Analyzing Molecular Traits of H9N2 Avian Influenza Virus Isolated from a Same Poultry Farm in West Java Province, Indonesia, in 2017 and 2023

Muhammad Ade Putra; Amin Soebandrio; I Wayan Teguh Wibawan; Christian Marco Hadi Nugroho Nugroho; Ryan Septa Kurnia; Otto Sahat Martua Silaen; Rifky Rizkiantino; Agustin Indrawati; Okti Nadia Poetri; Desak Gede Budi Krisnamurti

doi:10.12688/f1000research.150975.2

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Research Article

Revised

Analyzing Molecular Traits of H9N2 Avian Influenza Virus Isolated from a Same Poultry Farm in West Java Province, Indonesia, in 2017 and 2023

[version 2; peer review: 1 approved, 2 approved with reservations]

Muhammad Ade Putra¹, Amin Soebandrio², I Wayan Teguh Wibawan³, [...] Christian Marco Hadi Nugroho Nugroho⁴, Ryan Septa Kurnia⁴, Otto Sahat Martua Silaen⁴, Rifky Rizkiantino⁵, Agustin Indrawati³, Okti Nadia Poetri³, Desak Gede Budi Krisnamurti⁶

Muhammad Ade Putra¹, Amin Soebandrio², [...] I Wayan Teguh Wibawan³, Christian Marco Hadi Nugroho Nugroho⁴, Ryan Septa Kurnia⁴, Otto Sahat Martua Silaen⁴, Rifky Rizkiantino⁵, Agustin Indrawati³, Okti Nadia Poetri³, Desak Gede Budi Krisnamurti⁶

PUBLISHED 13 Sep 2024

Author details Author details

¹ Master of Animal Biomedical Sciences, School of Veterinary and Biomedical, IPB University, Bogor, West Java, 16680, Indonesia
² Department of Microbiology, Faculty of Medicine, University of Indonesia, Jakarta, Jakarta, 10320, Indonesia
³ Division of Medical Microbiology, School of Veterinary Medicine and Biomedical Sciences, IPB University, Bogor, West Java, 16680, Indonesia
⁴ Animal Health Diagnostic Unit, PT. Medika Satwa Laboratoris, Bogor, West Java, 16166, Indonesia
⁵ Division of Central Laboratory and Disease Research Center, Technology and Research Development, Central Proteina Prima (CP Prima) Inc., Tangerang, Banten, 15560, Indonesia
⁶ Department of Medical Pharmacy, Faculty of Medicine, University of Indonesia, Jakarta, Jakarta, 10430, Indonesia

Muhammad Ade Putra
Roles: Conceptualization, Formal Analysis, Methodology, Project Administration, Supervision, Visualization, Writing – Original Draft Preparation, Writing – Review & Editing

Amin Soebandrio
Roles: Conceptualization, Investigation, Supervision, Validation, Writing – Original Draft Preparation, Writing – Review & Editing

I Wayan Teguh Wibawan
Roles: Conceptualization, Investigation, Methodology, Validation

Christian Marco Hadi Nugroho Nugroho
Roles: Conceptualization, Data Curation, Formal Analysis, Visualization, Writing – Original Draft Preparation, Writing – Review & Editing

Ryan Septa Kurnia
Roles: Data Curation, Formal Analysis, Methodology, Resources, Supervision, Validation, Visualization

Otto Sahat Martua Silaen
Roles: Project Administration

Rifky Rizkiantino
Roles: Formal Analysis, Methodology, Resources, Software, Validation

Agustin Indrawati
Roles: Investigation, Methodology, Supervision, Validation, Writing – Original Draft Preparation

Okti Nadia Poetri
Roles: Conceptualization, Data Curation, Formal Analysis, Project Administration, Supervision, Writing – Original Draft Preparation, Writing – Review & Editing

Desak Gede Budi Krisnamurti
Roles: Conceptualization, Formal Analysis, Funding Acquisition, Methodology, Project Administration, Supervision, Writing – Review & Editing

OPEN PEER REVIEW

REVIEWER STATUS

This article is included in the Emerging Diseases and Outbreaks gateway.

Abstract

Background

Indonesia is one of the countries that is endemic to avian influenza virus subtype H9N2. This study aims to compare the molecular characteristics of avian influenza virus (AIV) subtype H9N2 from West Java.

Methods

Specific pathogen-free (SPF) embryonated chicken eggs were used to inoculate samples. RNA extraction and RT–qPCR confirmed the presence of H9 and N2 genes in the samples. RT–PCR was employed to amplify the H9N2-positive sample. Nucleotide sequences were obtained through Sanger sequencing and analyzed using MEGA 7. Homology comparison and phylogenetic tree analysis, utilizing the neighbor-joining tree method, assessed the recent isolate’s similarity to reference isolates from GenBank. Molecular docking analysis was performed on the HA1 protein of the recent isolate and the A/Layer/Indonesia/WestJava-04/2017 isolate, comparing their interactions with the sialic acids Neu5Ac2-3Gal and Neu5Ac2-6Gal.

Results

RT–qPCR confirmed the isolate samples as AIV subtype H9N2. The recent virus exhibited 11 amino acid residue differences compared to the A/Layer/Indonesia/WestJava-04/2017 isolate. Phylogenetically, the recent virus remains within the h9.4.2.5 subclade. Notably, at antigenic site II, the recent isolate featured an amino acid N at position 183, unlike A/Layer/Indonesia/WestJava-04/2017. Molecular docking analysis revealed a preference of HA1 from the 2017 virus for Neu5Ac2-3Gal, while the 2023 virus displayed a tendency to predominantly bind with Neu5Ac2-6Gal.

Conclusion

In summary, the recent isolate displayed multiple mutations and a strong affinity for Neu5Ac2-6Gal, commonly found in mammals.

Keywords

avian influenza, characterization, docking, H9N2, Indonesia, Mutation

Corresponding authors: Okti Nadia Poetri, Desak Gede Budi Krisnamurti

Competing interests: No competing interests were disclosed.

