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Multiple introductions of Avian influenza viruses (H5N1), Laos, 2009-2010.

Avian influenza viruses (H5N1) of clades 2.3.4.1, 2.3.4.2, and 2.3.2.1 were introduced into Laos in 20092010. To investigate these viruses, we conducted active surveillance of poultry during March 2010. We detected viruses throughout Laos, including several interclade reassortants and 2 subgroups of clade 2.3.4, one of which caused an outbreak in May 2010.

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Since 2003, highly pathogenic avian influenza virus (H5N1) has spread from southern China throughout Southeast Asia and to Europe and Africa (1,2). Since 2003, Laos has experienced outbreaks of clade 1 (2003), clade 2.3.4 (2006, 2007, 2008, 2009, 2010), and clade 2.3.2 viruses (twice in 2008) (3,4). Active surveillance of domestic ducks and chickens in Laos has been limited, but serum antibodies against subtypes H5 and H9 have been detected in ducks. In addition, subtype H5N1 virus was isolated from healthy ducks in 2006, and subtype H3N8 virus was detected in 2007 (4,5). To explore the diversity, extent, and endemicity of avian influenza viruses in Laos, we conducted a survey of healthy domestic poultry throughout the country in March 2010.

The Study

Serum samples were collected in 9 of 17 provinces in Laos from healthy ducks and chickens in live-bird markets, village backyard flocks, and layer duck farms. Cloacal, tracheal, and environmental (fecal and water) swab specimens were also collected and placed immediately in transport medium (Figure). Swab specimens were screened in pools of 4 by using a real-time reverse transcription PCR for the matrix (M) gene segment (d). Positive pools were reextracted individually, retested, tested for hemagglutinin 5 (H5) by real-time reverse transcription PCR (7), and injected into 10-11-day-old embryonated chicken eggs.

Sequencing was conducted by using an Illumina (San Diego, CA, USA) platform (swabs and isolates) (8,9) and conventional Sanger sequencing (isolates). Illumina reads were first mapped to a database of publicly available human, avian, and swine influenza virus reference sequences from the Western Hemisphere Americas and Eurasia, and then remapped against references with the highest number of reads and best average coverage. Final average coverage varied between samples and segments with ranges of 123-24,163 (9 samples), 21-18,671 (12 samples) and 9-436 for sample A/duck/Lao/670/10. Isolate genotypes were verified by using Sanger sequencing. Mixed infections could not be excluded for direct sequencing in the absence of an isolate.

Phylogenetic analysis (ClustalW [http://www.clustal. org/], neighbor-joining analysis, 1,000 bootstrap tests, maximum composite likelihood, pairwise deletions) was conducted by using MEGA version 5.02 (10). Sequences are available in GenBank (CY098294-CY098334, CY098336-CY098340, CY098342-CY098368, and CY098370-CY098464). Serum samples were screened by using an ELISA (FlockChek MultiS Screen; IDEXX Laboratories, Westbrook, ME, USA), and antibody-positive serum samples were tested by using a hemagglutinin inhibition assay for subtypes H3, H4, H5 (clades 2.3.2 and 2.3.4), H6, and H9 (lineages G1 and Y280) as described (11).

During March 2010, a total of 3,695 swab specimens were collected (1,928 duck and 279 chicken cloacal samples, 446 duck tracheal samples, 675 fecal samples, and 367 water samples). M gene prevalence was 4.0% (ducks), 1.8% (chickens), and 0.3% (environment samples). Five isolates were obtained (online Appendix Figure, wwwnc. cdc.gov/EID/article/18/7/11-1642-FA 1 .htm). All M gene-positive swab specimens were collected in 13 locations (8 backyards, 2 markets, and 3 farms) (Table 1; Figure) Sample protection was suboptimal, and only 21 samples could be subtyped by real-time RT-PCR and sequencing (Table 1).

