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

Virus Research 122 (2006) 194–199

Short communication

Genetic characterization of influenza A viruses (H5N1) isolated from 3rd wave of Thailand AI outbreaks Alongkorn Amonsin a , Salin Chutinimitkul b , Nuananong Pariyothorn a , Thaweesak Songserm c , Sudarat 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, Thailand c 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 2006 Available online 28 August 2006

Abstract Three major outbreaks of avian influenza (AI) occurred in Thailand. During the third episode in October 2005, we have isolated H5N1 viruses from one human case and three poultry cases. The whole genomes of AI viruses from human, chickens and quail from the outbreaks were characterized. Sequence analysis of eight gene segments revealed that the 2005 H5N1 viruses isolated in October 2005 were closely related to those recovered 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 site have been observed. © 2006 Elsevier B.V. All rights reserved. Keywords: H5N1; Characterization; Influenza A virus; Thailand

H5N1 influenza A virus causes avian influenza (AI), which poses a serious threat to public health, as it can be directly transmitted from poultry to humans. In Thailand, the AI outbreaks appeared to emerge in three major episodes. The first outbreak of H5N1 avian influenza was reported in Thailand in early January 2004. It lasted until March, leading to 12 cases of human infection with 8 fatalities. The second outbreak occurred in July 2004; it had run its course by the end of the year leaving in its wake five human cases with four fatalities (Tiensin, 2005). In October–December 2005, the third outbreak was reported resulting in five human cases with two fatalities. One of the fatalities reported in October 2005 was a 48-year-old chicken slaughterer in Kanchanaburi province, western part of Thailand. Moreover, his son was hospitalized and confirmed infected ∗

Correspondence to: Center of Excellence in Viral Hepatitis Research, Department of Pediatrics, Faculty of Medicine, Chulalongkorn University and Hospital, Rama IV Road, Patumwan, Bangkok 10330, Thailand, Tel.: +66 2 256 4909; fax: +66 2 256 4929. E-mail address: [email protected] (Y. Poovorawan). 0168-1702/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.virusres.2006.06.016

by H5N1. Another fatality, reported in December 2005, was a 5-year-old boy in the central province of Nakorn Nayok. In addition, two infected individuals were reported as a worker in a local chicken farm in Nontaburi province and an 18-month-old boy in the suburban of Bangkok (Fig. 1). During the third outbreak, a great number of poultry was reported dead in the villages of Kanchanaburi, Nakorn Pathom, Nontaburi and Nakorn Nayok provinces. Suspected samples from chickens and quail carcasses were collected and further processed for virus isolation by embryonated egg inoculation as the standard procedure recommended by OIE. In addition, the plasma sample from human patient in Nakorn Nayok reported by Chutinimitkul et al. (2006) was processed for virus isolation by using embryonated egg inoculation. The H5N1 virus could be isolated from plasma on the day 10 after patient developed symptoms. This case showed the live virus in the patient’s blood sample which could be raising a reminder of the necessity to carefully handle and transport serum or plasma samples of patients suspected of H5N1 AI infection. Subsequently, we applied hemagglutination (HA) and hemagglutination inhibition

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Fig. 1. Map of Thailand. The provinces with reported H5N1 outbreaks (Kanchanaburi, Nakorn Pathom, Nakorn Nayok and Nontaburi) are depicted.

