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Molecular analysis of hemagglutinin-1 fragment of avian influenza H5N1 viruses isolated from chicken farms in Indonesia from 2008 to 2010 by I Nyoman Suartha From Pubpangkat (pangkat)
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MAHARDIKA, G. N. K., N. DIBIA, N. S. BUDAYANTI, N. M. SUSILAWATHI, K. SUBRATA, A. E.
DARWINATA, F. S. WIGNALL, J. A. RICHT, W. A. VALDIVIA-GRANDA, and A. A. R. SUDEWI.
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Veterinary Microbiology 186 (2016) 52–58 Contents lists available at ScienceDirect Veterinary Microbiology journal homepage: www.elsevier.com/locate/vetmic
7Molecular analysis of hemagglutinin-1 fragment of avian influenza H5N1 viruses isolated from chicken farms in Indonesia from 2008 to 2010 Gusti N.
Mahardikaa,*, Melina Jonasb, Theresia Murwijatib, Nur Fitriab, I Nyoman Suarthaa, I Gusti A.A. Suartinia, I Wayan Teguh
Wibawanc a Animal Biomedical and Molecular Biology Laboratory, Udayana University, Sesetan-Markisa Street No. 6, Denpasar 80226, Bali, Indonesia b Research and Development Department, PT. Medion, Babakan Ciparay Street #282, Bandung, Indonesia c Faculty of Veterinary Medicine, Bogor Agricultural University, Bogor, West Java, Indonesia ARTICLE INFO Article history: Received 10 October 2015 Received in revised form 15 February 2016 Accepted 23 February 2016 Keywords: Avian influenza virus H5N1 Chicken industry Indonesia Vaccine ABSTRACT
4Highly pathogenic avian influenza virus of subtype H5N1 (AIV-H5N1) has been circulating in Indonesia since 2003. To understand the genetic diversity of these viruses, and to predict vaccine efficacy, the hemaglutinin-1 (HA-1) fragment of viruses
1isolated from chicken farms in Indonesia from 2008 to 2010
was sequenced and analyzed. The effects of these molecular changes were investigated in challenge experiments and HI assays of homologous and heterologous strains. Molecular analysis showed that these AIV-H5N1 isolates had evolved into three distinct sub-lineages from an ancestor circulating since 2003.
Although no significant positive selection of residues was detected, 12 negatively selected sites were identified (p < 0.05). Moreover, four sites showed evidence of significant episodic diversifying selection. The findings indicated complete protectivity and high HI titers with homologous strains, compared with
protectivity ranging from 40 to 100% and lower HI titers with heterologous strains resulting from
polymorphisms at antigenic sites. Our findings provide valuable insight into the molecular evolution of AIV and have important implications for vaccine efficacy and future vaccination strategies. ã 2016 Elsevier B.V.
All rights reserved. 1. Introduction
1Highly pathogenic avian influenza virus of H5N1 subtype (AIV- H5N1) has been circulating in Indonesia since 2003. This has led to
a countrywide epizootic and the death of millions of birds, either due to infection or culling to reduce the spread of the virus. AIV- H5N1 is also responsible for human fatalities, with Indonesia having the highest fatality rate in the world, until a recent increase in the number of human cases in Egypt (WHO, 2015).
Human infection is believed to result from transmission from infected poultry, as around 80% of cases in Indonesia have been linked with direct or indirect contact with sick poultry (Kandun et al., 2008).
Investigations into molecular changes in AIV-H5N1 circulating at commercial poultry farms, so-called sector 1, 2, and 3 facilities according to the FAO definition (FAO, 2013), are lacking. Most reports on AIV-H5N1 have dealt with domestic poultry from live bird markets (which may include some poultry from intensive * Corresponding author. E-mail addresses: [email protected], [email protected] (G.N.
Mahardika). http://dx.doi.org/10.1016/j.vetmic.2016.02.023 0378-1135/ã 2016 Elsevier B.V. All rights reserved. farms) or birds from backyard settings, and data on the commercial poultry industry are scarce.