Phylogenetic analysis identified 3 groups of viruses: clades 2.3.2.1,2.3.4.1, and 2.3.4.2 (online Appendix Figure). Two samples were closely related to A/chicken/Lao/ LH1/2010-like virus (outbreak in Vientiane in April-May 2010) and to A/chicken/Laos/C100209-194-PTK/2009--like virus (outbreak in Phongsaly in February 2009) (clade 2.3.4.1) (online Appendix Figure; Table 1). These viruses were closely related to A/Guizhou/1/2009 and A/chicken/ Vietnam/NCVD-404/2010 (online Appendix Figure).

Sixteen surveillance viruses of clade 2.3.4.2 were highly homogeneous (online Appendix Figure; Table 1) and closely related to A/environment/Guizhou/4/2009 and A/chicken/Vietnam/NCVD-394/2010-like viruses, although they contained the polymerase basic 2 (PB2) gene of a clade 2.3.2.1 donor virus and were therefore interclade reassortants (online Appendix Figure; Table 1). The genotype of the 4 isolates was verified by using Sanger sequencing. The PB2 gene was most closely related to A/grey_heron/Hong_Kong/1046/2008--like viruses. These reassortants were detected in 3 locations in northern (backyard), central (backyard), and southern (farm) Laos, suggesting multiple introductions of reassortants into Laos (online Appendix Figure; Table 1).

Three clade 2.3.2.1 viruses were detected in 2 ducks and 1 environmental sample (same trader). These viruses were interclade reassortants. Two (1 isolate and 1 direct sequence) contained 6 or 7 segments that were A/whooper swan/Mongolia/6/2009 like (clade 2.3.2.1), and the nucleoprotein gene was A/tree_sparrow/Jiangsu/1/08 like (clade 2.3.4). The environmental sample (direct sequence) contained A/whooper swan/Mongolia/6/2009--like hemagglutinin and M genes and A/Guizhou/1/2009--like PB2, nucleoprotein, and neuraminidase genes (clade 2.3.4.1) (Table 1). The genotype of the isolate was verified by using Sanger sequencing. Hemagglutinin segments of the reassortants were identical (100% nt identity). This identity and the source of the 3 samples (1 trader) suggest that reassortment occurred recently, likely in Laos.

Antibody titers to H5 and H9 and a subtype H3N8 virus isolate have been reported in Laos (4,5). Subtypes H4 and H6 also circulate in this region (1,2,12). For this study, 2,148 serum samples (1,899 from ducks, 200 from chickens, and 49 from unspecified species) were collected and 267 antibody-positive (seroprevalence 14%) duck and 15 (7.5%) chicken serum samples were detected. Hemagglutination inhibition testing for specific antibodies against H3, H4, H5, H6, and H9 detected all antibodies but to H3 (Table 2). Antibodies against H9 were detected at highest titers and most frequently (1.1% of ducks for each H9 lineage), although some cross-reactivity between G1 and Y280 likely occurred (Table 2). These ducks were most widely distributed (18 [19%] of 97 locations in all 9 provinces). Antibodies against H5 clade 2.3.4 were found at the detection limit in few serum samples (0.4% of ducks in 3 northern and 2 southern provinces). Antibodies against H5 clade 2.3.2 were detected in 0.4% of ducks in 4 northern provinces and the capital of Vientiane.

Among ducks, 34 (1.7%) were either shedding or had antibodies against avian influenza virus (H5N1), and there was [greater than or equal to] 1 virus-positive or antibody-positive duck in each of the 9 provinces sampled. One village had 36% of sampled ducks exposed to this virus; 18% shed 2.3.4 virus and 18% had antibody to 2.3.2 virus (Table 2). One duck in this village had antibodies against 2.3.2 and 2.3.4 clade viruses. Exposure to 2.3.2 and 2.3.4 viruses was evident in ducks from locations in 3 other provinces (1 district each in Champasak and Xiengkhoung, and several districts in Luang Prabang).