(HI) tests to identify influenza A virus and confirmed H5N1 influenza A virus by multiplex RT-PCR aimed at detecting the M, HA and NA genes as previously described (Payungporn et al., 2004). Four H5N1 viruses including one isolate derived from human, “A/Thailand/NK165/05” (NK-165), two isolates derived from chickens, “A/Chicken/Thailand/CK160/05” (CK-160), “A/Chicken/Thailand/CK162/05” (CK-162) and one isolate derived from quail “A/Quail/Thailand/QA161/05” (QA-161) were included in the present study. The chicken viruses (CK-160, CK-162) were isolated from the same districts as the human case (Fig. 1). Whole genome sequencing was performed as previously described (Viseshakul et al., 2004). Briefly, viral RNA was isolated from allantoic fluid using the RNAeasy mini kit (Qiagen). PCR products of each gene segment were generated using RTPCR with primers specific for each gene. The PCR products were purified using the Perfectprep Gel Cleanup Kit (Eppendrof). Direct PCR product sequencing was carried out using Big Dye Terminator V.3.0 Cycle Sequencing Ready Reaction (ABI, Foster City, CA) and the ABI-Prism 310 Genetic Analyzer (PerkinElmer, Norwalk, CT). The sequences were verified and assembled using computer program; Bioedit 5.0.9 (Ibis Therapeutics, Carlsbad, CA). The nucleotide sequences of eight gene segments of 2005 H5N1 viruses were submitted to GenBank under accession numbers, NK-165 (DQ372591–DQ372598), CK-160 (DQ334757–DQ334762), QA-161 (DQ334765–DQ334772) and CK-162 (DQ334773–DQ334780). Phylogenetic and genetic analyses were carried out using the computer program MegAlign (DNASTAR, Madison, WI). The analyses were performed to compare all genes of the 2005 H5N1 viruses (human (NK-165), chicken (CK-160), quail (QA-161) and chicken (CK-162)), with those of 2004 H5N1 viruses representing the 1st wave (chicken (CU-K2), human (SP-33) and tiger (Ti-1)) (Keawcharoen et al., 2004; Li et al., 2004; Viseshakul et al., 2004) and 2nd wave of the outbreaks (chicken (CU-23) and tiger (CU-T3)) (Amonsin et al., 2005;

Thanawongnuwech et al., 2005). In addition, the H5N1 viruses representing the current outbreaks in late 2005 to early 2006 in Qinghai, Western China (A/Goose/Qinghai/61/05(H5N1); China-61) (Liu et al., 2005), Astrakhan, Russia (A/Cygnus Olor/Astrakhan/Ast05-2-9/05(H5N1); Russia-2-9) and Africa (A/Chicken/Nigeria/641/06; Nigeria-641) were included in the analyses. Phylogenetic analysis of the HA and NA genes showed that H5N1 viruses from the 2005 AI outbreaks clustered in the same groups as H5N1 isolates from 2004, which had been identified as genotype Z (Fig. 2). On the other hand, the H5N1 isolates from Western China, Russia and Nigeria were grouped into separated cluster. In addition, phylogenetic analyses of the other six gene segments of the H5N1 isolates including PA, PB1, PB2, NP, NS and M, also showed high degrees of genetic relatedness to the human, chicken and tiger isolates from Thailand in 2004 (data not shown). Generally, phylogenetic analyses of all gene segments indicated that the 2005 H5N1 viruses were closely related and grouped with the H5N1 isolates responsible for the 2nd wave of the AI outbreaks in Thailand. Nucleotide identities between the genomes of the chicken viruses (CK-160) compared to those of the 2005–2006 H5N1 viruses (QA-161, CK-162, NK-165, China-61, Russia-2-9 and Nigeria-641), the 2004 H5N1 viruses (CU-K2, Ti-1, SP-33, CU23, CU-T3) and the 1996 H5N1 virus (GD-1), are shown as percentages in Table 1. Our results showed a striking similarity between the H5N1 viruses from the 2005 outbreaks and those from the 2004 outbreaks with percentages of nucleotide identity ranging from 97.8 to 100%. Conversely, the 2005 H5N1 isolate (CK-160) displayed a lower percentage of nucleotide identity with the 1996 H5N1 isolate from China (GD-1) as well as the current H5N1 isolates from Western China (China-61), Russia (Russia-2-9) and Nigeria (Nigeria-641), especially with regard to the NS gene (96.4–96.6%). This result supported the conclusion that the 2005 H5N1 viruses circulating in Thailand were genetically comparable with 2004 H5N1 isolates in Thailand and Vietnam, but distinct from the H5N1 virus strains circulat-

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Fig. 2. Phylogenetic analysis of the HA and NA genes of H5N1 viruses.

ing in Asia such as Hong Kong, China, Indonesia (1996–2005) and Europe (2005) as well as in Africa (2006). The 2005 H5N1 viruses harbor multiple insertions of basic amino acids at the HA cleavage site, which by definition is characteristic for highly pathogenic avian influenza (HPAI) (Claas et al., 1998). Interestingly, the HA cleavage site of 2005 H5N1 viruses in this study contained one basic amino acid “SPQREKRRKKR” differing from

that of 2004 H5N1 viruses in Thailand, Indonesia, Vietnam and Eastern China “SPQRERRRKKR” (arginine (R) to lysine (K)). It is noted that HA cleavage site of the 2005 H5N1 viruses from Vietnam (mallard/347/05) and Indonesia (duck/Parepare/BBVM/05) remain unchanged “SPQRERRRKK”. On the other hand, the HA cleavage site variability have been previously observed in some wild bird species during earlier outbreaks in Thailand in 2004 (open-bill/CU-