Intensive poultry production is a huge industry worldwide and will play a critical role in the spread and enhancement of the pathogenicity of AIVs (Olsen et al., 2006). An intensive poultry farm with a high degree of genetic uniformity between birds provides the optimum conditions for viral mutation and transmission. The cases in backyards and wild birds could represent propulsion from intensive farms, as has been found at Poyang Lake, southern China (Chen et al., 2006). Biosecurity is a major issue in controlling diseases such
as that caused by AIV, and inevitably breaches in biosecurity occur across the world as a result of the mass movement of people, materials and vehicles internationally. However, despite the need for surveillance, access to large commercial poultry farms is often strictly limited in Indonesia (Daniels et al., 2013) and other countries. Therefore, information on AIV-H5N1 in the integrated poultry enterprise sector is not publicly available. To control AIV-H5N1 in poultry, vaccination has been imple- mented in Indonesia. Various vaccine seeds have been registered, including a homologous seed based on H5N1 isolates, and heterologous seeds based on subtypes H5N2 and H5N9, prior to 2012. In 2012, the Livestock and Animal Health Authority issued a directorate decree, which allowed only the homologous vaccine seeds to be used in Indonesia.
Vaccination is claimed to have a masking effect on influenza evolution, as well as being a driving force for antigenic drift (Webster et al., 2006). To understand the molecular evolution of AIV,
4and to predict vaccine efficacy, analysis of the HA -1 fragment of
the hemagglutinin of AIV-H5N1 provides valuable information. HA-1 is the primary target for neutralizing antibodies and responsible for host receptor specificity; it also harbors the pathogenic domains of the virus (Horimoto and Kawaoka, 2001; Webby and Webster, 2001). Therefore, HA- 1 analysis may also provide important information on possible adaptations in circulating viruses that may facilitate human transmission.
Here we report the results of ongoing efforts to analyze molecularly AIV-H5N1 isolated from chicken farms in Indonesia. 2. Methods 2.1. Safety procedures All work with infectious material was carried out in a Bio- safety Class III facility (PT Medion, Bandung, Indonesia).
6RNA isolation and RT-PCR were performed
in the Animal Biomedical and Molecular Biology Laboratory of Udayana University, Denpasar, Bali, and the Research and Development Laboratory of PT Medion, Bandung, Indonesia. Sequencing was performed at the Research and Development Laboratory of PT Medion, Bandung, Indonesia. Ethical clearance for the study involving animal samples and experiments
6was granted by the Ethics Commission for the Use of Animals in Research and Education of the Faculty of Veterinary Medicine Udayana University, Bali, Indonesia,
in accordance with the
6Terrestrial Animal Health Code of the World Organization for Animal Health.
2.2. Virus transport and propagation Collection, transport, and propagation of field specimens were carried out in accordance with the Manual of Diagnostic Tests and Vaccines for Terrestrial Animals 2004 (FAO, 2014; WHO, 2002). The supernatant fluids of tissue specimens or fecal swabs were obtained by centrifugation at 1000g and ultrafiltration. The supernatants were then
3inoculated into the allantois sac of five fertile 10 -day-old specific-pathogen- free (SPF) chicken eggs. The eggs were incubated at 37 ?C for 5 days. Eggs
containing dead or dying embryos and all eggs remaining at the end of the incubation period were first chilled to 4 ?C and then the allantois fluids were
harvested. The clinical and epidemological history of each isolate is presented in Table 1. 2.3.
