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Identification of avian influenza virus subtype H9N2 in chicken farms in Indonesia
Preventive Veterinary Medicine 159 (2018) 99–105
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Preventive Veterinary Medicine journal homepage: www.elsevier.com/locate/prevetmed
Identification of avian influenza virus subtype H9N2 in chicken farms in Indonesia
T
Melina Jonasa, Aprilla Sahestia, Theresia Murwijatia, Christina Lilis Lestariningsiha, Ine Irinea, ⁎ Clara Sinta Ayesdaa, Wahyu Prihartinia, Gusti Ngurah Mahardikab, a b
PT Medion Farma Jaya, Jl. Babakan Ciparay #282, Bandung, Indonesia Faculty of Veterinary Medicine Udayana University, Jl. PB Sudirman, 80225, Denpasar, Bali, Indonesia
A R T I C LE I N FO
A B S T R A C T
Keywords: Influenza H9N2 subtype Indonesia
Avian influenza virus subtype H9N2 (AIV-H9N2) has become established in domestic poultry in Asia and Africa. AIV-H9N2 has not been reported previously in Indonesia. Here we describe the presence of AIV-H9N2 in chicken farms in Indonesia. Ninety-nine cases were observed in various provinces in Indonesia. Clinical signs, pathologic lesions and egg production were recorded. Confirmation was made using virus isolation, reverse transcriptase PCR (RT-PCR), and sequencing. To construct hemaglutinin (HA) phylogeny, the secondary data of Eurasian lineages were downloaded from GenBank. For neuraminidase, five sequences with the highest similarities with every sequence found in this study were downloaded. Phylogeny was inferred using Neighbor-Joining method in MEGA6 package. Forty-nine AIV-H9N2-positive cases were observed, of which 35 were tested positive for AIVH9N2 only. The age of the infected chickens was 43.17 ± 16.56 weeks, and their egg production was 35.85 ± 17.80% lower than before outbreak. BLAST search revealed that the nucleotide sequence of the HAencoding gene identified in this study shared 98% sequence identity with that of A/Muscovy duck/Vietnam/ LBM719/2014(H9N2), while its neuraminidase-encoding gene sequences shared 94%, 98%, and 100% identities with three different influenza viruses. The phylogeny shows that the HA of AIV-H9N2 found in this study forms distinct cluster with some Vietnam and China’s sequence data. The NA sequence data form three distinct clusters. We conclude that AIV-H9N2 is widespread in many provinces in Indonesia. To lessen economic losses to the poultry industry, flock biosecurity and vaccination against this virus subtype should be implemented rapidly. Thorough and rigid AIV surveillance is paramount to prevent further veterinary and public health consequences of the circulation of this virus in Indonesia.
1. Introduction Avian influenza virus of H9N2 subtype (AIV-H9N2) has become enzootic in domestic poultry in Asia and Africa (Guan et al., 2000; Sun and Liu, 2015). Although it is categorized as a low-pathogenic avian influenza (Davidson et al., 2013), the virus causes economic losses to the poultry industry (Nili and Asasi, 2003; Choi et al., 2008; Lee et al., 2008), and it is a possible threat to human health (Peiris et al., 1999; Butt et al., 2005). This subtype is also the source of internal proteinencoding gene segments that facilitate the emergence of new strains (Shehata et al., 2015). AIV-H9N2 has not been reported previously in Indonesia. By the end of 2016 and the beginning of 2017, chicken farm managers of various production sectors, so-called sectors 1, 2, and 3 (FAO, 2013), complained of increased mortality and decreased egg production. Efforts
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were made to detect infectious viral agents such as infectious bronchitis virus (IBV), Newcastle disease virus (NDV), and AIV of the H5N1 subtype (AIV-H5N1). These agents were detected in some, but not all, cases. We retested all the samples for AIV-H9N2 and found 50% of the cases were AIV-H9N2-positive. Here we describe the clinical cases and the identification of AIV-H9N2 from chicken farms in many provinces in Indonesia. 2. Material and methods Laboratory work was conducted in the BSL-3 facility of PT Medion Farma Jaya, Bandung, Indonesia. The pharyngeal and cloacal swabs were collected from each case. Sample collection, sample transport, virus isolation in specific-pathogenic-free (SPF) fertilized chicken eggs, RNA isolation, reverse transcriptase PCR (RT-PCR), and sequencing
Corresponding author. E-mail address: [email protected] (G.N. Mahardika).
https://doi.org/10.1016/j.prevetmed.2018.09.003 Received 19 September 2017; Received in revised form 11 February 2018; Accepted 4 September 2018 0167-5877/ © 2018 Published by Elsevier B.V.
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Table 1 Results of the RT-PCR detection of AIV-H5N1 and AIV-H9N2 in various chicken farms, as well as chicken age, egg production, and chicken mortality at various locations in Indonesia. No.