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

Region of comparison (bp)a

Nucleotide identity (%) China

PB2 PB1 PA HA NP NA M NS a b c d e f g h i j

82–2220 63–2226 28–2119 46–1623 58–1427 28–1296 1–952 36–815

Thailand: 1st wave of outbreaks c

d

Thailand: 2nd wave of outbreaks e

f

Thailand: 3rd wave of outbreaks

GD-1 , 1996 (goose)

CU-K2 , Jan-04 (chicken)

SP-33 , Jan-04 (human)

Ti-1 , Jan-04 (tiger)

CU-23, Jul-04 (chicken)

CU-T3 , Oct-04 (tiger)

QA-161, Oct-05 (quail)

93.9 93.2 93.2 96.5 92.4 91.2 96.5 69.3

99.2 99.2 99.4 97.9 99.4 98.9 99.9 99.1

98.9 99.2 99.2 99.0 99.4 98.9 99.8 99.4

99.3 99.4 99.5 99.2 99.4 99.0 99.8 99.5

99.0 99.2 99.3 99.2 99.2 98.6 99.4 99.5

98.8 99.1 99.2 99.0 99.2 98.5 99.5 99.1

99.7 99.6 99.8 99.5 99.9 99.6 99.7 99.7

CK-162, Oct-05 (chicken) 99.8 99.8 100 99.9 99.9 99.7 99.6 99.6

China g

Russia h

Nigeria i

NK-165 , Dec-05 (human)

China-61 , Oct-05 (goose)

Russia-2-9 , Dec-05 (Cygnus Olor)

Nigeria-641j , Jan-06 (chicken)

99.4 99.4 99.3 99.5 99.6 99.4 99.7 99.9

97.2 97.4 97.7 96.5 97.7 96.8 98.2 96.6

96.8 NA NA 96.1 97.4 96.8 97.7 96.4

NA NA NA 96.1 NA NA NA NA

Position of nucleotides is based on A/Chicken/Thailand/CK160/05(H5N1). A/Goose/Guangdong/1/96(H5N1). A/Chicken/Nakorn Pathom/Thailand/CU-K2/04(H5N1) (Viseshakul et al., 2004). A/Thailand/2(SP-33)/04(H5N1) (Li et al., 2004). A/Tiger/Suphanburi/Thailand/Ti-1/04(H5N1) (Keawcharoen et al., 2004). A/Chicken/Ayutthaya/Thailand/CU-23/04(H5N1) and A/Tiger/Thailand/CU-T3/04(H5N1) (Amonsin et al., 2005). 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). A/Goose/Qinghai/61/05(H5N1) (Liu et al., 2005). A/Cygnus Olor/Astrakhan/Ast05-2-9/05(H5N1). A/Chicken/Nigeria/641/06(H5N1).

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b

197

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Fig. 3. Alignment of HA1 amino acid sequences of 2005 AI viruses with those of H5N1 isolates from 1996 to 2004 and those of Western China, Europe and Nigeria from 2005 to 2006. The hemagglutinin cleavage site contained multiple basic amino acids of previous outbreaks (SPQRERRRKKR) and current outbreaks (SPQREKRRKKR).

