Skip directly to site content Skip directly to page options Skip directly to A-Z link Skip directly to A-Z link Skip directly to A-Z link
Volume 20, Number 4—April 2014

Genetic Characterization of Clade Avian Influenza A(H5N1) Viruses, Indonesia, 2012

Ni Luh Putu Indi DharmayantiComments to Author , Risza Hartawan, Pudjiatmoko, Hendra Wibawa, Hardiman, Amanda Balish, Ruben Donis, C. Todd Davis, and Gina Samaan
Author affiliations: Indonesian Research Center for Veterinary Science, Bogor, Indonesia (N.L.P.I. Dharmayanti, R. Hartawan, Hardiman); Ministry of Agriculture, Jakarta, Indonesia (Pudjiatmoko); Disease Investigation Center Wates, Jogjakarta, Indonesia (H. Wibawa); Centers for Disease Control and Prevention, Atlanta, Georgia, USA (A. Balish, R. Donis, C.T. Davis); Centers for Disease Control and Prevention, Jakarta (G. Samaan)

Cite This Article


After reports of unusually high mortality rates among ducks on farms in Java Island, Indonesia, in September 2012, influenza A(H5N1) viruses were detected and characterized. Sequence analyses revealed all genes clustered with contemporary clade viruses, rather than enzootic clade 2.1.3 viruses, indicating the introduction of an exotic H5N1 clade into Indonesia.

Highly pathogenic avian influenza A(H5N1) virus has circulated in poultry in Indonesia since 2003 (1,2). The phylogeny of A(H5N1) viruses detected during 2003–2011 indicated all genes descended from 1 ancestral virus with a clade 2.1 hemagglutinin (HA) introduced into Indonesia before 2003 (3). These viruses became enzootic and evolved into second-, third-, and fourth-order HA clades, leading to the recent dominance of clade viruses (4). Outbreaks in poultry typically caused high mortality rates among gallinaceous birds, especially layer, broiler, and native chickens. The virus seemed less pathogenic in aquatic birds (5). However, reports of duck deaths and a higher than usual mortality rate (100% in some outbreaks) in backyard farms in Central Java, Jogjakarta, and East Java Provinces, Indonesia, in September 2012 triggered a joint outbreak investigation by animal and public health authorities (6). We describe the genetic characteristics of viruses isolated from A(H5N1) infection outbreaks in these 3 provinces on Java Island, where a previously unrecognized clade was detected.

The Study

We investigated 9 small-holding duck farms that reported bird deaths during September 12–November 5, 2012 (6). Cloacal swab samples were collected from sick birds, placed in 1,000 µL of viral transport medium, and sent for testing at laboratories of the regional Ministry of Agriculture Disease Investigation Center, Jogjakarta. Seventeen A(H5N1)–positive samples were forwarded to the National Animal Health Laboratory, Indonesian Research Center for Veterinary Science (IRCVS), for virus isolation and genome sequencing.

In addition, IRCVS collected 122 cloacal swab samples from birds and 58 environmental swab samples (from defeathering machines) at 5 live-bird markets (LBMs) in East Java Province during November 5–8, 2012. RNA extracted from farm and LBM specimens was tested for influenza A matrix gene to identify presumptive A(H5N1)-positive samples (7). Select positive samples were inoculated in 9–11-day-old embryonated, specific pathogen–free eggs. Allantoic fluid was harvested 36 h postinfection and tested for HA with chicken erythrocytes to confirm virus isolation (8).


Thumbnail of Partial phylogenetic tree of influenza A(H5N1) hemagglutinin (HA) gene sequences. The phylogenetic tree was generated in MEGA version 4 (, using neighbor-joining analysis with 1,000 bootstrap replicates and the Kimura 2-parameter model. Viruses characterized in this study are indicated with a dot. The HA tree was rooted to A/goose/Guangdong/1/1996.

Figure. . . Partial phylogenetic tree of influenza A(H5N1) hemagglutinin (HA) gene sequences. The phylogenetic tree was generated in MEGA version 4 (, using neighbor-joining analysis with 1,000 bootstrap replicates and...

Samples showing suspected A(H5N1) infection were propagated in a Biosafety Level-3 laboratory at IRCVS in compliance with biosafety regulations. Ten virus isolates (7 from duck farms, 3 from LBMs) were chosen for full-length HA gene sequencing (GenBank accession nos. KC417271–KC417277, KC757643); 4 were selected for genome sequencing. Results of reverse transcription PCR and sequencing primers are available on request. Sequencing and consensus sequence generation were conducted as described (9). Phylogenetic trees were generated by using MEGA4 (10) (Figure; Technical Appendix 1).