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49
Farm location and Province in Indonesiaa
Palu (CSL) Banjarmasin (SK) Pare-pare (SSL) Sidrap (SSL) Sidrap (SSL) Sidrap (SSL) Subang (WJ) Sragen (CJ) Mojokerto (EJ) Medan (NSm) Tangerang (BA) Samarinda (EK) Gorontalo (GO) Sidrap (SSL) Sukabumi (WJ) Pare-pare (SSL) Pare-pare (SSL) Pare-pare (SSL) Binjai (NSM) Sukorejo (CJ) Citarum (WJ) Sukabumi (WJ) Sukabumi (WJ) Binjai (NSM) Binjai (NSM) Samarinda (EK) Samarinda (EK) Pare-pare (SSL) Kediri (EJ) Semarang (CJ) Sukabumi (WJ) Sukabumi (WJ) Bandung (WJ) Pare-pare (SSL) Blitar (EJ) Binjai (NSM) Binjai (NSM) Blitar (EJ) Kediri (EJ) Palembang (SSm) Bandung (WJ) Tangerang (BA) Malang (EJ) Purwokerto (CJ) Madiun (EJ) Tabanan (BL) Malang (EJ) Madiun (EJ) Serang (BA)
Type of chicken farm
Layer Layer Layer Layer Layer Layer Breeder Layer Layer Layer Layer Layer Layer Layer Breeder Layer Layer Layer Layer Layer Layer Breeder Breeder Layer Layer Broiler Broiler Layer Layer Layer Layer Layer Layer Layer Layer Layer Layer Layer Layer Layer Layer Layer Layer Layer Layer Layer Layer Layer Breeder
Chicken age (Weeks)
34 78 45 47 43 14 31 47 42 69 27 24 9 52 25 56 36 52 38 37 NI 50 48 NI 83 NI NI NI 48 60 NI NI 48 NI 30 51 67 33 36 50 28 30 80 41 28 38 53 31 31
Egg Production decrease (%)
50 20 10 50 50 50 0 47 NI 33 40 NI NI NI NI NI NI NI 62 NI NI NI NI NI NI NI NI NI 44 5 NI NI NI NI NI NI NI 55 43 20 NI 38 NI NI 30 20 50 NI NI
Information of increased mortalityb
Present NI Present NI NI NI Present NI 0% NI Present NI NI NI NI NI NI NI NI NI NI NI NI NI NI NI NI NI NI NI Present Present NI NI NI 4% 5% NI Present NI NI Present Present Present 0% 0% Present Present 0%
RT-PCR NDVc
IBVc
AIV-H5N1
AIV-H9N2
Negative ND Negative Negative Positive Negative Positive ND ND Positive Negative Positive Positive Positive Positive ND ND ND Positive Positive Positive ND Negative Negative Negative Negative Negative Negative ND Positive Negative Negative Negative Negative ND Negative Negative Negative ND ND ND Negative ND ND ND ND ND ND Negative
ND ND ND ND ND ND ND Negative Negative Positive ND ND ND ND ND Negative Negative Positive ND ND ND ND ND ND ND ND ND Negative Negative ND ND ND Negative Negative ND ND ND ND ND ND Negative ND ND ND ND ND ND ND Negative
Positive Negative Negative Negative Positive Negative Positive Negative Negative Negative Negative Positive Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Positive Negative Negative Positive Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative
Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive
a The province names were abbreviated from western to eastern part of Indonesia as NSM, SSM, BA, WJ, CJ, EJ, BL, EK, SK, SSL, CSL and GO for North Sumatra, South Sumatra, Banten, West Java, Central Java, East Java, Bali, East Kalimantan, South Kalimantan, South Sulawesi, Central Sulawesi and Gorontalo, respectively. b Information of increased mortality was based on farm baseline. c The NDV and IBV testing were conducted upon the request of farm managers, and they were based on differential diagnoses. NI, no information; ND, not determined.
sequences of each representative of the lineages were selected. For neuraminidase, five sequences of highest similarity with those found in this study were downloaded. Identical sequences were collapsed. Phylogeny was inferred using Neighbor-Joining method in MEGA6 package (Tamura et al., 2013).
were conducted as described previously (Mahardika et al., 2016). The detection of AIV-H5N1 was confirmed using a previously published protocol (Mahardika et al., 2016). The primers for RT-PCR and sequencing of the viral agents other than AIV-H5N1 were based on published primer sets for H9 (Shen et al., 2015), N2 (Chen et al., 2009), NDV (Nanthakumar et al., 2000) and IBV (Ariyoshi et al., 2010). The sequences were aligned using MEGA 6.0 software (Tamura et al., 2013) and subjected to the online BLAST search (www.blast.ncbi.nlm.nih. gov/Blast.cgi). To construct HA-phylogeny, the secondary data were downloaded from GenBank of the Eurasian G1, Y280, BJ94, Y439 and Korean lineages as previously indicated (Butt et al., 2010). Five most identical
3. Results The results of the RT-PCR detection AIV-H9N2 and other viral agents in various locations and provinces in Indonesia, as well as chicken age, egg production, and chicken mortality are presented in Table 1. The location and the province name of the farms are shown in 100
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Fig. 1. Map of Indonesia showing the province names of the location of each chicken farm, where the AIV H9N2 was detected. The province names are abbreviated as in Table 1. The national boundaries and major islands are indicated. The map was modified from http://www.maps-of-the-world.net/maps/maps-of-asia/maps-ofindonesia/large-detailed-elevation-map-of-indonesia.jpg.