Grant information: This study was funded by Universitas Indonesia through a PUTI Grant with contract number NKB-366/UN2.RST/HKP.05.00/2023.
The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Copyright: © 2024 Putra MA et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

How to cite: Putra MA, Soebandrio A, Wibawan IWT et al. Analyzing Molecular Traits of H9N2 Avian Influenza Virus Isolated from a Same Poultry Farm in West Java Province, Indonesia, in 2017 and 2023 [version 2; peer review: 1 approved, 2 approved with reservations]. F1000Research 2024, 13:571 (https://doi.org/10.12688/f1000research.150975.2) First published: 04 Jun 2024, 13:571 (https://doi.org/10.12688/f1000research.150975.1) Latest published: 05 Mar 2025, 13:571 (https://doi.org/10.12688/f1000research.150975.3)

Revised Amendments from Version 1

In this latest version of the manuscript, there are no changes to the title; however, there are minor adjustments in the abstract, such as aligning the terminology for "isolate" with the avian influenza nomenclature and including the name of the latest isolate to make it easier for readers to understand. In the introduction section, we made slight revisions to the wording of the avian influenza isolate to comply with the nomenclature. In the methods section, we clarified all the steps to ensure they are easy to follow, particularly the sample collection part, which we have completed. Some sentences in the methods section were also revised based on the reviewer's suggestions to make them clearer. In the results section, we removed several sentences that repeated information from the methods section, particularly those related to the PCR test results. All virus references have been updated according to the H9N2 nomenclature. Additionally, in the results section, we replaced the phylogenetic result image, specifically Figure 2, to reflect clade divisions based on Carnaccini and Perez (2020). In the discussion section, we removed overly theoretical explanations that did not directly address the research findings, making the discussion slightly more concise. Furthermore, we mentioned the limitations of this study in the discussion section. In this study, the binding ability to sialic acids of the 2017 virus and 2023 virus were just analyzed through molecular docking instead of direct experiment data. Conducting a receptor binding assay such as using analogues of different sialic acids will be more convincing in the next study.

To read any peer review reports and author responses for this article, follow the "read" links in the Open Peer Review table.

Introduction

Avian influenza is an acute viral infectious disease that can affect all types of birds of any age. Based on the differences in hemagglutinin (H1–18) and neuraminidase (N1–N11) components, avian influenza is divided into several subtypes.¹^,² Each subtype has varying levels of pathogenicity, ranging from low pathogenic avian influenza (LPAI) to high pathogenic avian influenza (HPAI). However, both LPAI and HPAI can affect bird health.³ The presence of gene mutations through antigenic drift and antigenic shift can result in a change in symptoms. Initially, LPAI may not cause significant harm, but with these mutations, it can become highly detrimental to the poultry industry.⁴

AIV is an RNA virus that consists of eight segments, each encoding viral protein genes. On the other hand, this virus is also more prone to mutation than DNA viruses.⁵ Diagnosis using polymerase chain reaction (PCR) and sequencing of the viral genome is necessary to determine the homology between the vaccine virus and the circulating field virus.⁶ This is because if the circulating virus has low homology with the vaccine, it can result in the ineffectiveness of the vaccine due to the continued shedding of the virus.⁷

In AIV, there is a crucial virus segment that needs to be characterized, which is hemagglutinin (HA), corresponding to the fourth segment of the AI genome.⁸^,⁹ HA functions at the early stage of infection by attaching the virus to the host receptor. Mutations in the HA gene can increase the virulence and pathogenicity of the virus.¹⁰ Avian influenza infects its target host by initiating the recognition of the HA protein by cellular proteases from the target infected cell. Cellular proteases activate HA0, splitting it into two parts: HA1 and HA2. HA1 binds to Neu5Ac, while HA2 plays a role in fusion between the viral envelope and the host endosomal membrane. Without this recognition, the virus cannot infect the cell.¹¹

One subtype AIV that poses a threat to the poultry industry is the H9N2 subtype.¹² In countries affected by avian influenza outbreaks, such as China, the H9N2 subtype of AIV causes significant losses due to its high morbidity rate of up to 100% in infected layer chicken farms.¹³ Infected layer chickens experience a decrease in appetite accompanied by a drastic decline in egg production. Viral infection is often exacerbated by secondary infections from bacteria or other viruses, resulting in high mortality among the infected layer chicken population.¹⁴ H9N2 virus infection may not be immediately visible at onset, but the virus spreads rapidly through the shedding of feces or nasal discharge from birds. Due to these characteristics, rapid and accurate detection is crucial to identify the presence of AIV infection on a farm, enabling appropriate measures to be taken in addressing H9N2 virus infection.¹⁵

West Java Province is a province in Indonesia that has several densely populated districts for layer chicken farming, including Bogor, Sukabumi, Cianjur, Ciamis, and several other districts. In 2017, one H9N2 isolate was successfully isolated and characterized, namely, strain A/Layer/Indonesia/WestJava-04/2017 (H9N2) (GenBank accession number MG957203), originating from a densely populated layer chicken farming area in West Java.¹⁶ The high density of layer chicken farms in a region facilitates the spread and mutation of AIVs. Therefore, this study aims to isolate and characterize the H9N2 subtype AIV from the layer chicken farm where the A/Layer/Indonesia/WestJava-04/2017 (H9N2) strain originated. This study also describes the molecular changes in the HA gene using molecular docking after implementing the H9N2 vaccination program, which has been ongoing for six years.

Methods

Sample collection

Sampling was conducted at a commercial layer chicken farm in West Java Province, the original location of the AIV subtype H9N2 strain A/Layer/Indonesia/WestJava-04/2017 (H9N2) (GenBank Accession No. MG957203). After five years of vaccination with a homologous commercial vaccine at the farm, a decrease in production and bird mortality occurred in February 2023. Samples consisting of brain, trachea, and oviduct organs from three deceased chickens with symptomatic of H9N2 infection. The samples were from flock of 100,000 chickens at a commercial egg production farm. All organs from all chickens combined in one sample and then was tested by virus isolation and PCR methods.

