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Genetic characterization of clade 2.3.2.1 avian influenza a(H5N1) viruses, Indonesia, 2012.

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 2.1.3.2 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 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 2.3.2.1 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 2.3.2.1 virus was identified as clade 2.1.3.2 (Figure), indicating likely cocirculation.

The 8 clade 2.3.2.1 HA genes analyzed possessed a multibasic amino acid cleavage site (Table 1). The cleavage site sequence of the clade 2.3.2.1 viruses from Indonesia (PQREdelRRRKRaG) differed from recent clade 2.1.3.2 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 2.3.2.1 viruses from Indonesia possessed 6 or 7 potential N-linked glycosylation sites (7 in clade 2.1.3.2 viruses), but unlike 2.1.3.2 viruses, all 2.3.2.1 viruses lacked the potential glycosylation site at position 154.

Up to 29 conserved amino acid changes occurred in the mature HA1 protein between clade 2.3.2.1 and clade 2.1.3.2 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 2.3.2.1 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 2.3.2.1 virus to the endemic clade 2.1.3.2 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 2.1.3.2 antiserum did not inhibit hemagglutination by a representative clade 2.3.2.1 virus from Indonesia, A/environment/East Java/LBM-LM13/2012. In contrast, this virus cross-reacted with antiserum to clade 2.3.2.1 viruses from other countries at heterologous titers generally within 2-fold of or equivalent to the homologous virus titer. The Indonesian clade 2.3.2.1 virus was most closely related antigenically to viruses that clustered genetically into the A/Hong Kong/6841/2010-like group of clade 2.3.2.1 (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 2.3.2.1 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 2.3.2.1 viruses and the previously identified clade 2.1.3.2 genotype virus in Indonesia.

Conclusions

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 2.3.2.1 and 2.1.3.2 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 2.3.2.1 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.

References

(1.) World Health Organization. Evolution of H5N1 avian influenza viruses in Asia. Emerg Infect Dis. 2005; 11:1515-21. http://dx.doi. org/10.3201/eid1110.050644

(2.) Lam TT, Hon CC, Pybus OG, Kosakovsky Pond SL, Wong RT, Yip CW, et al. Evolutionary and transmission dynamics of reassortant H5N1 influenza virus in Indonesia. PLoS Pathog. 2008; 4:e1000130. http://dx.doi.org/10.1371/journal.ppat.1000130

(3.) Vijaykrishna D, Bahl J, Riley S, Duan L, Zhang JX, Chen H, et al. Evolutionary dynamics and emergence of panzootic H5N1 influenza viruses. PLoS Pathog. 2008; 4:e1000161. http://dx.doi.org/10.1371/ journal.ppat.1000161

(4.) World Health Organization. Towards a unified nomenclature system for the highly pathogenic H5N1 avian influenza viruses. 2007 [cited 2011 Jul 8]. http://www.who.int/csr/disease/avian_influenza/ guidelines/nomenclature/en/.

(5.) Swayne DE. Understanding the complex pathobiology of high pathogenicity avian influenza viruses in birds. Avian Dis. 2007; 51 (Suppl):242-9. http://dx.doi.org/10.1637/7763-110706-REGR.1

(6.) Wibawa H, Prijono WB, Dharmayanti NLPI, Irianingsih SH, Miswati Y, Anieka R, et al. 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).

(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; 38:4096-101.

(8.) World Health Organization. WHO manual on animal influenza diagnosis and surveillance. 2002 [cited 2011 Jul 8]. http://www.who.int/ csr/resources/publications/influenza/en/whocdscsrncs20025rev.pdf

(9.) Younan M, Poh MK, Elassal E, Davis T, Rivailler P, Balish AL, et al. Microevolution of highly pathogenic avian influenza A(H5N1) viruses isolated from humans, Egypt, 2007-2011. Emerg Infect Dis. 2013; 19:43-50. http://dx.doi.org/10.3201/eid1901.121080

(10.) Tamura K, Dudley J, Nei M, Kumar S. MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0. Mol Biol Evol. 2007; 24:1596-9. http://dx.doi.org/10.1093/molbev/msm092

(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; 312:404-10. http://dx.doi.org/10.1126/science.1124513

(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]. http:// www.who.int/influenza/vaccines/virus/201302_h5h7h9_vaccine virusupdate.pdf

(13.) Schmolke M, Manicassamy B, Pena L, Sutton T, Hai R, Varga ZT, et al. 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. http://dx.doi.org/10.1371/ journal.ppat.1002186

(14.) World Organization for Animal Health. Outbreaks of highly pathogenic avian influenza (subtype H5N1) in poultry. 2013 [cited 2013 Feb 15]. http://www.oie.int/animal-health-in-the-world/updateon-avian-influenza/2013/

(15.) World Health Organization. Situation updates--avian influenza. 2013 [cited 2013 Feb 15]. http://www.who.int/influenza/human_ animal_interface/avian_influenza/archive/en/index.html

Address for correspondence: Ni Luh Putu Indi Dharmayanti, Virology Department, Indonesian Research Center for Veterinary Science, Jalan RE Martadinata 30, Bogor 16114, Indonesia; email: nlpdharmayanti@yahoo.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)

DOI: http://dx.doi.org/10.3201/eid2004.130517

Table 1. Genetic characteristics of influenza A(H5N1) clade 2.3.2.1
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 2.3.2.1 highly
pathogenic avian influenza A(H5N1) virus introduced into Indonesia,
2012 *

                                      Reference ferret antiserum

                                    1      2.2.1    2.3.4   2.1.3.2

                                                             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            2.1.3.2     10        10      40     1,280#
  CH/1 RG30            2.3.2.1     40        80      20       20
  BS/HK/1161           2.3.2.1     <10       40      10       <10
  BHG/MG/X53           2.3.2.1     10        80      20       20
  HK/6841              2.3.2.1     10        20      10       20
  DK/VN/1584           2.3.2.1     <10       40      10       20

Test strain
  A/environment/East   2.3.2.1     <10       40      10       10
  Java/LBM-LM13/2012

                         Reference ferret antiserum

                       2.3.2.1   2.3.2.1   2.3.2.1

                        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

                       2.3.2.1   2.3.2.1

                         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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Title Annotation:DISPATCHES
Author:Dharmayanti, Ni Luh Putu Indi; Hartawan, Risza; Pudjiatmoko; Wibawa, Hendra; Hardiman; Balish, Amand
Publication:Emerging Infectious Diseases
Geographic Code:9INDO
Date:Apr 1, 2014
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