4. Discussion
Fig. 1. Out of 99 cases, 49 were positive for AIV-H9N2. The virus affected layers, breeders, and broiler farms. The average age of the infected chickens was 43.17 ± 16.56 weeks, and egg production was 35.85 ± 17.80% lower than farm baselines. The mortality rate varied from > 1-2% (baseline level) to 5%. Of the 49 cases, 35 were tested positive for AIV-H9N2 only, while the others had mixed infections of AIV-H9N2 with one or two other viruses, namely NDV, IBV, and/or AIV-H5N1. Clinical signs of those AIV-H9N2-only-cases were decreased body weight, irregular egg shape, a thinner egg shell, appetite loss, pale to blue comb discoloration, a swollen head, snoring, nasal discharge, dyspnea, enlargement of the abdomen, wet stools, torticollis, and paralysis. Mild to severe lesions were observed as exudates in the nasal cavity, trachea, and bronchus, as well as petechial to focal hemorrhages in the pharynx, proventriculus, ventriculus, duodenum, ileum, cecal tonsil, kidney, ovarium, and oviduct. Diffused hemorrhages were found in the ovarium in some cases. Oviducts full of egg yolk were prominent in some cases, as were a soft liver, blackened spleen, enlarged heart, and congested brain. All hemagglutinin (HA) and neuraminidase (NA) coding sequences of the AIV-H9N2 isolates in this study are available in GenBank under the accession no. MF164878-MF164911. BLAST results and GenBank accession numbers of HA-H9 and NA-N2 of AIV-H9N2 detected from various provinces in Indonesia are presented in Table 2. We sequenced the HA-encoding gene of 25 cases. The lengths of the readable sequences ranged from 219 to 1626 bp. The NA-encoding gene from eight isolates was also sequenced, and its lengths ranged from 519 to 549 bp. BLAST search revealed that the highest nucleotide identity (98%) of all the long (1626) HA sequences was shared with the HA-encoding gene of the A/Muscovy duck/Vietnam/LBM719/2014(H9N2) virus (GenBank accession no. LC028176). The NA-encoding gene sequences of six isolates of the AIV-H9N2 virus shared 98% identity with that of the same strain. Two other NA sequences shared the highest identities of 94% and 100% with those of the A/gadwall/Altai/1202/2007(H5N2) and (A/duck/Peru/P114/2009(H3N2) viruses, respectively. The phylogeny shows that the HA of AIV-H9N2 found in this study (Fig. 2) forms distinct cluster with some Vietnam and China’s sequence data (bootstrap value 100%) that is diverging from BJ94 lineage (bootstrap value 97%). The NA sequence data (Fig. 3) form three distinct clusters (bootstrap values 100%).
To our knowledge, this is the first report to confirm the presence of AIV-H9N2 in Indonesia. Although the virus is co-circulating with its sister AIV-H5N1 virus in Asia and Africa (Aamir et al., 2007; Monne et al., 2013), the AIV-H9N2 was not detected in Indonesia until 2016, while AIV-H5N1 has been endemic in the country since 2004 (Webster et al., 2005; Smith et al., 2006). The epizootic pattern of AIV-H9N2 cases in many provinces in Indonesia indicates its recent introduction. We had conducted Indonesian-wide surveillance of AIV-H5N1 and AIV-H9N2 between 2005–2010, with no detection of AIV-H9N2. Additionally, before 2016, the virus had not been detected by The National Veterinary Research Institute, either (Dr. N. L. P. Indi Dharmayanti, DVM., MSi., Director, personal communication). That the Vietnam/LBM719 strain is dated 2014, which shares the highest similarity with most HA and NA sequenced in this study, is also an evidence of the recent introduction of this subtype to Indonesia. Our confirmation of the H9N2 subtype is convincing because we rigidly implemented the Food and Agriculture Organization of the United Nations standard protocol (FAO, 2014), albeit without the serological testing, which was not conducted because of the unavailability of standard reagents. However, the fact that the final confirmation was made using DNA sequencing validates our results. Unfortunately, a lack of resources limited our capacity to conduct full-genome sequencing, which would have provided a more complete picture of the dynamics of the spread of the AIV-H9N2 virus into and within Indonesia. We usually test for the presence of AIV-H5N1 as part of our standard protocol for any suspected AIV infection. Additional testing for the presence of NDV and IBV was conducted upon inquiries of farm managers and confirmation of possible differential diagnoses. All the testing was finalized by DNA sequencing, although the sequences of AIV-H5N1, NDV, and IBV are not shown. Of the 49 AIV-H9N2-positive cases, 35 were infected with AIV-H9N2 only, while the others were co-infected with one or two of the AIV-H5N1, NDV, and IBV. The detection of mixed infections is not unique to our study, as it has been reported elsewhere (Hassan et al., 2016). The presence of other infectious and non-infectious agents is not negligible. Fifty percent out of 99 suspected cases were not confirmed. The variation in clinical signs, the decrease in egg production, and the associated mortality are indicative of variations in viral pathogenicity 101
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South Sulawesi South Sulawesi South Sulawesi South Kalimantan South Sulawesi Central Sulawesi North Sumatera South Kalimantan East Java Central Java South Sulawesi West Java West Java Banten South Sulawesi South Sulawesi West Java North Sumatera North Sumatera Central Java East Java North Sumatera South Sulawesi Banten Central Java
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25
M92_01 M92_02 M92_03 M92_04 M92_05 M92_06 M92_07 M92_08 M92_10 M92_12 M92_13 M92_14 M92_15 M92_16 M92_18 M92_19 M92_20 M92_21 M92_22 M92_23 M92_24 M92_25 M92_26 M92_27 M92_28
Sample codes 01/01/2017 12/29/2016 12/29/2016 12/28/2016 01/05/2017 12/27/2016 02/01/2017 12/27/2016 02/02/2017 01/27/2017 02/09/2017 02/23/2017 01/26/2017 02/06/2017 02/24/2017 02/24/2017 03/09/2017 03/10/2017 03/09/2017 03/06/2017 03/16/2017 03/24/2017 03/27/2017 04/27/2017 05/02/2017
Case date Egg Egg Egg Egg Egg Egg Egg Egg Egg Egg Egg Egg Egg Egg Swab Swab Swab Swab Swab Swab Egg Egg Egg Swab Egg
Source 1626 1626 1626 219 219 1626 1626 219 219 219 1626 219 219 219 219 1626 219 219 1626 219 1626 1626 1626 1626 1626
HA sequence length V/LBM719 V/LBM719 V/LBM719 ND ND V/LBM719 V/LBM719 ND ND ND V/LBM719 ND ND ND ND V/LBM719 ND ND V/LBM719 ND V/LBM719 V/LBM719 V/LBM719 V/LBM719 V/LBM719
Highest homology 98 98 98 − − 98 98 − − − 98 − − − − 98 − − 98 − 98 98 98 98 98
Identity (%) 549 549 546 ND ND 549 546 ND ND 519 ND 546 549 ND ND ND ND ND ND ND ND ND ND ND ND
NA sequence length
V/LBM719 V/LBM719 V/LBM719 − − V/LBM719 V/LBM719 − − V/LBM719 − Altai/1202 Peru/P114 − − − − − − − − − − − −
Highest homology
98 98 98 − − 98 98 − − 98 − 94 100 − − − − − − − − − − − −
Identity (%)
The case dates and the source of the RNA used for the RT-PCR sequence determinations are indicated. Egg indicates that the RT-PCR was conducted from the allantoic fluid of specific-pathogen-free embryonated chicken eggs following the allantoic cavity inoculation of the specimen. Swab indicates that the RT-PCR was conducted from RNA isolated from pharyngeal and cloacal swabs. V/LBM719 = A/Muscovy duck/Vietnam/LBM719/ 2014(H9N2) with GenBank accession nos. LC028176 (the HA-encoding gene) and LC028178 (the NA-encoding gene). Altai/1202 = A/gadwall/Altai/1202/2007(H5N2) with the GenBank accession no. CY049758 (the NA-encoding gene), and Peru/P114 = (A/duck/Peru/P114/2009(H3N2) with the GenBank acc. no. KR824657 (the NA-encoding gene). ND, not determined.