Phylogenetic analysis revealed that A(H5N1) isolates from samples collected from duck farm outbreaks and an LBM were not related to isolates in long-established Indonesian clade 2.1; rather, the HA genes closely resembled those of clade viruses recently found in Vietnam, China, and Hong Kong (Figure). Full-length HA genes showed 97%–98%-nt identity with recent viruses from Vietnam and clustered in a larger group containing viruses from many Asian regions during 2009–2012. The environmental sample from an East Java LBM shared >99% nt similarity with viruses from samples at duck farms, indicating spread of this A(H5N1) clade into the marketing chain. A poultry sample from the same district as the virus was identified as clade (Figure), indicating likely cocirculation.

The 8 clade HA genes analyzed possessed a multibasic amino acid cleavage site (Table 1). The cleavage site sequence of the clade viruses from Indonesia (PQREdelRRRKR↓G) differed from recent clade viruses (PQRESRRKKR↓G) by a Ser deletion at position 325 and a K328R substitution. Like other serotype H5N1 HA proteins, all isolates possessed a conserved glutamine at position 222 (equivalent to H3 position 226) and glycine at position 224 (H3 position 228), indicating no substantial changes in avian receptor-binding specificity (Table 1) (11). The clade viruses from Indonesia possessed 6 or 7 potential N-linked glycosylation sites (7 in clade viruses), but unlike viruses, all viruses lacked the potential glycosylation site at position 154.

Up to 29 conserved amino acid changes occurred in the mature HA1 protein between clade and clade viruses found recently in Indonesia, indicating these A(H5N1) virus subgroups probably diverged substantially in antigenicity. In contrast, the HA1 of the new viruses collected in Indonesia differed by 8–10 aa from A/Hubei/1/2010, the most closely related clade A(H5N1) candidate vaccine virus recommended by the World Health Organization (Technical Appendix 2) (12).

To test the antigenic relationship of the clade virus to the endemic clade virus, we conducted a hemagglutination-inhibition test with ferret antiserum raised against viruses from these and other H5N1 clades (Table 2) (8). As the HA1 protein sequence differences suggest, clade antiserum did not inhibit hemagglutination by a representative clade virus from Indonesia, A/environment/East Java/LBM-LM13/2012. In contrast, this virus cross-reacted with antiserum to clade viruses from other countries at heterologous titers generally within 2-fold of or equivalent to the homologous virus titer. The Indonesian clade virus was most closely related antigenically to viruses that clustered genetically into the A/Hong Kong/6841/2010-like group of clade (Table 2).

All 4 isolates exhibited the typical 20-aa deletion in the stalk region (residue 48–68) of the neuraminidase gene (NA). Although 1 sample had an Ile203Val substitution in the NA, which has been associated with reduced susceptibility to oseltamivir, no other markers of resistance in the NA or M2 were identified (Table 1). All 4 viruses had NS1 protein sequences with the typical deletion at position 80–84 and an intact H5N1 consensus PDZ binding motif (ESEV). A truncated form (57 aa) of the PB1-F2 protein was found in all viruses characterized. Although the functional consequences of this truncation are unknown, this represents a change from the typical full-length 90-aa protein found in most A(H5N1) viruses (13). All other amino acid residues and motifs of interest in the internal genes of the 4 viruses sequenced in this study represented avian consensus sequences.

Phylogenetic comparison of the NA and internal gene segments revealed ancestral origins of the new viruses similar to those of the HA gene (Technical Appendix 1). Although partial nucleotide sequences from some genes were available for analysis (Table 1), sequence identities and phylogenetic comparisons to other clade genomes in GenBank and Global Initiative on Sharing Avian Influenza Data databases confirmed their relatedness to viruses circulating recently in China, Vietnam, and Hong Kong. Individual gene sequence analysis did not show reassortment between these clade viruses and the previously identified clade genotype virus in Indonesia.


Detection of a novel clade of A(H5N1) virus in Indonesia marks a potential turning point in the molecular epidemiology of this virus. Indonesia has the highest number of human A(H5N1) infections because of ongoing outbreaks in poultry (14,15). Whether this new virus will become entrenched, as did clade 2.1.3 viruses over the past decade, remains to be seen, as do its effects on the incidence of human infection. Potential cocirculation of subtypes of 2 different clades warrants review of diagnostic methods and vaccination strategy to maximize effectiveness of disease control interventions. The lack of antigenic relatedness between the clade and viruses must be considered when evaluating A(H5N1) serologic diagnostic reagents used in Indonesia. This change also may have implications in selecting prepandemic candidate vaccine virus for the region. Furthermore, poultry vaccines may need to be matched antigenically to circulating virus if clade virus continues to circulate in Indonesia. Introduction of this virus is a stark reminder of the value of control measures to reduce the spread of subtype H5N1 and the need for enhanced surveillance of humans and poultry to monitor changes in its genetic and immunologic features.