Virus isolation

The sample was multiplied using standard laboratory procedures. Tissue of pooled organs of three chickens was combined with sterile phosphate-buffered saline (pH 7.4) from ThermoFisher (28348) containing antibiotics (200 g/ml penicillin and 100 g/ml streptomycin) and then centrifuged at 1,000 g and 4 °C for fifteen minutes. The remaining supernatant was filtered through a 0.45 μm membrane filter and injected intra-allantoically into 9-day-old SPF eggs. The SPF eggs were incubated at 37 °C for 48–72 hours, and daily monitoring of embryo mortality was performed.¹⁷ After 72 hours of incubation, all ECEs, regardless of their viability, were subsequently preserved at a refrigerated temperature of 8 °C overnight and then harvested. The collected specimens consisted of allantoic fluid, which was subsequently utilized for conducting a rapid HA assay to promptly detect AIV in early stages.¹⁶

Viral RNA was identified using a reverse transcription-polymerase chain reaction (RT–PCR) assay

The RNA was further extracted from the allantoic fluid that was collected using a total RNA mini kit reagent from Geneaid^®, Taiwan (RPD050). The RNA obtained was then analyzed for the presence of the H9N2 virus using the SensiFASTTM SYBR Lo-ROX Kit from Bioline^®, Taunton (BIO-94005). For the RT–qPCR assay, the primers H9-F: 5′-ATCGGCTGTTAATGGAATGTGTT-3′, H9-R: 5′-TGGGCGTCTTGAATAGGGTAA-3′¹⁸ and N2-F: 5′-CTCCAATAGACCCGTACTAT-3′, N2-R: 5′-CCTGAAGTCCCACAAAATAC-3′¹⁹ were utilized with the LongGene Q2000C (China). The thermal profile for gene amplification included two minutes of polymerase activation at 95 °C, which was followed by a total of 45 cycles of denaturation at 95 °C for 5 seconds, annealing at 60 °C for 10 seconds, and extension at 72 °C for 15 seconds. When the cycle threshold (Ct) value falls below 40, a positive result is indicated. In addition, RT–PCR assays were conducted to detect avian influenza subtype H9N2, Newcastle disease virus (NDV), and infectious bronchitis virus (IBV). RT–PCR was conducted according to the protocol used in a previous study.²⁰

Sequencing

The confirmed HA of the H9 gene from RT–PCR was amplified for sequencing using the MyTaq One-Step RT–PCR kit from Bioline^®, Taunton (BIO-65049), and the primers HAp1-F: 5′-TCCACGGAAACTGTAGACACA-3′, HAp1-R: 5′-TTCTGTGGCTCTCTCCTGAAA-3′ and Hap2-F: 5′-AGGCCTCTTGTCAACGGTTT-3′, Hap2-R: 5′-CCAACGCCCTCTTCACTTTA-3′ were used.²¹ The Sanger sequencing method was performed by First BASE Laboratories, Malaysia, which separated the PCR products using electrophoresis and purified the desired band for sequencing. Using Bioedit v.7 (https://bioedit.software.informer.com/7.0/), the nucleotide and amino acid sequences of a recently isolated strain’s HA gene were determined, and ClustalW was used for alignment. A modern virus phylogenetic tree was constructed using MEGA v 7.0 (https://www.megasoftware.net/), the neighbor-joining method, and 1,000 alignment repetitions. The genetic distance between isolates and the topology of the phylogenetic tree were used to designate strains.²²

A comparison was made between the recent study isolate, a previous isolate A/Layer/Indonesia/WestJava-04/2017 (H9N2) (GenBank Accession No. MG957203) and other H9N2 isolates from GenBank. Several key regions were investigated in this investigation, including the receptor-binding sites (RBS) on the left and right edges of the binding pocket, as well as the cleavage site. In addition, the correlation between particular amino acid residues (54, 80, 106, 109, 113, 123, 125, 129, 130, 135, 137, 146, 147, 149, 150, 152, 164, 165, 178, 179, 182, 183, 188, 189, 194, and 216) and H9N2 virus antigenicity was analysed. The amino acid sequence of the HA gene from the most recent H9N2 virus was uploaded to http://www.cbs.dtu.dk/services/NetNGlyc/to evaluate potential N-glycosylation sites.²¹

Molecular Docking of Protein HA1 (2017 and 2023 isolates) with Ligands Neu5Ac2-3Gal and Neu5Ac2-6Gal

The simulation was conducted on a computer hardware system with the following specifications: 8.00 GB RAM and an Intel^® Core i5-2520 M CPU with a clock speed of 2.50 GHz. The software used in the study included Google Chrome v114.0.5735.199 for accessing the SwissDock website, which served as the protein–ligand docking server and ran automatically (http://swissdock.ch/docking). Additionally, BIOVIA Discovery Studio Visualizer v21.1.0.20298 (https://discover.3ds.com), Chimera v1.17.3 (https://www.cgl.ucsf.edu/chimera/download.html), AutoDock Tools v1.5.7 (https://autodock.scripps.edu/), PyMol v2.5.2 (https://pymol.org/), and LigPlot+ v2.2.8 (https://www.ebi.ac.uk/thornton-srv/software/LigPlus/download.html) were used for data analysis and visualization.

The simulation begins with the creation of a 3D model of the HA1 protein from the AIV, specifically the recent study isolate (HA1-2023) and the A/Layer/Indonesia/WestJava-04/2017 strain (HA1-2017). This is achieved using the PHYRE2 Protein Fold Recognition Server (http://www.sbg.bio.ic.ac.uk/). The ligands used in the simulation were obtained from https://pubchem.ncbi.nlm.nih.gov/. They are Neu5Ac2-3Gal (compound CID: 13832708) and Neu5Ac2-6Gal (compound CID: 53262334).