Province
No.
Table 2 BLAST results of the HA-H9 and NA-N2 coding sequences of AIV-H9N2 isolates from various provinces in Indonesia.
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Fig. 2. Rooted bootstrap-tested Neighborjoining tree of the HA of AIV-H9N2 isolated in this study with previously described Eurasian G1, Y280, BJ94, Y439 and Korean lineages as previously indicated (Butt et al., 2010). The tree was rooted to Missouri/10OS4670/2010. The accession numbers are shown in the taxa. The length of sequence was 1620 base pair, corresponding nucleotide position of no. 61 – 1680 of HA gene of A/Muscovy duck/ Vietnam/LBM719/2014(H9N2). Phylogeny was inferred using Neighbor-Joining method applying Kimura-2 parameter and tested using bootstrap method with 1000 replicates in MEGA6 package (Tamura et al., 2013). The Indonesian sequences isolated in this study are marked with filled square. The bootstrap value of < 90% are not shown. The proposed novel CVI lineage is indicated.
of the NA-encoding gene of one isolate shared 100% similarity to a South American virus (A/duck/Peru/P114/2009(H3N2), this might be an indication of the mixing of Asian and American viruses. Determination of the whole-genome sequence of each isolate might provide a greater understanding of the genetic heterogeneity of the introduced virus, as well mixing events that accompanied its introduction. The phylogenies of HA and NA support the existence of multiple ancestries of AIV-H9N2 in Indonesia. The sequences of the HA-encoding gene of the samples in this study form a distinct cluster with some sequence data of AIV-H9N2 from China and Vietnam. A novel China-
and the presence of predisposing factors. We isolated AIV-H9N2 from two broiler farms that had chickens exhibiting decreased body weight. AIV-H9N2 has been reported to cause clinical disease in broilers (Nili and Asasi, 2002), and co-infection of broilers with Ornithobacterium rhinotracheale and AIV-H9N2 has been described previously (Pan et al., 2012). From the limited data available in this study, it is likely that multiple ancestries of AIV-H9N2 are present in Indonesia. The sequences of the HA-encoding gene of the viruses isolated in this study were closely related to each other. However, the sequences of the NA-encoding gene show high similarity to three different sequence data. As the sequence 103
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Fig. 3. Un-rooted bootstrap Neighbor joining tree of the NA of AIV-H9N2 isolated in Indonesia with closest sequence data available in GenBank. The length of the sequences was 515 base pair corresponding nucleotide position 358–872 of NA of A/ Muscovy duck/Vietnam/LBM719/2014(H9N2). Accession numbers, phylogenetic analysis and sequence identifier in the tree are as of in Fig. 2.
rigid surveillance, is paramount for preventing further veterinary and public health consequences of this virus in Indonesia.
Vietnam-Indonesia (CVI) lineage is proposed for the well-supported divergence from BJ94 (Fig. 2). However, the sequences of the NA-encoding gene vary significantly, which are located in three well-supported distinct clusters (Fig. 3). As evidenced by the 35.85 ± 17.80% decrease in egg production, based on the claim of farm management, AIV-H9N2 is a major threat to layer and breeder farms, which is confirmed by the economic losses in other countries in which AIV-H9N2 is endemic (Nili and Asasi, 2003; Choi et al., 2008; Lee et al., 2008). The massive and rapid spread of AIV-H9N2 in many provinces in Indonesia prompts, that rigid flock biosecurity and vaccination program should be implemented rapidly. Flock biosecurity should be especially focused on restricting risk factors such as selling of eggs/birds directly to live bird retail stalls, being near case/infected farms and the history of immunosuppressive disease in the farm. These factors have been demonstrated in Pakistan (Chaudhry et al., 2015). Vaccination is a favored strategy for controlling AIV-H9N2 in various countries (Lu et al., 2001; Lee and Song, 2013). Considering experiences in producing an anti-AIV-H5N1 vaccine, vaccine producers should be ready to develop an anti-AIV-H9N2 vaccine for Indonesia. The wide co-circulation of low-pathogenic AIV-H9N2 with highpathogenic AIV-H5N1 in chicken farms (Mahardika et al., 2016) enhances the need for rigid AIV surveillance in Indonesia. The presence of other AIV subtypes, such as H7N9, which are endemic in Asia, is a threat to Indonesia too. Moreover, it has been proven that AIV-H9N2 contributed to the emergence of new strains (Yu et al., 2015), which have serious consequences for animal and human health. We conclude that AIV-H9N2 has spread in many provinces in Indonesia and affected layer, breeder, and broiler farms. The main clinical sign was decreased egg production. Additionally, our data suggest that multiple AIV-H9N2 ancestries have been introduced into Indonesia. To lessen the economic loss to the poultry industry, flock biosecurity and vaccination measures should be implemented. Determining the whole-genome sequences of AIV isolates, as well as