Hemagglutination (HA) and hemagglutination inhibition (HI) assay The allantois fluids were tested for HA activity. In the HA assay, 25 mL of allantoic fluid were titrated in two-fold serial dilutions and reacted with 1%
chicken red blood cells. In the HI assay, 0.25 mL positive control sera (Animal Biomedical and Molecular Biology Laboratory, Udayana University, Bali) were two-fold serially diluted in PBS, then an equal volume of 4HA units of allantois fluid or control virus were added and incubated at room Table 1 Characteristics of the AIV subtype H5N1 isolates from Indonesia, isolated from 2008 to 2010, and the farms from which they originated. No Simplified isolate name Estimated population size Type of farm Vaccination history Age of outbreak (weeks) 1 West Java/M04/2008 2
2West Java/M05/2008 3 West Java/M06/2008 4 West Java/M07/2008
5 East Java/M08/2008 6 East Java/M09/2008 7 North Sumatra/M10/2008 8 East Java/M11/2008 9 East Java/M12/2009 10 West Java/M13/2009 11 East Java/M14/2009 12 Banten/M15/2009 14 West
Java/M16/2009 15 East Java/M17/2009 16 Central Java/M18/2009 17 East Java/M19/2009 18 Central Java/M20/2009 19 South Sulawesi/M21/2009 20 Central Java/M22/2009 21 East Java/M23/2009 22 West Java/M24/2009 23 West Java/M25/2009 25 Bali/M26/2009 24 North Sumatra/M27/2009 26 South
Kalimantan/M28/2010 27 South Kalimantan/M29/2010 28 West Java/M30/2010 29 Central Java/M31/2010 30 Central Sulawesi/M32/2010 31 South Sumatra/M33/2010 32 West Java/M34/2010 33 West
Java/M35/2010 50.000 Layer 100.000 Layer 100.000 Breeder 50.000 Layer 10.000 Layer 10.000 Broiler 40.000 Layer 50.000 Layer 10.000 Broiler 30.000 Layer 10.000 Broiler 50.000 Broiler 100.000 Broiler 100.000 Broiler 10.000 Broiler 10.000 Broiler 50.000 Broiler 10.000 Broiler 10.000 Broiler 100.000 Layer 50.000 Broiler N/A Broiler 5.000 Broiler N/A Layer 10.000 Broiler 100.000 Broiler 20.000 Broiler 10.000 Broiler 10.000 Layer 10.000 Broiler N/A Breeder 100.000 Layer Vaccinated Vaccinated Vaccinated Vaccinated Vaccinated Unvaccinated Vaccinated Unknown Unvaccinated Unvaccinated Unvaccinated Vaccinated N/A N/A Vaccinated N/A N/A No No Vaccinated No Vaccinated No Vaccinated No No Vaccinated Vaccinated Vaccinated No Vaccinated Vaccinated Unknown Unknown Unknown Unknown 70 Unknown Unknown 87 4.71 6.14 4.86 3.71 4.57 4.14 3.5 5 4.86 3.5 4.29 58 4.86 4.57 4 3.86 5 4 3.71 5.71 10 3.29 32 20 temperature for 30 min, along with 1% chicken red blood cells. Positive HI reactions indicated that samples were AIV H5 positive. 2.4. Reverse transcriptase-polymerase chain reaction (RT-PCR)
Confirmation of the H5N1 subtype was achieved using a standard RT-PCR. Genomic RNAs were isolated from allantoic fluid, after digestion with proteinase K in 0.1% SDS, using the RNeasy Mini Kit (Qiagen). RT- PCR was carried out using the SuperScriptTM III One-Step RT-PCR System with Platinum1 Taq DNA Polymerase (Invitrogen). Reactions included 0.2 mM dNTPs, 1.6 mM MgSO4 in the provided buffer, and 600 mM of each of the standard primers, Matrix, HA-H5 and NA-N1, described previously (WHO, 2005). After the addition of 1–3 mL of RNA sample and Taq polymerase, PCR tubes were placed in a thermocycler (GenAmp PCR System 9700). The
5RT-PCR cycle included incubation at 45 ?C for 60 min, followed by
a preheating step of 7 min at 95 ?C, then 40 cycles of 45 s at 94 ?C, 45 s at 50–55 ?C and 60 s at 72 ?C, and a final extension step at 72 ?C for 5 min. Following PCR, 10–20% of the product was added to 1–2 mL of loading dye (Bromphenol-blue dan Cyline Cyanol) and subjected to electrophoresis
5in 1% agarose. The gel was stained with 25 mg/mL ethidium bromide and visualized under UV
light. 2.5. Sequence analysis RT-PCR products for sequencing were prepared using primer pairs: HA_1F and HA_486BR, HA _245F and HA_787R, and HA Fig. 1. Phylogenetic analysis of the HA
1-1 fragment of AIV -H5N1 isolated from chicken farms in Indonesia from 2008 to 2010.
Available secondary data for HA-1 of isolates from Indonesia and other countries are included in the analysis as references. The tree was rooted to Gs/Guandong/2006. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) is shown next to the branches. Only values higher than 90% are shown. The evolutionary distances were computed using the Kimura 2-parameter method. _680F and HA _1218R, available at http://msc.jcvi.org/infl_a_virus/
primers.shtml, as previously published (Salzberg et al., 2007).