Conclusions

This study showed that 3 groups of avian influenza viruses (H5N1) were likely introduced into Laos in 2009-2010, one of which resulted in 2 outbreaks (2009, 2010). In all 9 provinces where surveillance was conducted, ducks had been exposed to this virus. Evidence of clades 2.3.2 and 2.3.4 virus activity was detected in 4 provinces. Several interclade reassortants were identified, demonstrating the high genetic mobility of these viruses in the region. Since 2004, Laos has had repeated outbreaks of highly pathogenic avian influenza viruses, which have also been detected in China and Vietnam. There is no evidence that a particular virus lineage has established itself in Laos. The frequency of introduction, diversity, and extent of these viruses in Laos suggests considerable movement of viruses into the country from surrounding territories (China and Vietnam, but not Cambodia) and within the country.

Acknowledgments

We thank Jennifer DeBeauchamp, Scott Krauss, Mariette Ducatez, David Walker, Jerry Parker, Richard Elia, Betsy Williford, and David Galloway for technical assistance, data management, and help in preparing the figures and manuscript; and the Australian Animal Health Laboratory (Geelong, Victoria, Australia) for provision of Lao H5 sequences of avian influenza virus.

This study was supported by US Agency International Development project OSRO/RAS/604/USA Baby 3, National Institutes of Health contract HHSN266200700005C, and American Lebanese Syrian Associated Charities.

References

(1.) Alexander DJ. Summary of avian influenza activity in Europe, Asia, Africa, and Australasia, 2002-2006. Avian Dis. 2007;51 (Suppi): 161-6. http://dx.doi.org/10.1637/7602-041306R.1

(2.) Brown IH. Summary of avian influenza activity in Europe, Asia, and Africa, 2006-2009. Avian Dis. 2010;54(Suppl):187-93. http:// dx.doi.org/10.1637/8949-053109-Reg.1

(3.) World Organisation for Animal Health. Update on highly pathogenic avian influenza in animals (type H5 and H7) [cited 2012 May 1], http://www.oie.int/en/animal-health-in-the-world/update-on-avian-influenza/2011/

(4.) Boltz DA, Douangngeun B, Phommachanh P, Sinthasak S, Mondry R, Obert C, et al. Emergence of H5N1 avian influenza viruses with reduced sensitivity to neuraminidase inhibitors and novel reassortants in Lao People's Democratic Republic. J Gen Virol. 2010;91:949-59. http://dx.doi.org/10.1099/vir.0.017459-0

(5.) Boltz DA, Douangngeun B, Sinthasak S, Phommachanh P, Rolston S, Chen H, et al. H5N1 influenza vimses in Lao People's Democratic Republic. Emerg Infect Dis. 2006;12:1593-5. http://dx.doi. org/10.3201/eidl 210.060658

(6.) World Health Organization. CDC protocol of real-time RT-PCR for swine influenza A (HINI) [cited 2012 May 1], http://www.who.int/ csr/resources/publications/swineflu/CDCrealtimeRTPCRprotocol_ 20090428.pdf

(7.) Centers for Disease Control and Prevention (CDC) CDC Realtime RT-PCR. (rRTPCR) protocol for detection and characterization of influenza (version 2007). CDC ref. no. 1-007-05. Atlanta: The Centers; 2007.

(8.) Zhou B, Donnelly ME, Scholes DT, St George K, Hatta M, Kawaoka Y, et al. Single-reaction genomic amplification accelerates sequencing and vaccine production for classical and swine origin human influenza A viruses. J Virol. 2009;83:10309-13. http://dx.doi. org/10.1128/JVI.01109-09

(9.) Hoffmann E, Stech J, Guan Y, Webster RG, Perez DR. Universal primer set for the full-length amplification of all influenza A vimses. Arch Virol. 2001;146:2275-89. http://dx.doi.org/10.1007/ s007050170002

(10.) Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S. MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol. 2011;28:2731-9. http://dx.doi.org/10.1093/molbev/ msr121

(11.) Palmer DF, Dowdle WR, Coleman MT, Schild GC. Advanced laboratory techniques for influenza diagnosis. Immunology Series no. 6. Atlanta: Center for Disease Control; 1975.