The docking was performed using the automated server facility on SwissDock, where the uploaded documents were in PDB format (.pdb) for the protein acting as the receptor and SYBYL MOL2 format (.mol2) for the molecules acting as ligands. Docking was carried out for the protein HA1-2017 with the Neu5Ac2-3Gal ligand, HA1-2017 with the Neu5Ac2-6Gal ligand, HA1-2023 with the Neu5Ac2-3Gal ligand, and HA1-2023 with the Neu5Ac2-6Gal ligand. Only models with ligands attached to the Leu216 residue region are observed and selected for further analysis.

The selection of the residue is based on the fact that the amino acid at position 216 determines the virus’s tendency to infect mammalian cells dominated by Neu5Ac2-6Gal or avian cells dominated by Neu5Ac2-3Gal.²⁰ The docking results were downloaded in the Chimera Web Data format (.xml) and run in Chimera software v1.17.3 to select the best docking model. The best model was chosen based on the Gibbs free energy (ΔG) value, where ΔG represents the stability parameter of the bond formed between the protein and the ligand. A more negative value indicates a more stable bond. The protein–ligand interactions are then evaluated, observing hydrogen bonding, hydrogen bond distances (Å), interacting residues and functional groups, and residues that interact with the ligand noncovalently.²³

Nucleotide sequence accession numbers

The partial CDS of the HA gene of A/chicken/Indonesia/MSL0123/2023 (H9N2) in this study was deposited in GenBank and received the accession number OR243721.

Results

H9N2 subtype AI virus detection

The presence of viral RNA was identified by RT–qPCR specifically targeting H9 and N2. The results shown in Figure 1a and Figure 1b indicate that the recent isolate of this study is an AIV subtype H9N2. The H9 gene in a recent isolate was amplified with a Ct value of 28.50, while the N2 gene was amplified at a Ct value of 34.69.

Figure 1. Curves of H9 (a) and N2 (b) RT–qPCR results. The sample revealed single band (c).

Amplification of the Hemagglutinin (HA) gene

The results of HA gene amplification using primers HApar1 and HApar2 on a recent sample from this investigation are shown in Figure 1c. The electrophoresis results revealed the presence of DNA bands at the 736 bp (for HApar1) and 712 bp (for HApar2) sites. Marker 100-2000 bp is the size of the molecular marker displayed in the illustration.

Amino acid analysis

The partial CDS of the HA gene from this study was deposited in GenBank, with the accession number OR243721 as A/chicken/Indonesia/MSL0123/2023 (H9N2). The amino acid sequence at the cleavage site of the recent isolate is PSRSSR↓GLF, while the receptor binding site has the motif PWTNTLY (Table 1). Additionally, at position 217 on the left side of the receptor binding site (RBS), the recent isolate contained the amino acid M (methionine). It should be noted that this amino acid sequence is identical to A/Layer/Indonesia/WestJava-04/2017, which originated from the same farm.

Table 1. Amino acid analysis of important sites in the HA gene of H9N2 viruses.

A. Receptor-binding pockets, cleavage sites and antigenic site of recent isolate of H9N2 compared to A/Layer/Indonesia/WestJava-04/2017 (H9N2)^a
Virus	Lineage	Receptor binding site	Left-edge of binding pocket	Right-edge of binding pocket	Cleavage site	Antigenic site		Acession Number
Virus	Lineage	Receptor binding site	Left-edge of binding pocket	Right-edge of binding pocket	Cleavage site	Site I	Site II	Acession Number
A/Layer/Indonesia/WestJava-04/2017 (H9N2)	h9.4.2.5	PWTNTLY	NGLMGR	GTSKA	PSRSSR↓GLF	SKP	DDL	MG957203
A/chicken/Indonesia/MSL0123/2023 (H9N2)	h9.4.2.5	PWTNTLY	NGLMGR	GTSKA	PSRSSR↓GLF	SKP	DNL	OR243721

B. Potential N-glycosylation sites₂₎
					HA1
Virus	11–13^b	123–125	127–202	178–180	188–190	200–202	280–282	287–289	295–297
A/Layer/Indonesia/WestJava-04/2017 (H9N2)	NST	NVS	–	–	–	NRT	NTT	NVS	NCS
A/chicken/Indonesia/MSL0123/2023 (H9N2)	NST	NVS	–	–	–	NRT	NTT	NVS	NCS

C Other mutations of recent isolate
					HA1
Virus	23	34	53	69	72	74	114	120	163	179
A/Layer/Indonesia/WestJava-04/2017 (H9N2)	N	H	D	P	E	R	I	K	E	T
A/chicken/Indonesia/MSL0123/2023 (H9N2)	D	Q	E	S	G	K	T	T	G	D

a The receptor-binding site (RBS) includes residues at positions 92, 143, 145, 173, 180, 184, and 185. The left-edge of the binding pocket is located at positions 214-219, while the right-edge of the binding pocket is situated at positions 128-132. The cleavage site is positioned at positions 315-323. The antigenic site is divided into two regions: site I, which includes positions 125, 147, and 152, and site II, which encompasses positions 135, 183, and 216.

b The arrangement of amino acid residues in the HA genes of AIV subtype H9N2.These residue placements are based on the H9 numbering system.

In a recent study, the antigenic site I of the HA gene in the recent sample displayed the same motif as A/Layer/Indonesia/WestJava-04/2017 (H9N2). At site I, amino acids S (serine), K (lysine), and P (proline) were present. For site II, the motif consisted of amino acids D (aspartate), N (asparagine), and L (leucine) at positions 135, 183, and 216. In contrast, A/Layer/Indonesia/WestJava-04/2017 (H9N2) exhibited D (aspartate) at position 183. The analysis of the HA gene in the recent sample included an examination of potential glycosylation sites (PGS). The HA1 gene segment revealed the NXT/S motif (where X represents any amino acid except proline) at positions 11-13 (NST), 123-125 (NVS), 200-202 (NRT), 280-282 (NTT), 287-289 (NVS), and 295-297 (NCS). There were also several other amino acid differences observed at positions 23, 34, 53, 69, 72, 74, 114, 120, 163, and 179 (Table 1).