5RT- PCR products were purified using the QIAquick PCR Purification Kit (Qiagen). Sequencing reactions were carried out using a Big Dye Terminator v3.
1Cycle Sequencing Kit
using universal M13 primers and were run in an automatic DNA sequencer (Applied Biosystems 3130/3130x Genetic Analyzer). Trace identification was performed using Sequence Scanner Ver. 1.0 (Applied
Biosystems: http://www. appliedbiosystems.com). Nucleotide and deduced amino acid sequence alignments were completed using Mega4 software (Tamura et al., 2007). The sequence of each isolate was judged based on at least two independent RT-PCR products. In some cases, a third product was required to confirm the consensus sequence. Evolutionary relationships were inferred using the neighbor- joining method (Saitou and Nei, 1987) with Kimura-2 parameters (Kimura, 1980). In the inference, HA sequences of some Indonesian avian isolates dating from 2003 to 2006 available in the GenBank database were included representing groups A, B, and C as formerly proposed (Smith et al., 2006), i.e. Ck/Wajo/2005 (Group A; Acc.
No. DQ320933), Ck/BL/2003 (Group B; Acc. No. AY651321), and Tk/ Kedaton/2004 (Group C; Acc. No.
DQ497664), as well as the first official vaccine seed of Ck/West Java/2003 (Acc. No. KR078216). The A/Influenza/Goose/Guandong/1996 (Acc. No. F148678) was ap- plied as the root for all phylogenetic analyses. Screening of positively and negatively selected sites was conducted using the Single-Likelihood Ancestor Counting method (Pond and Frost, 2005), and evidence of episodic diversifying selection was traced using the Mixed Effects Model of Evolution (Murrell et al., 2012). All analyses were performed using the Datamonkey webserver and the HyPhy package (Delport et al., 2010; Pond and Frost, 2005; Pond et al., 2005). 2.6. Determination of the embryo infectious dose 50 (EID50) and the chicken lethal dose (CLD50) The virus infectivity titer was measured by inoculating 10-day- old embryonated SPF eggs with a 10-fold serial dilution of virus suspension. Each serial dilution was inoculated into the allantoic cavity of five eggs.
After incubation for 5 days, the EID50 was calculated using the Reed-Muench method. The CLD50 of each isolate was determined by inoculating five 3-week-old SPF chickens intranasally with 10-fold dilutions of virus suspension. Mortality of the chickens was recorded and the IED50 was calculated. 2.7. Challenge experiments A representative of each clade (M06, M12, and M13) was grown in embryonated SPF eggs.
Following 48 h incubation at 37 ?C, the allantoic fluid was harvested. The infectivity titer was determined
using the protocol described above. Each harvested virus suspen- sion was then inactivated by adding formaldehyde at a final concentration of 0.05%. The inactivation process was confirmed by inoculating three embryonated SPF eggs for each suspension. The inactivated virus suspension was further mixed with oil emulsion. The final titer of the oil emulsion vaccine was 108 EID50. Each vaccine was injected
subcutaneously into 10 of the 3-week-old SPF chickens. Unvaccinated SPF chickens served as a control.