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Identification of avian influenza virus subtype H9N2 in chicken farms in Indonesia
T
Melina Jonasa, Aprilla Sahestia, Theresia Murwijatia, Christina Lilis Lestariningsiha, Ine Irinea, ⁎ Clara Sinta Ayesdaa, Wahyu Prihartinia, Gusti Ngurah Mahardikab, a b
PT Medion Farma Jaya, Jl. Babakan Ciparay #282, Bandung, Indonesia Faculty of Veterinary Medicine Udayana University, Jl. PB Sudirman, 80225, Denpasar, Bali, Indonesia
A R T I C LE I N FO
A B S T R A C T
Keywords: Influenza H9N2 subtype Indonesia
Avian influenza virus subtype H9N2 (AIV-H9N2) has become established in domestic poultry in Asia and Africa. AIV-H9N2 has not been reported previously in Indonesia. Here we describe the presence of AIV-H9N2 in chicken farms in Indonesia. Ninety-nine cases were observed in various provinces in Indonesia. Clinical signs, pathologic lesions and egg production were recorded. Confirmation was made using virus isolation, reverse transcriptase PCR (RT-PCR), and sequencing. To construct hemaglutinin (HA) phylogeny, the secondary data of Eurasian lineages were downloaded from GenBank. For neuraminidase, five sequences with the highest similarities with every sequence found in this study were downloaded. Phylogeny was inferred using Neighbor-Joining method in MEGA6 package. Forty-nine AIV-H9N2-positive cases were observed, of which 35 were tested positive for AIVH9N2 only. The age of the infected chickens was 43.17 ± 16.56 weeks, and their egg production was 35.85 ± 17.80% lower than before outbreak. BLAST search revealed that the nucleotide sequence of the HAencoding gene identified in this study shared 98% sequence identity with that of A/Muscovy duck/Vietnam/ LBM719/2014(H9N2), while its neuraminidase-encoding gene sequences shared 94%, 98%, and 100% identities with three different influenza viruses. The phylogeny shows that the HA of AIV-H9N2 found in this study forms distinct cluster with some Vietnam and China’s sequence data. The NA sequence data form three distinct clusters. We conclude that AIV-H9N2 is widespread in many provinces in Indonesia. To lessen economic losses to the poultry industry, flock biosecurity and vaccination against this virus subtype should be implemented rapidly. Thorough and rigid AIV surveillance is paramount to prevent further veterinary and public health consequences of the circulation of this virus in Indonesia.
1. Introduction Avian influenza virus of H9N2 subtype (AIV-H9N2) has become enzootic in domestic poultry in Asia and Africa (Guan et al., 2000; Sun and Liu, 2015). Although it is categorized as a low-pathogenic avian influenza (Davidson et al., 2013), the virus causes economic losses to the poultry industry (Nili and Asasi, 2003; Choi et al., 2008; Lee et al., 2008), and it is a possible threat to human health (Peiris et al., 1999; Butt et al., 2005). This subtype is also the source of internal proteinencoding gene segments that facilitate the emergence of new strains (Shehata et al., 2015). AIV-H9N2 has not been reported previously in Indonesia. By the end of 2016 and the beginning of 2017, chicken farm managers of various production sectors, so-called sectors 1, 2, and 3 (FAO, 2013), complained of increased mortality and decreased egg production. Efforts
⁎
were made to detect infectious viral agents such as infectious bronchitis virus (IBV), Newcastle disease virus (NDV), and AIV of the H5N1 subtype (AIV-H5N1). These agents were detected in some, but not all, cases. We retested all the samples for AIV-H9N2 and found 50% of the cases were AIV-H9N2-positive. Here we describe the clinical cases and the identification of AIV-H9N2 from chicken farms in many provinces in Indonesia. 2. Material and methods Laboratory work was conducted in the BSL-3 facility of PT Medion Farma Jaya, Bandung, Indonesia. The pharyngeal and cloacal swabs were collected from each case. Sample collection, sample transport, virus isolation in specific-pathogenic-free (SPF) fertilized chicken eggs, RNA isolation, reverse transcriptase PCR (RT-PCR), and sequencing
Corresponding author. E-mail address: [email protected] (G.N. Mahardika).
https://doi.org/10.1016/j.prevetmed.2018.09.003 Received 19 September 2017; Received in revised form 11 February 2018; Accepted 4 September 2018 0167-5877/ © 2018 Published by Elsevier B.V.
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Table 1 Results of the RT-PCR detection of AIV-H5N1 and AIV-H9N2 in various chicken farms, as well as chicken age, egg production, and chicken mortality at various locations in Indonesia. No.