Homology comparison

The results of the homology comparison are shown in Table 2. The recent isolate has a similarity of 95.84% with A/Layer/Indonesia/WestJava-04/2017 (H9N2), which was isolated from the same farm but in a different year. When analyzed against several representatives of the H9N2 subtype clades, the recent isolate has a similarity of approximately 72.24% with clade h9.1, 72.56% with clade h9.2, 74.72% with clade h9.3, and 81.80% with h9.4.1. These results are lower compared to the similarity of the recent isolate with viruses classified under clade h9.4.2. Based on homology comparison, a recent isolate, A/chicken/Indonesia/MSL0123/2023 (H9N2), belonged to subclade h9.4.2.5.

Table 2. Nucleotide sequence similarities between the current strain and several H9N2 isolates in GenBank.

No.	Strain	Accession No.	Clade	Similarity (%)
1	A/turkey/California/189/66 (H9N2)	AF156390	h9.1	72.24
2	A/Shorebird/Delaware/9/96 (H9N2)	AF156386	h9.2	72.56
3	A/Duck/Hong_Kong/Y439/97 (H9N2)	AF156377	h9.3	74.72
4	A/Quail/Hong_Kong/G1/97 (H9N2)	AF156378	h9.4.1	81.80
5	A/Chicken/Shanghai/F/98 (H9N2)	AY743216	h9.4.2.1	86.05
6	A/guineafowl/HongKong/NT184/03 (H9N2)	AY664674	h9.4.2.2	83.66
7	A/Chicken/Guangdong/SS/94 (H9N2)	AF384557	h9.4.2.3	86.95
8	A/Duck/Hong_Kong/Y280/97 (H9N2)	AF156376	h9.4.2.4	88.30
9	A/chicken/Henan/LY-36/2013 (H9N2)	KF638574	h9.4.2.5	90.95
10	A/Layer/Indonesia/WestJava-04/2017 (H9N2)	MG957203	h9.4.2.5	95.84
11	A/muscovy_duck/Vietnam/LBM719/2014 (H9N2)	LC028176	h9.4.2.5	95.55
12	A/chicken/Guangdong/FZH/2011 (H9N2)	JF715024	h9.4.2.6	84.21

Phylogenetic tree

The phylogenetic tree showed that the samples were together with other isolates from Indonesia such as A/Layer/Indonesia/WestJava-04/2017 (H9N2), A/chicken/East_Java/M92_10/2017 (H9N2), A/chicken/East_Java/M92_24/2017 (H9N2), Vietnam A/muscovy_duck/Vietnam/LBM719/2014 (H9N2) and China such as A/chicken/Zhejiang/HE6/2009 (H9N2), A/chicken/Henan/LY-36/2013 (H9N2), A/chicken/Guangdong/LGQ02/2014 (H9N2) so that these were included in China, Vietnam, and Indonesia (CVI) clades (Figure 2). Recent viruses belonged to subclade h9.4.2.5, according to a phylogenetic study.

Figure 2. Phylogenetic tree of the hemagglutinin (HA) gene of the AIV H9N2 virus was constructed.

Docking analysis

The target HA1 proteins of AIV subtype H9N2 isolated in 2017 (HA12017) and 2023 (HA12023) were chosen for the study because of their importance in viral attachment for the docking analysis, as there were no 3D structures of the protein, and its modeling was carried out. After submission of their amino acid sequences on Phyre2, the modeled proteins obtained were downloaded and visualized in Discovery Studio Visualizer. The Neu5Ac2-3Gal (Sia3) and Neu5Ac2-6Gal (Sia6) ligands used in this study are shown in Figure 3.

Figure 3. Modeled protein HA1 of H9N2 predicted by the Robetta server, visualized in Discovery Studio Visualizer.

Based on the docking results, the data obtained are presented in Table 3. The obtained results represent the best model with the most negative ∆G and the ligand positioned and bound near the Leu216 residue. The interactions between each protein and ligand are shown in Figure 4, with hydrogen bonding data and residues involved in hydrogen bonding interactions presented in Table 4. The interactions that occur can be either hydrogen bonding or nonbonding interactions that strengthen the ligand’s affinity with the protein.

Table 3. Docking results of proteins HA12017 and HA12023 with ligands Sia3 and Sia6 selected.

Protein	Ligand	Cluster	Delta G ( $∆$ G) (Kcal/mol)
HA12017	Sia3	2	-7.98304
HA12017	Sia6	11	-7.79904
HA12023	Sia3	2	-7.42221
HA12023	Sia6	8	-7.84408

Figure 4. Interactions occurring in HA12017 and HA12023 with ligand Sia3 and Sia6.

Table 4. Hydrogen bonds and interacting residues.

Protein	Ligand	Hydrogen bond distance (Å)	Residues and functional groups that are binding	Residues that have nonbonding interactions with the ligand
HA12017	Sia3	3.22	Asn173 (O-O)	Thr129, Pro178, Leu184, Pro175, Gly218, Thr179, Leu216
		3.23	Met217 (O-N)
		3.18	Thr177 (O-O)
		3.17	Thr180 (O-O)
HA12023	Sia3	2.93	Thr180 (O-O)	Thr177, Gly215, Leu216, Pro176, Ser130, Lys131
HA12023	Sia3	3.09	Met217 (O-N)	Thr177, Gly215, Leu216, Pro176, Ser130, Lys131
HA12017	Sia6	3.21	Lys131 (O-N)	Gly218, Pro175, Trp143, Pro176, Tyr91, Leu216, Met217
		2.85	Gly215 (O-O)
		2.85	Ser130 (O-O)
		3.20	Asn173 (O-N)
HA12023	Sia6	3.18	Lys 131 (O-N)	Ser130, Leu216, Pro176, Tyr91, Gly218, Asn173, Trp143
		3.19	Lys 131 (O-N)
		3.19	Gly215 (O-O)
		3.10	Thr180 (O-O)
		2.87	Thr129 (O-O)

