Genetic characterization of clade 126.96.36.199 avian influenza a(H5N1) viruses, Indonesia, 2012.
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 liL 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).
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; online Technical Appendix 1, wwwnc.cdc.gov/EID/article/20/4/13-0517-Techapp1.pdf).
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 188.8.131.52 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 184.108.40.206 virus was identified as clade 220.127.116.11 (Figure), indicating likely cocirculation.
The 8 clade 18.104.22.168 HA genes analyzed possessed a multibasic amino acid cleavage site (Table 1). The cleavage site sequence of the clade 22.214.171.124 viruses from Indonesia (PQREdelRRRKRaG) differed from recent clade 126.96.36.199 viruses (PQRESRRKKRaG) 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 188.8.131.52 viruses from Indonesia possessed 6 or 7 potential N-linked glycosylation sites (7 in clade 184.108.40.206 viruses), but unlike 220.127.116.11 viruses, all 18.104.22.168 viruses lacked the potential glycosylation site at position 154.
Up to 29 conserved amino acid changes occurred in the mature HA1 protein between clade 22.214.171.124 and clade 126.96.36.199 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 188.8.131.52 A(H5N1) candidate vaccine virus recommended by the World Health Organization (online Technical Appendix 2, wwwnc.cdc. gov/EID/article/20/4/13-0517-Techapp2.pdf) (12).
To test the antigenic relationship of the clade 184.108.40.206 virus to the endemic clade 220.127.116.11 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 18.104.22.168 antiserum did not inhibit hemagglutination by a representative clade 22.214.171.124 virus from Indonesia, A/environment/East Java/LBM-LM13/2012. In contrast, this virus cross-reacted with antiserum to clade 126.96.36.199 viruses from other countries at heterologous titers generally within 2-fold of or equivalent to the homologous virus titer. The Indonesian clade 188.8.131.52 virus was most closely related antigenically to viruses that clustered genetically into the A/Hong Kong/6841/2010-like group of clade 184.108.40.206 (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 (online Technical Appendix 1). Although partial nucleotide sequences from some genes were available for analysis (Table 1), sequence identities and phylogenetic comparisons to other clade 220.127.116.11 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 18.104.22.168 viruses and the previously identified clade 22.214.171.124 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 126.96.36.199 and 188.8.131.52 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 184.108.40.206 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.
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Address for correspondence: Ni Luh Putu Indi Dharmayanti, Virology Department, Indonesian Research Center for Veterinary Science, Jalan RE Martadinata 30, Bogor 16114, Indonesia; email: email@example.com
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); and Centers for Disease Control and Prevention, Jakarta (G. Samaan)
Table 1. Genetic characteristics of influenza A(H5N1) clade 220.127.116.11 viruses found in Indonesia, 2012 * ([dagger]) PB2 PB1-F2 aa aa PB1-F2 aa Strain name 627 701 truncation 66 A/Hubei/1/2010 E D 90 aa N A/Hong Kong/ E D 90 aa N 6841/2010 A/env/East Java/ E D 57 aa N LBM-LM13/2012 A/duck/Sukoharjo/ E D 57 aa N BBVW-1428-9/2012 A/duck/Bantul/ E D 57 aa N BBVW-1443-9/2012 A/duck/Sleman/ E D 57 aa N BBVW-1463-10/2012 A/md/Tegal/ ND ND ND ND BBVW-1732-11/2012 A/dk/Blitar/ ND ND ND ND BBVW-1731-11/2012 A/dk/Tegal/ ND ND ND ND BBVW-1727-11/2012 A/dk/Wonogiri/ ND ND ND ND BBVW-1730-11/2012 HA ([double dagger]) aa aa Strain name 222 224 Cleavage site A/Hubei/1/2010 Q G PQRERRRKR([down arrow])G A/Hong Kong/ Q G PQRERRRKR([down arrow])G 6841/2010 A/env/East Java/ Q G PQRERRRKR([down arrow])G LBM-LM13/2012 A/duck/Sukoharjo/ Q G PQRERRRKR([down arrow])G BBVW-1428-9/2012 A/duck/Bantul/ Q G PQRERRRKR([down arrow])G BBVW-1443-9/2012 A/duck/Sleman/ Q G PQRERRRKR([down arrow])G BBVW-1463-10/2012 A/md/Tegal/ Q G PQRERRRKR([down arrow])G BBVW-1732-11/2012 A/dk/Blitar/ Q G PQRERRRKR([down arrow])G BBVW-1731-11/2012 A/dk/Tegal/ Q G PQRERRRKR([down arrow])G BBVW-1727-11/2012 A/dk/Wonogiri/ Q G PQRERRRKR([down arrow])G BBVW-1730-11/2012 NA M2 NS sequence aa PDZ aa aa aa 80-84 binding Strain name 203 27 31 del ligand A/Hubei/1/2010 I I S Yes ESEV A/Hong Kong/ I I S Yes ESEV 6841/2010 A/env/East Java/ V I S Yes ESEV LBM-LM13/2012 A/duck/Sukoharjo/ I I S Yes ESEV BBVW-1428-9/2012 A/duck/Bantul/ I I S Yes ESEV BBVW-1443-9/2012 A/duck/Sleman/ ND I S Yes ESEV BBVW-1463-10/2012 A/md/Tegal/ ND ND ND ND ND BBVW-1732-11/2012 A/dk/Blitar/ ND ND ND ND ND BBVW-1731-11/2012 A/dk/Tegal/ ND ND ND ND ND BBVW-1727-11/2012 A/dk/Wonogiri/ ND ND ND ND ND BBVW-1730-11/2012 * HA, hemagluttinen; NA, neuraminidase; M2, matrix 2; NS, nonstructural; ND, not determined. ([dagger]) Numbering of the first and last nucleotide position of the gene that was sequenced is as follows: PB2, 1618-2192; PB1, 130-632; PA, 34-429; HA, 1-1710; NP, 268-755; NA, 55-1314; M, 55-919; NS, 25-690. ([double dagger]) Glycosylation motif at 154 was absent for all strains. Table 2. Hemagglutination-inhibition assay of clade 18.104.22.168 highly pathogenic avian influenza A(H5N1) virus introduced into Indonesia, 2012 * Reference ferret antiserum 1 2.2.1 2.3.4 22.214.171.124 IND/ Antigent Clade VN/1203 EG/321 ANH/1 12379 Reference strains VN/1203 1 320 *# 20 40 <10 EG/321 2.2.1 80 1,280# 80 20 ANH/1 2.3.4 160 80 640# 80 IND/12379 126.96.36.199 10 10 40 1,280# CH/1 RG30 188.8.131.52 40 80 20 20 BS/HK/1161 184.108.40.206 <10 40 10 <10 BHG/MG/X53 220.127.116.11 10 80 20 20 HK/6841 18.104.22.168 10 20 10 20 DK/VN/1584 22.214.171.124 <10 40 10 20 Test strain A/environment/East 126.96.36.199 <10 40 10 10 Java/LBM-LM13/2012 Reference ferret antiserum 188.8.131.52 184.108.40.206 220.127.116.11 CH/1 BS/HK/ BHG/MG/ Antigent RG30 1161 X53 Reference strains VN/1203 10 <10 10 EG/321 40 10 80 ANH/1 <10 <10 <10 IND/12379 <10 <10 <10 CH/1 RG30 640# 40 160 BS/HK/1161 320 80# 160 BHG/MG/X53 320 40 320# HK/6841 160 20 320 DK/VN/1584 320 20 320 Test strain A/environment/East 160 40 320 Java/LBM-LM13/2012 Reference ferret antiserum 18.104.22.168 22.214.171.124 HK/ DK/VN/ Antigent 6841 1584 Reference strains VN/1203 10 10 EG/321 80 40 ANH/1 40 10 IND/12379 40 40 CH/1 RG30 640 160 BS/HK/1161 640 80 BHG/MG/X53 640 320 HK/6841 640# 160 DK/VN/1584 320 160# Test strain A/environment/East 320 160 Java/LBM-LM13/2012 * Homologous titers of reference antigen to ferret antiserum are indicated in boldface. ([dagger]) Strains: VN/1203, A/Vietnam/1203/2004; EG/321, A/Egypt/2321-NAMRU3/2007; ANH/1, A/Anhui/1/2005; IND/12379, A/Indonesia/NIHRD12379/2012; CH/1 RG30, A/Hubei/1/2010 IDCDC-RG30; BS/HK/1161, A/barn swallow/Hong Kong/1161/2010; BHG/MG/X53, A/barheaded goose/Mongolia/X53/2009; HK/6841, A/Hong Kong/6841/2010; DK/VN/1584, A/duck/Vietnam/NCVD-1584/2012. Note: ferret antiserum of Homologous titers of reference antigen are indicated by #.
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|Author:||Dharmayanti, Ni Luh Putu Indi; Hartawan, Risza; Pudjiatmoko; Wibawa, Hendra; Hardiman; Balish, Amand|
|Publication:||Emerging Infectious Diseases|
|Date:||Apr 1, 2014|
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