(12.) Global Initiative on Sharing All Influenza Data. EpiFlu [cited 2012 May 1], http://platform.gisaid.org

Address for correspondence: Stephanie Sonnberg, Division of Virology, St. Jude Children's Research Hospital, MS 330, Memphis, TN 38105, USA; email: stephanie.sonnberg@stjude.org

DOI: http://dx.doi.org/10.3201/eid1807.111642

Author affiliations: St. Jude Children's Research Hospital, Memphis, Tennessee, USA (S. Sonnberg, D. Darnell, A.M. Rubrum, R.J. Webby, R.G. Webster); National Animal Health Centre, Vientiane, Laos (P. Phommachanh, P. Meeduangchanh, B. Douangngeun); Food and Agriculture Organization of the United Nations, Rome, Italy (T.S.P. Naipospos, J. McKenzie, C. Chanthavisouk, S. Pathammavong); and Ministry of Agriculture and Forestry, Vientiane (M. Souriya, B. Khambounheuang)

Dr Sonnberg is a postdoctoral research associate at St. Jude Children's Research Hospital, Memphis, Tennessee. Her research interests are surveillance, transmission propensity, and reassortment of influenza viruses.

Table 1. Genotypes of 21 surveillance samples of avian influenza virus
(H5N1) and outbreak virus A-chicken-Lao-LH1-2010 Laos 2009-2010 *

                                                        Gene ([dagger])
Sample      Sample                          Sampling
ID           type     Location ([dagger])     site      PB2   PB1   PA

A/ck/LH1#   Chicken        Vientiane          Farm      (A)   (A)   (A)
210317       Duck       Xiengkhouang-1       Market     (A)   NA    NA
210265       Duck       Luang Namtha-1      Backyard    (A)   NA    NA
210281        Env         Champasak-1        Market     (A)   NA    NA
210287       Duck         Champasak-1        Market     (B)   NA    (B)
210289#      Duck         Champasak-1        Market     (B)   (B)   (B)
210252       Duck       Luang Namtha-1      Backyard    (B)   (D)   (D)
210253       Duck       Luang Namtha-1      Backyard    (B)   (D)   (D)
210255#      Duck       Luang Namtha-1      Backyard    (B)   (D)   (D)
210358       Duck       Xiengkhouang-2      Backyard    (B)   (D)   (D)
210360       Duck       Xiengkhouang-2      Backyard    (B)   (D)   NA
210361#      Duck       Xiengkhouang-2      Backyard    (B)   (D)   (D)
210363        Env       Xiengkhouang-2      Backyard    (B)   (D)   (D)
210367       Duck       Xiengkhouang-2      Backyard    (B)   (D)   NA
210374#      Duck       Xiengkhouang-2      Backyard    (B)   (D)   (D)
210376#       Env       Xiengkhouang-2      Backyard    (B)   (D)   (D)
210378        Env       Xiengkhouang-2      Backyard    (B)   (D)   (D)
210379       Duck       Xiengkhouang-2      Backyard    (B)   (D)   (D)
210380       Duck       Xiengkhouang-2      Backyard    (B)   (D)   (D)
210385       Duck       Xiengkhouang-2      Backyard    (B)   (D)   (D)
210386       Duck       Xiengkhouang-2      Backyard    (B)   (D)   (D)
210349       Duck        Savanakhet-1         Farm      (B)   (D)   (D)

                         Gene ([dagger])
Sample
ID          H5 ([section])   NP    N1     M    NS

A/ck/LH1#     2.3.4 (A)      (A)   (A)   (A)   (A)
210317        2.3.4 (A)      (A)   (A)   (A)   (A)
210265        2.3.4 (A)      (A)   (A)   (A)   NA
210281        2.3.2 (B)      (A)   (A)   (A)   NA
210287        2.3.2 (B)      (C)   (A)   (A)   (A)
210289#       2.3.2 (B)      (C)   (A)   (A)   (A)
210252          2.3.4        (D)   (D)   (D)   (D)
210253          2.3.4        (D)   (D)   (D)   (D)
210255#         2.3.4        (D)   (D)   (D)   (D)
210358          2.3.4        (D)   (D)   (D)   (D)
210360          2.3.4        (D)   (D)   (D)   (D)
210361#         2.3.4        (D)   (D)   (D)   (D)
210363          2.3.4        (D)   (D)   (D)   (D)
210367          2.3.4        (D)   (D)   (D)   (D)
210374#         2.3.4        (D)   (D)   (D)   (D)
210376#         2.3.4        (D)   (D)   (D)   (D)
210378          2.3.4        (D)   (D)   (D)   (D)
210379          2.3.4        (D)   (D)   (D)   (D)
210380          2.3.4        (D)   (D)   (D)   (D)
210385          2.3.4        (D)   (D)   (D)   (D)
210386          2.3.4        (D)   (D)   (D)   (D)
210349          2 3 4        (D)   (D)   NA    (D)

