laas, E.C., Osterhaus, A.D., van Beek, R., De Jong, J.C., Rimmelzwaan, G.F.,Senne, D.A., Krauss, S., Shortridge, K.F., Webster, R.G., 1998. Humaninfluenza A H5N1 virus related to a highly pathogenic avian influenza virus.Lancet 351, 472–477.

eawcharoen, J., Oraveerakul, K., Kuiken, T., Fouchier, R.A., Amonsin, A.,Payungporn, S., Noppornpanth, S., Wattanodorn, S., Theambooniers, A.,Tantilertcharoen, R., Pattanarangsan, R., Arya, N., Ratanakorn, P., Oster-

haus, D.M., Poovorawan, Y., 2004. Avian influenza H5N1 in tigers andleopards. Emerg. Infect. Dis. 10, 2189–2191.

eawcharoen, J., Amonsin, A., Oraveerakul, K., Wattanodorn, S., Papravasit,T., Karnda, S., Lekakul, K., Pattanarangsan, R., Noppornpanth, S., Fouchier,R.A., Osterhaus, A.D., Payungporn, S., Theamboonlers, A., Poovorawan,

V

rch 122 (2006) 194–199 199

Y., 2005. Characterization of the hemagglutinin and neuraminidase genes ofrecent influenza virus isolates from different avian species in Thailand. ActaVirol. 49, 277–280.

e, Q.M., Kiso, M., Someya, K., Sakai, Y.T., Nguyen, T.H., Nguyen, K.H., Pham,N.D., Ngyen, H.H., Yamada, S., Muramoto, Y., Horimoto, T., Takada, A.,Goto, H., Suzuki, T., Suzuki, Y., Kawaoka, Y., 2005. Avian flu: isolation ofdrug-resistant H5N1 virus. Nature 437, 1108.

i, K.S., Guan, Y., Wang, J., Smith, G.J., Xu, K.M., Duan, L., Rahardjo, A.P.,Puthavathana, P., Buranathai, C., Nguyen, T.D., Estoepangestie, A.T., Chais-ingh, A., Auewarakul, P., Long, H.T., Hanh, N.T., Webby, R.J., Poon, L.L.,Chen, H., Shortridge, K.F., Yuen, K.Y., Webster, R.G., Peiris, J.S., 2004.Genesis of a highly pathogenic and potentially pandemic H5N1 influenzavirus in Eastern Asia. Nature 430, 209–213.

iu, J., Xiao, H., Lei, F., Zhu, Q., Qin, K., Zhang, X.W., Zhang, X.L., Zhao,D., Wang, G., Feng, Y., Ma, J., Liu, W., Wang, J., Gao, G.F., 2005. Highlypathogenic H5N1 influenza virus infection in migratory birds. Science 309,1206.

atrosovich, M., Zhou, N., Kawaoka, Y., Webster, R., 1999. The surface glyco-proteins of H5 influenza viruses isolated from humans, chickens, and wildaquatic birds have distinguishable properties. J. Virol. 73, 1146–1155.

ayungporn, S., Phakdeewirot, P., Chutinimitkul, S., Theamboonlers, A.,Keawcharoen, J., Oraveerakul, K., Amonsin, A., Poovorawan, Y., 2004.Single-step multiplex reverse transcription-polymerase chain reaction (RT-PCR) for influenza A virus subtype H5N1 detection. Viral Immunol. 17,588–593.

hutinimitkul, S., Bhattarakosol, P., Srisuratanon, S., Eiamudomkan, A., Kong-somboon, K., Damrongwatanapokin, S., Chaisingh, A., Suwannakarn,K., Chieochansin, T., Theamboonlers, A., Poovorawan, Y., 2006. H5N1influenza A virus and infected human plasma. Emerg. Infect. Dis. 12,1041–1043.

hinya, K., Hamm, S., Hatta, M., Ito, H., Ito, T., Kawaoka, Y., 2004. PB2 aminoacid at position 627 affects replicative efficiency, but not cell tropism, ofHong Kong H5N1 influenza A viruses in mice. Virology 320, 258–266.

hanawongnuwech, R., Amonsin, A., Tantilertcharoen, R., Damrong-watanapokin, S., Theamboonlers, A., Payungporn, S., Nanthapornphiphat,K., Ratanamungklanon, S., Tunak, E., Songserm, T., Vivatthanavanich, V.,Lekdumrongsak, T., Kesdangsakonwut, S., Tunhikorn, S., Poovorawan, Y.,2005. Probable tiger-to-tiger transmission of avian influenza H5N1. Emerg.Infect. Dis. 11, 699–701.

iensin, T., 2004. Highly pathogenic avian influenza H5N1, Thailand. Emerg.Infect. Dis. 11, 1664–1672.

iseshakul, N., Thanawongnuwech, R., Amonsin, A., Suradhat, S., Payungporn,S., Keawchareon, J., Oraveerakul, K., Wongyanin, P., Plitkul, S., Theam-boonlers, A., Poovorawan, Y., 2004. The genome sequence analysis of H5N1avian influenza A virus isolated from the outbreak among poultry popula-tions in Thailand. Virology 328, 169–176.