2/04 “SPQREKRRKKR”, white peafowl/CU-16/04 and Kalij pheasant/CU-18/04 “SPQRERKRKKR”) (Keawcharoen et al., 2005). This finding showed evidence of amino acid substitution at the pathogenic site of the HA gene of H5N1 viruses circulating in Thailand. In addition, the genetic change at one amino acid upstream of the HA cleavage site of 2005–2006 H5N1 viruses in Western China, Russia and Africa have also been observed “SPQGERRRKKR” (arginine (R) to glycine (G)) as shown in Fig. 3. The 2005 H5N1 viruses also contained the same amino acids (Q222 and G224) at the receptor binding site of the HA gene (positions 222–224) as has been previously reported with chicken, human and tiger isolates (Amonsin et al., 2005). This finding demonstrated that the 2005 H5N1 isolates still possess avian-specific receptor binding properties (Matrosovich et al., 1999). In this study, a 20-amino acid deletion at the NA stalk region (positions 49–68) was discovered in all 2005–2006 H5N1 isolates as well as 2004 H5N1 isolates from Thailand, yet could not be found in the 1996 H5N1 isolates from China (GD-1). This shortening of the NA stalk region presumably represents an adaptation of the H5N1 viruses (Matrosovich et al., 1999). Amino acids conveying oseltamivir resistance were not discernible among the conserved residues (E119V, H274Y, R292K, N294S) at the NA active site of any 2005 H5N1 isolates from Thailand as well as the isolates from Western China, Europe and Africa, whereas oseltamivir resistant H5N1 isolates had been reported in Vietnam (Le et al., 2005). In our investigation of more than 100 H5N1 isolates, we have never encountered oseltamivir resistant amino acids in those particular residues (data not shown). Alterations of the amino acid at position 31 (serine (S)) of the M2 protein can result in amantadine resistance. All

H5N1 viruses isolated in 2004–2005 contained asparagine (N) at residue 31 of the M2 protein and therefore, the viruses were presumably resistant to the amantadines. Five amino acid deletions at positions 79–83 of the NS gene were observed in all 2004–2005 H5N1 viruses in Thailand, but were not found in the H5N1 virus from China (GD-1). The purpose of this deletion remains as yet unknown. In addition, another virulence marker in NS gene, the mutation of aspartic acid (D) to glutamic acid (E) at position 92 of the NS1 protein, associated with high virulence of the virus in mammalian species, was not observed in this study. We further analyzed the amino acids of the polymerase (PA, PB1, PB2) and nucleoprotein (NP) genes. In none of the 2004/2005 H5N1 isolates from poultry did we discover any single amino acid substitutions at position 627 of the PB2 protein (glutamic acid (E) to lysine (K)). In contrast, we observed a change of the amino acid at position 627 from glutamic acid (E) to lysine (K) in human H5N1 isolate in this study (NK-165) as well as H5N1 isolates from tigers and human in Thailand from previous analyses (Amonsin et al., 2005). It is interesting to note that all avian isolates from the current outbreaks in Western China, Russia and Nigeria contain Lysine (K) at position 627 of the PB2 protein. Lysine (K) at position 627 of the PB2 gene is deemed responsible for an increase in virus replication efficiency especially in mammals (Shinya et al., 2004). Since H5N1 viruses circulating in Thailand, particularly the human and tiger isolates, harbor both lysine (K) and glutamic acid (E) at PB2 627 (Amonsin et al., 2005; Li et al., 2004), it is important to comprehensively monitor the PB2 627 position, especially in chicken isolates. To summarize, this study highlights the significance of genetic characterization presenting as examples of 4 H5N1

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viruses isolated from human, chickens and quail which isolated from Thailand in 2005. Sequence analysis of eight gene segments revealed that the 2005 H5N1 viruses isolated in October 2005 were related to those recovered from chicken, tiger(s) and human(s) in January and July 2004 but not related to the viruses from the recent outbreaks in Western China, Eurasia and Africa. Interestingly, amino acid substitution especially at the HA cleavage site has been observed. However, the amino acid substitution still basic protein which can cause highly pathogenic to host. It shows that H5N1 viruses have continued to evolve since early 2004–2005 of Thailand with minor change, alternatively unchanged in pathogenicity. Acknowledgements This study was supported by a grant from the National Research Council of Thailand, the Thailand Research Fund, Senior Research Scholar and the funding from Chulalongkorn University. The authors would like to thank Dr. Surangrat Srisuratanon, Department of Pediatrics, Faculty of Medicine, Srinakharinwirot University, Nakorn Nayok, for her assistance. References Amonsin, 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 zoo tigers in Thailand. Virology 344, 480–491. Claas, 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. Human influenza A H5N1 virus related to a highly pathogenic avian influenza virus. Lancet 351, 472–477. Keawcharoen, 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., Osterhaus, D.M., Poovorawan, Y., 2004. Avian influenza H5N1 in tigers and leopards. Emerg. Infect. Dis. 10, 2189–2191. Keawcharoen, 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,

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