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49
Farm location and Province in Indonesiaa
Palu (CSL) Banjarmasin (SK) Pare-pare (SSL) Sidrap (SSL) Sidrap (SSL) Sidrap (SSL) Subang (WJ) Sragen (CJ) Mojokerto (EJ) Medan (NSm) Tangerang (BA) Samarinda (EK) Gorontalo (GO) Sidrap (SSL) Sukabumi (WJ) Pare-pare (SSL) Pare-pare (SSL) Pare-pare (SSL) Binjai (NSM) Sukorejo (CJ) Citarum (WJ) Sukabumi (WJ) Sukabumi (WJ) Binjai (NSM) Binjai (NSM) Samarinda (EK) Samarinda (EK) Pare-pare (SSL) Kediri (EJ) Semarang (CJ) Sukabumi (WJ) Sukabumi (WJ) Bandung (WJ) Pare-pare (SSL) Blitar (EJ) Binjai (NSM) Binjai (NSM) Blitar (EJ) Kediri (EJ) Palembang (SSm) Bandung (WJ) Tangerang (BA) Malang (EJ) Purwokerto (CJ) Madiun (EJ) Tabanan (BL) Malang (EJ) Madiun (EJ) Serang (BA)
Type of chicken farm
Layer Layer Layer Layer Layer Layer Breeder Layer Layer Layer Layer Layer Layer Layer Breeder Layer Layer Layer Layer Layer Layer Breeder Breeder Layer Layer Broiler Broiler Layer Layer Layer Layer Layer Layer Layer Layer Layer Layer Layer Layer Layer Layer Layer Layer Layer Layer Layer Layer Layer Breeder
Chicken age (Weeks)
34 78 45 47 43 14 31 47 42 69 27 24 9 52 25 56 36 52 38 37 NI 50 48 NI 83 NI NI NI 48 60 NI NI 48 NI 30 51 67 33 36 50 28 30 80 41 28 38 53 31 31
Egg Production decrease (%)
50 20 10 50 50 50 0 47 NI 33 40 NI NI NI NI NI NI NI 62 NI NI NI NI NI NI NI NI NI 44 5 NI NI NI NI NI NI NI 55 43 20 NI 38 NI NI 30 20 50 NI NI
Information of increased mortalityb
Present NI Present NI NI NI Present NI 0% NI Present NI NI NI NI NI NI NI NI NI NI NI NI NI NI NI NI NI NI NI Present Present NI NI NI 4% 5% NI Present NI NI Present Present Present 0% 0% Present Present 0%
RT-PCR NDVc
IBVc
AIV-H5N1
AIV-H9N2
Negative ND Negative Negative Positive Negative Positive ND ND Positive Negative Positive Positive Positive Positive ND ND ND Positive Positive Positive ND Negative Negative Negative Negative Negative Negative ND Positive Negative Negative Negative Negative ND Negative Negative Negative ND ND ND Negative ND ND ND ND ND ND Negative
ND ND ND ND ND ND ND Negative Negative Positive ND ND ND ND ND Negative Negative Positive ND ND ND ND ND ND ND ND ND Negative Negative ND ND ND Negative Negative ND ND ND ND ND ND Negative ND ND ND ND ND ND ND Negative
Positive Negative Negative Negative Positive Negative Positive Negative Negative Negative Negative Positive Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Positive Negative Negative Positive Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative
Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive
a The province names were abbreviated from western to eastern part of Indonesia as NSM, SSM, BA, WJ, CJ, EJ, BL, EK, SK, SSL, CSL and GO for North Sumatra, South Sumatra, Banten, West Java, Central Java, East Java, Bali, East Kalimantan, South Kalimantan, South Sulawesi, Central Sulawesi and Gorontalo, respectively. b Information of increased mortality was based on farm baseline. c The NDV and IBV testing were conducted upon the request of farm managers, and they were based on differential diagnoses. NI, no information; ND, not determined.
sequences of each representative of the lineages were selected. For neuraminidase, five sequences of highest similarity with those found in this study were downloaded. Identical sequences were collapsed. Phylogeny was inferred using Neighbor-Joining method in MEGA6 package (Tamura et al., 2013).
were conducted as described previously (Mahardika et al., 2016). The detection of AIV-H5N1 was confirmed using a previously published protocol (Mahardika et al., 2016). The primers for RT-PCR and sequencing of the viral agents other than AIV-H5N1 were based on published primer sets for H9 (Shen et al., 2015), N2 (Chen et al., 2009), NDV (Nanthakumar et al., 2000) and IBV (Ariyoshi et al., 2010). The sequences were aligned using MEGA 6.0 software (Tamura et al., 2013) and subjected to the online BLAST search (www.blast.ncbi.nlm.nih. gov/Blast.cgi). To construct HA-phylogeny, the secondary data were downloaded from GenBank of the Eurasian G1, Y280, BJ94, Y439 and Korean lineages as previously indicated (Butt et al., 2010). Five most identical
3. Results The results of the RT-PCR detection AIV-H9N2 and other viral agents in various locations and provinces in Indonesia, as well as chicken age, egg production, and chicken mortality are presented in Table 1. The location and the province name of the farms are shown in 100
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Fig. 1. Map of Indonesia showing the province names of the location of each chicken farm, where the AIV H9N2 was detected. The province names are abbreviated as in Table 1. The national boundaries and major islands are indicated. The map was modified from http://www.maps-of-the-world.net/maps/maps-of-asia/maps-ofindonesia/large-detailed-elevation-map-of-indonesia.jpg.
4. Discussion
Fig. 1. Out of 99 cases, 49 were positive for AIV-H9N2. The virus affected layers, breeders, and broiler farms. The average age of the infected chickens was 43.17 ± 16.56 weeks, and egg production was 35.85 ± 17.80% lower than farm baselines. The mortality rate varied from > 1-2% (baseline level) to 5%. Of the 49 cases, 35 were tested positive for AIV-H9N2 only, while the others had mixed infections of AIV-H9N2 with one or two other viruses, namely NDV, IBV, and/or AIV-H5N1. Clinical signs of those AIV-H9N2-only-cases were decreased body weight, irregular egg shape, a thinner egg shell, appetite loss, pale to blue comb discoloration, a swollen head, snoring, nasal discharge, dyspnea, enlargement of the abdomen, wet stools, torticollis, and paralysis. Mild to severe lesions were observed as exudates in the nasal cavity, trachea, and bronchus, as well as petechial to focal hemorrhages in the pharynx, proventriculus, ventriculus, duodenum, ileum, cecal tonsil, kidney, ovarium, and oviduct. Diffused hemorrhages were found in the ovarium in some cases. Oviducts full of egg yolk were prominent in some cases, as were a soft liver, blackened spleen, enlarged heart, and congested brain. All hemagglutinin (HA) and neuraminidase (NA) coding sequences of the AIV-H9N2 isolates in this study are available in GenBank under the accession no. MF164878-MF164911. BLAST results and GenBank accession numbers of HA-H9 and NA-N2 of AIV-H9N2 detected from various provinces in Indonesia are presented in Table 2. We sequenced the HA-encoding gene of 25 cases. The lengths of the readable sequences ranged from 219 to 1626 bp. The NA-encoding gene from eight isolates was also sequenced, and its lengths ranged from 519 to 549 bp. BLAST search revealed that the highest nucleotide identity (98%) of all the long (1626) HA sequences was shared with the HA-encoding gene of the A/Muscovy duck/Vietnam/LBM719/2014(H9N2) virus (GenBank accession no. LC028176). The NA-encoding gene sequences of six isolates of the AIV-H9N2 virus shared 98% identity with that of the same strain. Two other NA sequences shared the highest identities of 94% and 100% with those of the A/gadwall/Altai/1202/2007(H5N2) and (A/duck/Peru/P114/2009(H3N2) viruses, respectively. The phylogeny shows that the HA of AIV-H9N2 found in this study (Fig. 2) forms distinct cluster with some Vietnam and China’s sequence data (bootstrap value 100%) that is diverging from BJ94 lineage (bootstrap value 97%). The NA sequence data (Fig. 3) form three distinct clusters (bootstrap values 100%).