Three weeks after vaccination, the vaccinated and unvaccinated groups were then challenged with 104 CLD50 of M06, M10, M12, and M13 infectious viruses. Clinical signs and mortality of the chickens were recorded for an observation period of 10 days. 2.8. Cross-reactivity HI assay Primary antibodies against the original vaccine seed of M03 and three other isolates, M05, M06, and M10, were produced in SPF chickens using the above protocol. Three weeks after vaccination, sera were harvested and inactivated at 56 ?C for 30 min. The sera were then subjected to a HI assay using various isolates. 3. Results The HA-1 fragments of 32 isolates of AIV-H5N1 from poultry farms in Indonesia were analyzed. The analysis focused on the area from nucleotide 49-1195 (counted from the start codon of the HA gene). All sequence data acquired in this study were submitted to the GenBank database (Acc. No. KR078216-KR078247). To ensure coverage of all geographical areas, viruses that represent each previously reported geographic group of AIV-H5N1 that were available in the GenBank database, i.e. Groups A, B, and C (Smith et al., 2006), were also included in phylogenetic analysis. At the nucleotide level, the results of phylogenetic analysis showed that all
1viruses isolated from chicken farms in Indonesia from 2008 to 2010
formed three unique clusters (bootstrap value >94%). The clusters were annotated as M10, M13, and M12 (Fig. 1). The clusters were rooted to isolate Ck/Wajo/2005 with 100% bootstrap confident limits. The M10- group was isolated from North Sumatra, South Sulawesi, and Central Sulawesi. The origins of the M13- group were West, Central, and East Java as well as Bali Provinces. Most isolates belonged to the M12- group, which were isolated from Banten, West Java, Central Java, East Java, South Kalimantan, and South Sumatra Provinces. The overall mean distance of all sequence pairs was 0.039 (SD = 0.003). The group distances for HA-1 of AIV-H5N1 from chicken farms when comparing 2008–2009, 2008–2010 and 2009–
2010 were 0.042 (SD = 0.003), 0.047 (SD = 0.004), and 0.039 (SD = 0.003), respectively. The value derived from Tajima’s neutrality test (D) was ?1.157. Analysis of polymorphic sites in the amino acid sequences of all 32 isolates identified 72 sites. The polymorphic residues are presented in Supplementary Material S1, and were located at amino acids spanning positions 2–320. Deletions were detected at residues 74 and 129.
Both deletions were found in isolate Ck/West Java/M04/2008. Deletion of residue S129 was also found in isolates
2Ck/West Java/ M06 /2008, Ck/West Java/ M07 /2008, and Ck/West Java/ M08 /2008.
Compared with the original vaccine seed, some isolates gained new N-linked glycosylation motifs at residues 84, 86, 156 and 238, while other substitutions caused a loss of glycosylation at residues 165 and 195 in three isolates. Besides deletions, amino acid serine (S) at position 129 was substituted to leucine (L) in two isolates. Amino acid residues Q222 and Table 2 Cross-protectivity (%) of AIV H5N1
1isolated from chicken farms in Indonesia from 2006 to 2010.
Challenge virus Experimental vaccine seed M06 M12 M13 Control
2A/Ck/West Java/ M06 /2008 A/Ck/ North Sumatra/M10 /2008 A/Ck/ East Java/
M12/2009 A/Ck/Central Java/M13/2009 100 60 70 40 80 80 100 80 40 100 80 100 0 0 0 0 G224 were invariably present in the isolates. However, amino acids proximal to these residues were highly polymorphic.
Substitutions of S217T, N220H, and E227D were also identified in three isolates, i.e. M05, M06, and M07.
Other polymorphic variants of S217P, M226I, and E227 G were detected in one isolate. The cleavage site of all isolates was ‘RESRRKKR’, which differs from the vaccine seed sequence of ‘RERRRKKR’. Screening of positively and negatively selected sites indicated that there were no sites under positive selection, while 12 sites were found to be under negative selection (p < 0.05), i.e. residues 34, 85, 135, 201, 235, 298, 299, 324, 327, 363, 367, and 376. Four sites displaying evidence of episodic diversifying selection (p < 0.05) were residues 73, 94, 121, and 239. The protectivity of vaccines against homologous and heterolo- gous isolates is presented in Table 2. The results showed that homologous challenge resulted in 100%
protectivity, whereas heterologous challenge resulted in 40–100% protectivity. An example of 100%
protectivity resulting from heterologous chal- lenge was in an M13-vaccinated chicken challenged with M10.
The results of the cross-reactivity HI assay are presented in Fig. 2. These findings demonstrated that the HI titers with heterologous isolates were 1–4 log2 lower than with homologous isolates. 4. Discussion The main finding of the current study is that HA-1 gene fragments of highly pathogenic AIV-H5N1 isolates from
chicken farms in Indonesia show high levels of genetic variation, leading to limited cross-protection between strains. This demonstrates that the long-term circulation of AIV-H5N1 has resulted in the establishment of novel sub-lineages. The data clearly showed genetic drift in the circulating virus. Phylogenetic analysis of the AIV-H5N1 sequences available in the GenBank database (Fig. 1) showed that the recent isolates had clearly evolved from an Indonesian group A ancestor (Smith et al., 2006). We found no strong evidence of HA-1 antigenic drift due to vaccination. The observed variation seemed to simply express non- sterilizing immunity, that vaccinated birds may excrete low levels of virus after challenge infection (Webster et al., 2006). We expected significant positive selection of amino acids of HA-1, as has been previously reported for AIV-H5N1 (Duvvuri et al., 2009; Shi et al., 2008). However, instead, 12 residues were found to be under purifying selection. Positive selection did occur in low numbers of isolates at four sites with evidence of episodic diversifying selection. These results were supported by the negative Tajima’s test of neutrality (D), which explains purifying selection or recent population expansion (Tajima, 1989). We identified novel viruses that have two new deletion patterns. One pattern was deletion of residues 74 and 129 and occurred in one isolate Ck/West Java/M04/2008. The second pattern was deletion of residue 129 only and was detected in three isolates, i.e.