* Boldface indicates outbreak virus isolate and closely related
isolates. ID, identification; PB, polymerase basic; PA, polymerase
acidic; H, hemagglutinin; NP, nucleoprotein; N, neuraminidase; M,
matrix; NS, nonstructural; NA, not available; env, environmental.

([dagger]) Identical numbers indicate same sampling site.

([double dagger]) Pink shading, A/Guizhou/1/2009-like clade 2.3.4.1;
blue shading, A/whooper swan/Mongolia/6/2009-like clade 2.3.2.1;
purple shading, A/tree_sparrow/Jiangsu/1/08-like clade 2.3.4; gray
shading, A/environment/Guizhou/4/2009-like clade 2.3.4.2.

([section]) Clade is indicated.

Noe: Pink shading, A/Guizhou/1/2009-like clade 2.3.4.1 indicated with
(A); blue shading, A/whooper swan/Mongolia/6/2009-like clade 2.3.2.1
indicated with (B); purple shading, A/tree_sparrow/Jiangsu/1/08-like
clade 2.3.4 indicated with (C); gray shading, A/environment/Guizhou/
4/2009-like clade 2.3.4.2 indicated with (D).

Note:  Boldface indicates outbreak virus isolate and closely related
isolates indicated with #.

Table 2 Serum HI titers in ducks against avian influenza virus (H5N1)
H3, H4, H5, H6, and H9 antigens, Laos, 2009-2010 *

Serum          Location       Sampling
sample ID     ([dagger])        site

790           Phongsaly-1     Backyard
797           Phongsaly-1     Backyard
798           Phongsaly-1     Backyard
925           Phongsaly-2     Backyard
11A         Luang Namtha-1    Backyard
11B         Luang Namtha-1    Backyard
12          Luang Namtha-1    Backyard
14          Luang Namtha-1    Backyard
15          Luang Namtha-1    Backyard
1686        Luang Namtha-2    Backyard
1688        Luang Namtha-2    Backyard
1696        Luang Namtha-2    Backyard
1699        Luang Namtha-2    Backyard
1736        Luang Namtha-3     Market
757         Luang Namtha-3     Market
1314          Oudomxay-1      Backyard
1319          Oudomxay-1      Backyard
1322          Oudomxay-2       Market
1324          Oudomxay-2       Market
1325          Oudomxay-2       Market
1350          Oudomxay-2       Market
1362          Oudomxay-3       Market
1372          Oudomxay-3       Market
1459          Oudomxay-4      Backyard
1470          Oudomxay-5        Farm
24          Luang Prabang-1   Backyard
36          Luang Prabang-1   Backyard
37          Luang Prabang-1   Backyard
38          Luang Prabang-1   Backyard
951         Luang Prabang-2   Backyard
958         Luang Prabang-2   Backyard
721         Xiengkhouang-3    Backyard
1673        Xiengkhouang-4      Farm
1378          Vientiane-1     Backyard
1379          Vientiane-1     Backyard
1381          Vientiane-1     Backyard
1382          Vientiane-1     Backyard
1386          Vientiane-1     Backyard
1389          Vientiane-1     Backyard
1393          Vientiane-1     Backyard
1526          Vientiane-2       Farm
463           Vientiane-1      Market
464           Vientiane-1      Market
467           Vientiane-1      Market
468           Vientiane-1      Market
1245          Vientiane-2     Backyard
1249          Vientiane-2     Backyard
1277          Vientiane-2     Backyard
1419          Vientiane-3      Market
657          Savanakhet-2       Farm
990          Savanakhet-3       Farm
589           Champasak-2     Backyard
606           Champasak-3     Backyard