Discussion

While numerous genes are involved in avian influenza pathogenesis, the HA surface glycoprotein remains critical.²⁴ Hemagglutinin regulates the parameters that influence the virulence and pathogenicity of avian influenza virus. The amino acid makeup of the HA gene includes the cleavage site, receptor binding site (RBS), antigenic site, and whether or not glycosylation sites surround the receptor binding sites.²⁵

The cleavage site, which is located between the HA1 and HA2 subunits of the HA gene, is critical in determining the pathogenicity of AIV.²⁶ Viral evolution is responsible for the formation of H9N2 virus variants with several residues (dibasic/tribasic) at the cleavage site.²⁷

The presence of an RSSR motif at a virus’s cleavage site influences viral propagation, host tissue tropism, and viral pathogenicity.²⁸ This specific motif, PSRSSR↓GLF, was previously identified in A/Layer/Indonesia/WestJava-04/2017 (H9N2), A/Layer/Indonesia/WestJava-06/2017 (H9N2), A/Layer/Indonesia/WestJava-09/2017 (H9N2), A/Layer/Indonesia/WestJava-13/2017 (H9N2), A/Layer/Indonesia/WestJava-14/2017 (H9N2), A/Layer/Indonesia/WestJava-15/2017 (H9N2), and A/Layer/Indonesia/CentralJava-01/2017 (H9N2).¹⁶ The H9.4.2.5 clade of the H9N2 virus has a PSRSSRGLF pattern in cleavage, indicating that it is a low pathogenic avian influenza (LPAI) virus. This motif selectively responds to the trypsin enzyme, which is mostly released by the digestive and respiratory systems, limiting the breadth of the infection.²⁹

The current isolate in this study has the PWTNTLY amino acid motif of the receptor-binding site (RBS). RBS position 180 has been reported to influence the amount of bond affinity to sialyl-2,6-galactose (human cell receptor). Threonine (T) at position 180 had the highest binding affinity.³⁰ The motif of the H9.4.2.5 subclade is amino acid M (methionine) at position 217 on the left side of the RBS.³¹

Threonine (T) is found at position 129 on the right side of the receptor binding site (RBS), and lysine (K) is found at position 131. The H9.4.2 subclade is the only subclade with these unique I129T and R131K amino acid changes.²¹ A single modification to an amino acid within the receptor binding pocket can have a major impact on the AI virus’s ability to infect and disseminate. This element is critical in influencing AIV host diversity.³² Notably, changed receptor binding avidity of H9N2 viruses, including higher binding to human-like receptors, results in antigenic diversity in AIVs, raising the possibility of spreading zoonotic agents.³³

The antigenic site motif detected in recent isolate was similar to that of A/Layer/Indonesia/WestJava-04/2017 (H9N2) and other Indonesian isolates. The residue at position 216 inside this antigenic region is important in receptor-binding interactions with the host. Human influenza viruses that contain the amino acid L216 preferentially bind to Neu5Ac2-6Gal receptors, whereas AIVs that contain the amino acid Q216 preferentially bind to Neu5Ac2-3Gal receptors.³⁴ Additionally, the change from Q216L can improve the virus’s capacity for infecting people.³⁵

H9N2 pathogens are prevalent and have rarely caused severe illnesses in people. Although massive chicken vaccination has been beneficial in suppressing H9N2, it may have also contributed to the virus’s evolutionary alterations.³⁶ As a result, it is critical to track the evolution of circulating H9N2 strains and assess their potential dangers to veterinary medicine and public health. We discovered numerous mutations that could have been impacted by vaccination in this study.

Regarding the eleven amino acid variations among a recent isolate and A/Layer/Indonesia/WestJava-04/2017 (H9N2), position 183 was discovered to alter H9N2 avian influenza infection. Residue N in this location encodes the preference for human-like receptors. One change in amino acids at this position significantly impacted H9N2 antigenicity, as measured by monoclonal antibodies and antisera.³⁷ in vivo studies demonstrated that changes influenced viral replication and transmission of H9N2 in chickens. D183-containing viruses were able to multiply in the lungs of infected hens. Variations in antigenic site II locations 183 and 216 decrease viral interactions with epitope-specific antibodies and can result in mutant virus escape.¹⁶^,³⁸

The possibility of a glycosylation site (PGS) pattern in the sample isolate matched those observed in H9.4.2.5, including A/Layer/Indonesia/WestJava-04/2017 (H9N2) and other Indonesian isolates. A viral method involves changing the amino acid sequence at the glycosylation site to conceal or reveal the antigenic area, preventing it from being recognized by the immune system. The addition of PGS to the sample at residue positions 295-297 may improve the virus’s ability to evade neutralization by host antibodies.³¹^,³⁹

According to phylogeography, the H9 subtype of AIV can be separated into two basic lineages: American and Eurasian.⁴⁰ The H9 subtype is divided into four clades based on HA gene segments.⁴¹ The H9.1 clade is derived from the A/turkey/Wisconsin/1/1966 (H9N2) virus found in bird populations on the continent of North America. The H9.2 clade of the American lineage is linked to the A/quail/Arkansas/29209-1/93 (H9N2) strain from the Arkansas region. The Eurasian lineage, on the other hand, is composed of two primary gene lines: Y439-H9.3 (A/duck/HongKong/Y439/1997 (H9N2)) and G1-H9.4 (A/Quail/Hong Kong/G1/1997 (H9N2)). The Y439 lineage, which has spread in wild bird species across Asia, Africa, and Europe, is likely to be the closest to the origin of the AI virus. The G1 lineage has additionally been reported, which is most typically found in commercial poultry and live poultry markets.⁴² The H9.4 clade has been divided into two subclades: H9.4.1 and H9.4.2. Since 2013, the H9.4.2 subclade of the H9N2 AI virus has been discovered in China.¹³ Based on a genetic distance analysis, the recent sample in this study shared genetic similarities with A/muscovy duck/Vietnam/LBM719/2014 (H9N2).¹⁶