2Ck/West Java/M05/2008, Ck/West Java/M06/2008, and Ck/West Java/M07/2008.
The significance of a codon deletion at position 74 has not been reported. Amino acid 129 has been
reported to interact structurally with cellular xylosidase receptors (Ha et al., 2001), and may therefore have a functional role in receptor binding. To date, the predominant change found at this position is a serine to leucine substitution (S129L) (Smith et al., 2006; WHOGIPSN, 2005). The deletion of this amino acid might have a major impact on the affinity of respective isolates towards the avian or mammalian receptor (Table 3).
All isolates retain the arginine (R) and lysine (K) rich cleavage site, a defining feature of highly pathogenic AIV (FAO, 2014; WHOGIPSN, 2005). However, all exhibit serine (S) instead of arginine (R) at position 325.
The fact that this variant is present in all of the most recent isolates from chicken farms in Indonesia
suggests that the cleavage site motif is not a signature of the human virus. However, the R325S substitution might enhance cleavability of the infecting strain in humans as well as in birds, heightening the severity of
the disease. Further studies are needed to elucidate the possible impact of this substitution. Analysis of all of the polymorphic sites in the isolates collected in this study suggests that almost all isolates are avian
viruses. With the exception of A/Ck/East Java/M19/2009, all isolates possess amino acid residues Q222 and G224 at the receptor binding pocket that preferentially bind to the avian receptor of Fig. 2. Cross-HI assay of AIV-H5N1
1from chicken farms in Indonesia from 2008 to 2010.
Each antibody was from 10 SPF chicken at 3 weeks post-vaccination. The experimental vaccine seeds were M03 (Legok), M05, M06, and M10. The HI assay was conducted using all isolates as antigen. Table 3 Summary of polymorphic amino acids of HA-1 of highly pathogenic AIV-H5N1
1isolated from chicken farms in Indonesia from 2008 to 2010
in relation to known antigenic sites. Antigenic sites Proposed locations or residues involveda Polymorphic amino acids Site A 119–121, 138–141 and 161–162 Site B 124, 129–133, 151–156, 183–185 and 188–189 Site C 40, 45, 277 and 282 Site D 94 and 226–227 Site E 84–86 and 263–269 CB 70–75 119, 121, 138, 140, 141, 162 129, 133, 151, 152, 155, 156, 183–185, 188, 189 None 94, 226–227 84, 86, 263, 266, 72–74 a Extended from Zou et al. (2012) and Shore et al. (2013) and taking into account the findings of Wu et al.
(2008),Cattoli et al. (2011), and Koel et al. (2014). a-2,3-NeuAcGal (Ha et al., 2001). However, some might acquire the additional capability to bind to the mammalian receptor. The amino acids surrounding residues Q222 and G224 are highly polymorphic. Moreover, A/Ck/East Java/M19/2009 has a Q222K substitution. The genetic drift and deletion events identified in this study are signals for the possible emergence of new strains, which may acquire the capacity to be transmitted across species barriers, including possible human- to-human transmission. Moreover, polymorphic site analysis revealed changes that might alter the biological properties of the viruses. Some changes, such as A156T, T188I, R189G/M, and R325S, affect residues reported to play a role in increasing virulence, as well as modulation of receptor binding and HA cleavage efficiency (WHOGIPSN, 2005). Referring to antigenic sites based on the extended map (Shore et al., 2013;
Zou et al., 2012), isolates from chicken farms in Indonesia in 2008–2010 have presented molecular changes in the A, B, D, E, and CB antigenic sites. The residues in site C at positions 40, 45, 277, and 282 are
homologous. The molecular antigenic profile seems to explain the loss of immune-potency of the original vaccine seed. Most 2008 viruses analyzed in this study originated from vaccinated commercial chicken flocks that displayed high mortality rates and/or a sudden and drastic drop in egg production. The variety of antigenic structures among the AIV isolates was revealed in the cross-HI and challenge experiments. The degree of protectivity of vaccines against heterologous isolates varied from 40 to 100%. Furthermore, the loss of protectivity seemed to be clade independent. Decreased protectivity even occurred in the chal- lenge experiment between M12 and M06, which belong to the same group phylogenetically (Fig. 1 and Table 2). In another case, the group of chickens that had been vaccinated with M13 was completely protected against M10 challenge. The results of the cross-HI assay therefore reveal the antigenic structure heteroge- neity.