To our knowledge, this is the first report to confirm the presence of AIV-H9N2 in Indonesia. Although the virus is co-circulating with its sister AIV-H5N1 virus in Asia and Africa (Aamir et al., 2007; Monne et al., 2013), the AIV-H9N2 was not detected in Indonesia until 2016, while AIV-H5N1 has been endemic in the country since 2004 (Webster et al., 2005; Smith et al., 2006). The epizootic pattern of AIV-H9N2 cases in many provinces in Indonesia indicates its recent introduction. We had conducted Indonesian-wide surveillance of AIV-H5N1 and AIV-H9N2 between 2005–2010, with no detection of AIV-H9N2. Additionally, before 2016, the virus had not been detected by The National Veterinary Research Institute, either (Dr. N. L. P. Indi Dharmayanti, DVM., MSi., Director, personal communication). That the Vietnam/LBM719 strain is dated 2014, which shares the highest similarity with most HA and NA sequenced in this study, is also an evidence of the recent introduction of this subtype to Indonesia. Our confirmation of the H9N2 subtype is convincing because we rigidly implemented the Food and Agriculture Organization of the United Nations standard protocol (FAO, 2014), albeit without the serological testing, which was not conducted because of the unavailability of standard reagents. However, the fact that the final confirmation was made using DNA sequencing validates our results. Unfortunately, a lack of resources limited our capacity to conduct full-genome sequencing, which would have provided a more complete picture of the dynamics of the spread of the AIV-H9N2 virus into and within Indonesia. We usually test for the presence of AIV-H5N1 as part of our standard protocol for any suspected AIV infection. Additional testing for the presence of NDV and IBV was conducted upon inquiries of farm managers and confirmation of possible differential diagnoses. All the testing was finalized by DNA sequencing, although the sequences of AIV-H5N1, NDV, and IBV are not shown. Of the 49 AIV-H9N2-positive cases, 35 were infected with AIV-H9N2 only, while the others were co-infected with one or two of the AIV-H5N1, NDV, and IBV. The detection of mixed infections is not unique to our study, as it has been reported elsewhere (Hassan et al., 2016). The presence of other infectious and non-infectious agents is not negligible. Fifty percent out of 99 suspected cases were not confirmed. The variation in clinical signs, the decrease in egg production, and the associated mortality are indicative of variations in viral pathogenicity 101
102
South Sulawesi South Sulawesi South Sulawesi South Kalimantan South Sulawesi Central Sulawesi North Sumatera South Kalimantan East Java Central Java South Sulawesi West Java West Java Banten South Sulawesi South Sulawesi West Java North Sumatera North Sumatera Central Java East Java North Sumatera South Sulawesi Banten Central Java
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25
M92_01 M92_02 M92_03 M92_04 M92_05 M92_06 M92_07 M92_08 M92_10 M92_12 M92_13 M92_14 M92_15 M92_16 M92_18 M92_19 M92_20 M92_21 M92_22 M92_23 M92_24 M92_25 M92_26 M92_27 M92_28
Sample codes 01/01/2017 12/29/2016 12/29/2016 12/28/2016 01/05/2017 12/27/2016 02/01/2017 12/27/2016 02/02/2017 01/27/2017 02/09/2017 02/23/2017 01/26/2017 02/06/2017 02/24/2017 02/24/2017 03/09/2017 03/10/2017 03/09/2017 03/06/2017 03/16/2017 03/24/2017 03/27/2017 04/27/2017 05/02/2017
Case date Egg Egg Egg Egg Egg Egg Egg Egg Egg Egg Egg Egg Egg Egg Swab Swab Swab Swab Swab Swab Egg Egg Egg Swab Egg
Source 1626 1626 1626 219 219 1626 1626 219 219 219 1626 219 219 219 219 1626 219 219 1626 219 1626 1626 1626 1626 1626
HA sequence length V/LBM719 V/LBM719 V/LBM719 ND ND V/LBM719 V/LBM719 ND ND ND V/LBM719 ND ND ND ND V/LBM719 ND ND V/LBM719 ND V/LBM719 V/LBM719 V/LBM719 V/LBM719 V/LBM719
Highest homology 98 98 98 − − 98 98 − − − 98 − − − − 98 − − 98 − 98 98 98 98 98
Identity (%) 549 549 546 ND ND 549 546 ND ND 519 ND 546 549 ND ND ND ND ND ND ND ND ND ND ND ND
NA sequence length
V/LBM719 V/LBM719 V/LBM719 − − V/LBM719 V/LBM719 − − V/LBM719 − Altai/1202 Peru/P114 − − − − − − − − − − − −
Highest homology
98 98 98 − − 98 98 − − 98 − 94 100 − − − − − − − − − − − −
Identity (%)
The case dates and the source of the RNA used for the RT-PCR sequence determinations are indicated. Egg indicates that the RT-PCR was conducted from the allantoic fluid of specific-pathogen-free embryonated chicken eggs following the allantoic cavity inoculation of the specimen. Swab indicates that the RT-PCR was conducted from RNA isolated from pharyngeal and cloacal swabs. V/LBM719 = A/Muscovy duck/Vietnam/LBM719/ 2014(H9N2) with GenBank accession nos. LC028176 (the HA-encoding gene) and LC028178 (the NA-encoding gene). Altai/1202 = A/gadwall/Altai/1202/2007(H5N2) with the GenBank accession no. CY049758 (the NA-encoding gene), and Peru/P114 = (A/duck/Peru/P114/2009(H3N2) with the GenBank acc. no. KR824657 (the NA-encoding gene). ND, not determined.