Dr Dharmayanti is a researcher in the Virology Department at the Indonesian Research Center for Veterinary Science, Ministry of Agriculture. Her primary research interest is avian influenza.



  1. World Health Organization. Evolution of H5N1 avian influenza viruses in Asia. Emerg Infect Dis. 2005;11:151521 . DOIPubMedGoogle Scholar
  2. Lam  TT, Hon  CC, Pybus  OG, Kosakovsky Pond  SL, Wong  RT, Yip  CW, Evolutionary and transmission dynamics of reassortant H5N1 influenza virus in Indonesia. PLoS Pathog. 2008;4:e1000130. DOIPubMedGoogle Scholar
  3. Vijaykrishna  D, Bahl  J, Riley  S, Duan  L, Zhang  JX, Chen  H, Evolutionary dynamics and emergence of panzootic H5N1 influenza viruses. PLoS Pathog. 2008;4:e1000161 . DOIPubMedGoogle Scholar
  4. World Health Organization. Towards a unified nomenclature system for the highly pathogenic H5N1 avian influenza viruses. 2007 [cited 2011 Jul 8].
  5. Swayne  DE. Understanding the complex pathobiology of high pathogenicity avian influenza viruses in birds. Avian Dis. 2007;51(Suppl):2429 . DOIPubMedGoogle Scholar
  6. Wibawa  H, Prijono  WB, Dharmayanti  NLPI, Irianingsih  SH, Miswati  Y, Anieka  R, Disease outbreak investigation in ducks in Central Java, Jogjakarta and East Java: identification of a new clade of avian influenza A(H5N1) virus in Indonesia [in Indonesian]. Buletin Laboratorium Veteriner. 2012;12(4). DOIPubMedGoogle Scholar
  7. Fouchier  RA, Bestebroer  TM, Herfst  S, Van Der Kemp  L, Rimmelzwaan  GF, Osterhaus  AD. Detection of influenza A viruses from different species by PCR amplification of conserved sequences in the matrix gene. J Clin Microbiol. 2000;38:4096101 .PubMedGoogle Scholar
  8. World Health Organization. WHO manual on animal influenza diagnosis and surveillance. 2002 [cited 2011 Jul 8].
  9. Younan  M, Poh  MK, Elassal  E, Davis  T, Rivailler  P, Balish  AL, Microevolution of highly pathogenic avian influenza A(H5N1) viruses isolated from humans, Egypt, 2007–2011. Emerg Infect Dis. 2013;19:4350 . DOIPubMedGoogle Scholar
  10. Tamura  K, Dudley  J, Nei  M, Kumar  S. MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0. Mol Biol Evol. 2007;24:15969. DOIPubMedGoogle Scholar
  11. Stevens  J, Blixt  O, Tumpey  TM, Taubenberger  JK, Paulson  JC, Wilson  IA. Structure and receptor specificity of the hemagglutinin from an H5N1 influenza virus. Science. 2006;312:40410. DOIPubMedGoogle Scholar
  12. World Health Organization. Antigenic and genetic characteristics of zoonotic influenza viruses and development of candidate vaccine viruses for pandemic preparedness. 2013 [cited 2013 Mar 4].
  13. Schmolke  M, Manicassamy  B, Pena  L, Sutton  T, Hai  R, Varga  ZT, Differential contribution of PB1–F2 to the virulence of highly pathogenic H5N1 influenza A virus in mammalian and avian species. PLoS Pathog. 2011;7:e1002186. DOIPubMedGoogle Scholar
  14. World Organization for Animal Health. Outbreaks of highly pathogenic avian influenza (subtype H5N1) in poultry. 2013 [cited 2013 Feb 15].
  15. World Health Organization. Situation updates—avian influenza. 2013 [cited 2013 Feb 15].




Cite This Article

DOI: 10.3201/eid2004.130517

Table of Contents – Volume 20, Number 4—April 2014

EID Search Options
presentation_01 Advanced Article Search – Search articles by author and/or keyword.
presentation_01 Articles by Country Search – Search articles by the topic country.
presentation_01 Article Type Search – Search articles by article type and issue.



Please use the form below to submit correspondence to the authors or contact them at the following address:

Ni Luh Putu Indi Dharmayanti, Virology Department, Indonesian Research Center for Veterinary Science, Jalan RE Martadinata 30, Bogor 16114, Indonesia

Send To

10000 character(s) remaining.


Page created: March 20, 2014
Page updated: March 20, 2014
Page reviewed: March 20, 2014
The conclusions, findings, and opinions expressed by authors contributing to this journal do not necessarily reflect the official position of the U.S. Department of Health and Human Services, the Public Health Service, the Centers for Disease Control and Prevention, or the authors' affiliated institutions. Use of trade names is for identification only and does not imply endorsement by any of the groups named above.