                      HI test antigen subtype ([double dagger])
                          (clade or lineage) ([section])
Serum
sample ID    H4    H5 (2.3.2)   H5 (2.3.4)    H6    H9 (G1)   H9 (Y280)

790         <10       <10           <5       <10      10         <10
797         <10       <10           5        <10      <10        <10
798         <10       <10           <5       <10      10         <10
925         <10       <10           5        <10      <10        <10
11A         <10       <10           <5       <10      10         10
11B         <10        40           <5       <10      <10        10
12          <10       160           <5       <10      <10        <10
14          <10       160           40       <10      <10        <10
15          <10       320           <5       <10      <10        <10
1686        <10       <10           <5       <10      <10        640
1688        <10       <10           <5       <10      10         <10
1696        <10       <10           <5       <10      10         <10
1699        <10       <10           <5       <10      <10        320
1736        <10       <10           <5       <10      10         <10
757         <10       <10           10       <10      <10        <10
1314         80       <10           <5        80      <10        <10
1319         40        10           <5       320      <10        <10
1322        <10       <10           <5       <10      <10        40
1324        <10       <10           <5       <10      <10        10
1325        <10       <10           <5       <10      <10        40
1350        <10       <10           <5       <10      <10        160
1362         80       <10           <5       <10      <10        <10
1372        <10       <10           <5       <10      <10        40
1459        <10       <10           <5       640      320        320
1470        <10       <10           <5       <10      <10        10
24          <10        10           <5       <10      <10        <10
36          <10       <10           <5       <10      10         <10
37          <10       <10           <5       <10      10         <10
38          <10       <10           <5       <10      10         <10
951         <10       <10           5        <10      <10        <10
958         <10       <10           5        <10      10         <10
721         <10        10           <5       <10      <10        <10
1673        <10       <10           <5       <10      <10        10
1378        640       <10           <5       <10      20         40
1379        <10       <10           <5       <10      10         <10
1381         40       <10           <5       <10      20         <10
1382        <10       <10           <5       <10      10         <10
1386         40       <10           <5       <10      <10        40
1389        <10       <10           <5       <10      10         <10
1393         40       <10           <5       <10      <10        <10
1526        <10       <10           <5       <10      <10        10
463         <10       <10           <5       <10      160        320
464         <10       <10           <5       <10    >1,280     >1,280
467         <10       <10           <5       <10      160        160
468         <10       <10           <5       <10      <10        80
1245        <10       <10           <5       <10      <10        10
1249        <10        10           <5       <10      <10        <10
1277        <10        10           <5       <10      <10        <10
1419         20       <10           <5       <10      <10        <10
657         <10       <10           <5       <10      10         10
990         <10       <10           10       <10      <10        <10
589         <10       <10           <5       <10      40         <10
606         <10       <10           20       <10      10         10

* H, hemagglutinin; ID, identification; HI, hemagglutination
inhibition.

[dagger]) Identical numbers indicate same sampling site.

[double dagger]) All titers for H3 (A/duck/Laos-Xaythany/A0573/2007)
were <10. H4, A/duck/Czech/1956; H5 clade 2.3.2, A/common magpie/Hong
Kong/5052/2007; H5 clade 2.3.4, A/duck/Laos/3295/2006; H6,
A/turkey/Massachusetts/1965; H9 lineage G1, A/Hong Kong/33982/2009; H9
lineage Y280, A/duck/Hong Kong/Y280/1997.

([section]) Values represent HI titers reciprocal to the highest
dilution of serum that inhibited haemagglutination of 4
hemagglutination units of antigen by using 0.5% chicken erythrocytes.
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Title Annotation:DISPATCHES
Author:Sonnberg, Stephanie; Phommachanh, Phouvong; Naipospos, Tri Satya Putri; McKenzie, Joanna; Chanthavis
Publication:Emerging Infectious Diseases
Article Type:Report
Geographic Code:9LAOS
Date:Jul 1, 2012
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