Province
No.
Table 2 BLAST results of the HA-H9 and NA-N2 coding sequences of AIV-H9N2 isolates from various provinces in Indonesia.
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Fig. 2. Rooted bootstrap-tested Neighborjoining tree of the HA of AIV-H9N2 isolated in this study with previously described Eurasian G1, Y280, BJ94, Y439 and Korean lineages as previously indicated (Butt et al., 2010). The tree was rooted to Missouri/10OS4670/2010. The accession numbers are shown in the taxa. The length of sequence was 1620 base pair, corresponding nucleotide position of no. 61 – 1680 of HA gene of A/Muscovy duck/ Vietnam/LBM719/2014(H9N2). Phylogeny was inferred using Neighbor-Joining method applying Kimura-2 parameter and tested using bootstrap method with 1000 replicates in MEGA6 package (Tamura et al., 2013). The Indonesian sequences isolated in this study are marked with filled square. The bootstrap value of < 90% are not shown. The proposed novel CVI lineage is indicated.
of the NA-encoding gene of one isolate shared 100% similarity to a South American virus (A/duck/Peru/P114/2009(H3N2), this might be an indication of the mixing of Asian and American viruses. Determination of the whole-genome sequence of each isolate might provide a greater understanding of the genetic heterogeneity of the introduced virus, as well mixing events that accompanied its introduction. The phylogenies of HA and NA support the existence of multiple ancestries of AIV-H9N2 in Indonesia. The sequences of the HA-encoding gene of the samples in this study form a distinct cluster with some sequence data of AIV-H9N2 from China and Vietnam. A novel China-
and the presence of predisposing factors. We isolated AIV-H9N2 from two broiler farms that had chickens exhibiting decreased body weight. AIV-H9N2 has been reported to cause clinical disease in broilers (Nili and Asasi, 2002), and co-infection of broilers with Ornithobacterium rhinotracheale and AIV-H9N2 has been described previously (Pan et al., 2012). From the limited data available in this study, it is likely that multiple ancestries of AIV-H9N2 are present in Indonesia. The sequences of the HA-encoding gene of the viruses isolated in this study were closely related to each other. However, the sequences of the NA-encoding gene show high similarity to three different sequence data. As the sequence 103
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Fig. 3. Un-rooted bootstrap Neighbor joining tree of the NA of AIV-H9N2 isolated in Indonesia with closest sequence data available in GenBank. The length of the sequences was 515 base pair corresponding nucleotide position 358–872 of NA of A/ Muscovy duck/Vietnam/LBM719/2014(H9N2). Accession numbers, phylogenetic analysis and sequence identifier in the tree are as of in Fig. 2.
rigid surveillance, is paramount for preventing further veterinary and public health consequences of this virus in Indonesia.
Vietnam-Indonesia (CVI) lineage is proposed for the well-supported divergence from BJ94 (Fig. 2). However, the sequences of the NA-encoding gene vary significantly, which are located in three well-supported distinct clusters (Fig. 3). As evidenced by the 35.85 ± 17.80% decrease in egg production, based on the claim of farm management, AIV-H9N2 is a major threat to layer and breeder farms, which is confirmed by the economic losses in other countries in which AIV-H9N2 is endemic (Nili and Asasi, 2003; Choi et al., 2008; Lee et al., 2008). The massive and rapid spread of AIV-H9N2 in many provinces in Indonesia prompts, that rigid flock biosecurity and vaccination program should be implemented rapidly. Flock biosecurity should be especially focused on restricting risk factors such as selling of eggs/birds directly to live bird retail stalls, being near case/infected farms and the history of immunosuppressive disease in the farm. These factors have been demonstrated in Pakistan (Chaudhry et al., 2015). Vaccination is a favored strategy for controlling AIV-H9N2 in various countries (Lu et al., 2001; Lee and Song, 2013). Considering experiences in producing an anti-AIV-H5N1 vaccine, vaccine producers should be ready to develop an anti-AIV-H9N2 vaccine for Indonesia. The wide co-circulation of low-pathogenic AIV-H9N2 with highpathogenic AIV-H5N1 in chicken farms (Mahardika et al., 2016) enhances the need for rigid AIV surveillance in Indonesia. The presence of other AIV subtypes, such as H7N9, which are endemic in Asia, is a threat to Indonesia too. Moreover, it has been proven that AIV-H9N2 contributed to the emergence of new strains (Yu et al., 2015), which have serious consequences for animal and human health. We conclude that AIV-H9N2 has spread in many provinces in Indonesia and affected layer, breeder, and broiler farms. The main clinical sign was decreased egg production. Additionally, our data suggest that multiple AIV-H9N2 ancestries have been introduced into Indonesia. To lessen the economic loss to the poultry industry, flock biosecurity and vaccination measures should be implemented. Determining the whole-genome sequences of AIV isolates, as well as
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