The specimens utilized in this study are closely linked to previously reported isolates from Indonesia, notably Yogyakarta and Central Java,¹⁶ Sulawesi,⁴³ Banten and North Sumatra,¹⁶ based on the phylogenetic tree. Furthermore, the isolates are similar to those found in China and Vietnam. Wild birds serve as natural reservoirs for all AIV subtypes and play a crucial role in the virus’s ecology and spread. The global spread of the AI virus is assisted by the trade in chicken and poultry products, as well as seasonal bird movement.⁴⁴ The passage of wild birds from East Asia to Australia has a substantial impact on the spread of the H9N2 AI virus in Chinese layer farms, and this movement can result in viral transmission between different places.⁴⁵

Based on the docking results of HA12017 and HA12023 with ligand Sia 3, a lower ∆G value was obtained for the binding of HA12017-Sia3 compared to HA12023-Sia3. This prediction suggests that the A/Layer/Indonesia/WestJava-04/2017 (H9N2) isolate has a better affinity for Neu5Acα(2,3)-Gal than the recent isolate from 2023. The Gibbs free energy (∆G) is one of the parameters for the stability of the ligand–receptor conformation. In living organisms, metabolic reactions are evaluated based on thermodynamics, which can be either endergonic or exergonic.⁴⁶^–⁴⁸

The isolate A/Layer/Indonesia/WestJava-04/2017 (H9N2) forms more hydrogen bonds compared to the latest isolate. Hydrogen bonds are specific and essential interactions in the process of ligand–receptor interactions.⁴⁹ Therefore, hydrogen bonds contribute to the affinity of a molecule toward the target protein, forming electrostatic interactions between hydrogen donors and acceptors. The analysis of hydrogen bond interactions considers a bond distance of 3.9 Å between the donor and acceptor.⁵⁰ However, in the docking results between HA12017 and HA12023 with ligand Sia 6, a lower ∆G value was obtained for the binding of HA12023-Sia6 compared to HA12017-Sia6. The hydrogen bond interactions observed in this case also support the notion that the latest isolate has a stronger binding affinity for Neu5Acα(2,3)-Gal than the A/Layer/Indonesia/WestJava-04/2017 (H9N2) isolate. The number of hydrogen bonds identified in the docking results for HA12023-Sia6 is five, while HA12017-Sia6 forms four hydrogen bonds. More hydrogen bonds indicate a stronger ligand–receptor interaction. In summary, the A/Layer/Indonesia/WestJava-04/2017 (H9N2) isolate forms more hydrogen bonds, indicating its higher affinity for Neu5Acα(2,3)-Gal. Conversely, the latest isolate, HA12023, shows a lower ∆G value and forms more hydrogen bonds with the ligand Sia 6, suggesting its increased strength in binding to Neu5Acα(2,3)-Gal compared to the A/Layer/Indonesia/WestJava-04/2017 isolate.

In addition to the ∆G parameter and the number of hydrogen bonds, nonbonding residue interactions between the protein and ligand, commonly known as hydrophobic interactions, also play a role in determining the stability of Neu5Acα with HA12017 and HA12023. Hydrophobic interactions are characterized by avoiding the aqueous environment and tend to cluster within the interior of the globular protein structure, minimizing interactions of nonpolar residues with water.⁵¹^,⁵² In this study, all docking results indicated the presence of a hydrophobic residue, Leu216, which is a nonpolar amino acid. Nonpolar amino acid residues tend to form chain-like clusters in the interior part of the protein. Residues involved in hydrophobic interactions are located in the interior region of the protein, contributing to the stability of the protein’s tertiary structure. This indicates that position 216 plays a crucial role in the binding interaction of the HA1 protein with Neu5Acα. Besides, the binding ability to sialic acids of the 2017 virus and 2023 virus were just analyzed through molecular docking instead of direct experiment data. Conducting a receptor binding assay such as using analogues of different sialic acids will be more convincing in the next study.

In conclusion, this study investigated the molecular characteristics of avian influenza virus subtype H9N2 in Indonesia, focusing on a farm in West Java province in 2017 and 2023. Molecular analysis revealed 11 amino acid residue differences between the recent virus isolate and the 2017 reference isolate, yet both viruses still fell within the h9.4.2.5 subclade phylogenetically. At critical AIV sites, the recent isolate exhibited a similar arrangement to the 2017 isolate, with the exception of amino acid N at position 183 in site II of the antigenic site. Furthermore, molecular docking study indicated that the HA1 protein from the 2017 virus had a preference for binding with Neu5Ac2-3Gal, while the 2023 virus displayed a tendency to predominantly bind with Neu5Ac2-6Gal, a sialic acid receptor that commonly found in mammals. These findings highlight the evolution of avian influenza virus subtype H9N2 in the region, with notable mutations and changes in receptor binding preferences. The insights are crucial for understanding the potential risks associated with the virus’s adaptability and its interactions with host species, including humans, emphasizing the importance of continued surveillance and research efforts in managing avian influenza outbreaks.

Ethical statement

The present research adheres to the guidelines outlined in the Indonesian Law on Animal Health Research (UU/18/2009, article 80). Due to the absence of live animals in this study, ethical approval was not necessary.

Data availability statement

GenBank: The Nucleotide database. Accession number MG957203; https://www.ncbi.nlm.nih.gov/nuccore/MG957203.¹⁶ The ligands used in the simulation were obtained from https://pubchem.ncbi.nlm.nih.gov/. They are Neu5Ac2-3Gal (compound CID: 13832708) and Neu5Ac2-6Gal (compound CID: 53262334).

Acknowledgment

The authors thank Dr. Sudarisman and Adin Priadi, dvm for discussion and kind advice.