The HI assay with homogenous isolates as antigens always resulted in the highest titer. This result
highlights the fact that when using the HI assay to determine antibody levels, closely related isolates should be used; otherwise the results may be misleading as to the interpretation of protectivity of vaccine seeds. In other words, when it comes to judging the protectivity of any vaccine formulation, the assay should be performed using the most dominant isolate(s). For more accurate antigenic structure determination, cross- reactivity should be validated with the serum neutralization assay. The HI assay is known to have limitations, and the results of HI testing should be confirmed by a microneutralization test (Chen et al., 2006). In light of the finding that all three clusters might circulate throughout Indonesia, we propose the use of a polyvalent
vaccine, comprising a member of each novel sub-lineage, to overcome the problem of antigenic drift. A cocktail vaccine of this kind would offer protection against field viruses with various antigenic profiles.
Moreover, to overcome the ‘masking’ effect of vaccination, vaccines and vaccine delivery systems should be developed that can induce systemic as well as local mucosal immunity. This would help to reduce or
eliminate virus shedding in vaccinated birds. To our knowledge, this is the first report on the partial genetic characterization of H5N1 viruses isolated from sector 1 and sector 2 poultry farm systems in Indonesia. We also managed to obtain isolates from broiler farms, which have never been reported previously. Although the HA-1 fragment analyzed here is indeed the most biologically important peptide, especially regarding
neutralizing antigenic and pathogenic determinants, further genotyping studies are needed to elucidate possible genotype shifts or reassortment events in H5N1 viruses, as reported in Vietnam (Smith et al., 2006), that characterize AIVs (Review by (Horimoto and Kawaoka, 2001; Webster et al., 2006)). Data from antigenic cartography (Koel et al., 2014; Smith et al., 2004), as well as cross-protection assays, will also be invaluable in judging the most appropriate vaccine seed(s) to be used in Indonesia. Moreover, a biological study of isolates harboring K/P/Q74 and S129 deletions, as well as S217T, N220H, and E227D substitutions, needs to be conducted to determine whether these residues are important in pathogenicity and/or
adaptability towards mamma- lian hosts, which might constitute a molecular signature of the emergence of a new pandemic strain. Our findings demonstrate that surveillance in chicken farms is essential for
understanding overall epizootic events. Studies focused completely on human viruses in Indonesia do not provide any insight into the possible origin of these viruses, as the potential animal counterparts at the time were not reported (Kandun et al., 2010). 5. Conclusion All viruses from chicken farms in Indonesia in 2008–
2010 form distinct sublineages that have evolved from an Indonesian ancestor. No strong evidence was found that HA-1 antigenic drift was due to vaccination. The genetic variation observed is thought to result from non-sterilizing immunity. Positive selection did occur in a low number of isolates, with evidence of episodic diversifying selection. Molecular variation was found to mediate partial cross-protectivity. The array of antigenic site variations was determined, explaining the loss of immune-potency of a single vaccine seed.
Virus surveillance in poultry farms is essential for pinpointing appropriate vaccine seeds, and these data will be invaluable in understanding human infection. Conflicts of interest None. Acknowledgements We would like to thank the United States Department of State Biosecurity Engagement Program for providing most of the equipment needed for isolation and characterization of viruses in this study. GNM, INS, IGAAS, and IWTW were supported by a Project Research Grant for the National Strategic Research Batch I from the Directorate General of the Higher Education Department of the National Education Government of
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