References

1. Balkhy H, Al-Hajjar S: Avian influenza: are our feathers ruffled? Ann. Saudi Med. 2006 May-Jun; 26(3): 175–82. eng. PubMed Abstract | Publisher Full Text | Free Full Text
2. Nugroho C, Pawitan J: Production of Antibody for Direct Fluorescence Antibody Assay against Avian Influenza H9N2. Hosts and Viruses. 2020; 7(28): 7. Publisher Full Text
3. Kanaujia R, Bora I, Ratho RK, et al.: Avian influenza revisited: concerns and constraints. Virusdisease. 2022 Dec; 33(4): 456–65. Epub 20221028. eng. PubMed Abstract | Publisher Full Text | Free Full Text
4. Shao W, Li X, Goraya MU, et al.: Evolution of Influenza A Virus by Mutation and Re-Assortment. Int. J. Mol. Sci. 2017 Aug 7; 18(8). Epub 20170807. eng. PubMed Abstract | Publisher Full Text | Free Full Text
5. Lee DH, Song CS: H9N2 avian influenza virus in Korea: evolution and vaccination. Clin Exp. Vaccine Res. 2013 Jan; 2(1): 26–33. Epub 20130115. eng. PubMed Abstract | Publisher Full Text | Free Full Text
6. Gao X, Wang N, Chen Y, et al.: Sequence characteristics and phylogenetic analysis of H9N2 subtype avian influenza A viruses detected from poultry and the environment in China, 2018. PeerJ. 2021; 9: e12512. Epub 20211220. eng. PubMed Abstract | Publisher Full Text | Free Full Text
7. Li C, Yu K, Tian G, et al.: Evolution of H9N2 influenza viruses from domestic poultry in Mainland China. Virology. 2005 2005/09/15/; 340(1): 70–83. Publisher Full Text
8. Bouvier NM, Palese P: The biology of influenza viruses. Vaccine. 2008 Sep 12; 26 Suppl 4(Suppl 4): D49–53. eng. PubMed Abstract | Publisher Full Text | Free Full Text
9. Sriwilaijaroen N, Suzuki Y: Molecular basis of the structure and function of H1 hemagglutinin of influenza virus. Proc. Jpn. Acad. Ser. B Phys. Biol. Sci. 2012; 88(6): 226–49. Epub 2012/06/26. eng. PubMed Abstract | Publisher Full Text | Free Full Text
10. Neumann G: H5N1 influenza virulence, pathogenicity and transmissibility: what do we know? Future Virol. 2015; 10(8): 971–80. eng. PubMed Abstract | Publisher Full Text | Free Full Text
11. Taubenberger JK: Influenza virus hemagglutinin cleavage into HA1, HA2: no laughing matter. Proc. Natl. Acad. Sci. USA. 1998 Aug 18; 95(17): 9713–5. eng. PubMed Abstract | Publisher Full Text | Free Full Text
12. Xu H, Qian J, Song Y, et al.: The adaptability of H9N2 avian influenza A virus to humans: A comparative docking simulation study. Biochem. Biophys. Res. Commun. 2020 2020/09/03/; 529(4): 963–9. PubMed Abstract | Publisher Full Text
13. Gu M, Xu L, Wang X, et al.: Current situation of H9N2 subtype avian influenza in China. Vet. Res. 2017 12/01; 48: 49. PubMed Abstract | Publisher Full Text | Free Full Text
14. Bonfante F, Mazzetto E, Zanardello C, et al.: A G1-lineage H9N2 virus with oviduct tropism causes chronic pathological changes in the infundibulum and a long-lasting drop in egg production. Vet. Res. 2018 Aug 29; 49(1): 83. Epub 20180829. eng. PubMed Abstract | Publisher Full Text | Free Full Text
15. Iqbal M, Yaqub T, Mukhtar N, et al.: Infectivity and transmissibility of H9N2 avian influenza virus in chickens and wild terrestrial birds. Vet. Res. 2013 Oct 17; 44(1): 100. Epub 20131017. eng. PubMed Abstract | Publisher Full Text | Free Full Text
16. Nugroho CMH, Silaen OSM, Kurnia RS, et al.: Isolation and molecular characterization of the hemagglutinin gene of H9N2 avian influenza viruses from poultry in Java, Indonesia. J. Adv. Vet. Anim. Res. 2021 Sep; 8(3): 423–34. Epub 2021/11/02. eng. PubMed Abstract | Publisher Full Text | Free Full Text
17. Kurnia RS, Tarigan S, Nugroho CMH, et al.: Potency of bacterial sialidase Clostridium perfringens as antiviral of Newcastle disease infections using embryonated chicken egg in ovo model. Vet. World. 2022 Aug; 15(8): 1896–905. Epub 20220806. eng. PubMed Abstract | Publisher Full Text | Free Full Text
18. Chaharaein B, Omar AR, Aini I, et al.: Detection of H5, H7 and H9 subtypes of avian influenza viruses by multiplex reverse transcription-polymerase chain reaction. Microbiol. Res. 2009; 164(2): 174–9. Epub 2007/03/06. eng. PubMed Abstract | Publisher Full Text
19. Abid M, Yaqub T, Mehboob A, et al.: Characterization and Phylogenetic Analysis of Avian Influenza Virus Subtype h9n2 in Pakistan.2017 08/24.
20. Nugroho CMH, Kurnia RS, Tarigan S, et al.: Screening and purification of NanB sialidase from Pasteurella multocida with activity in hydrolyzing sialic acid Neu5Acα(2-6) Gal and Neu5Acα(2-3)Gal. Sci. Rep. 2022 Jun 8; 12(1): 9425. Epub 2022/06/09. eng. PubMed Abstract | Publisher Full Text | Free Full Text
21. Maharani NR, Susetya H, Wibowo MH: Characterization of Hemagglutinin Gene Fragment of H9N2 Avian Influenza Virus Isolated from Environmental Live Bird Market in the Greater Jakarta Area. Trop. Anim. Sci. J. 2022 05/18; 45(2): 141–153. Publisher Full Text
22. Silaen OSM, Murtini S, Pamungkas J, et al.: Isolation and molecular characterization of fowl aviadenovirus associated with inclusion body hepatitis from poultry in Banten and West Java, Indonesia. Vet. World. 2020 Sep; 13(9): 1940–6. Epub 20200922. eng. PubMed Abstract | Publisher Full Text | Free Full Text
23. Syahputra G: Docking Simulation of Curcumin and Its Analogs as Inhibitors on 12-Lipoxygenase Enzyme.2014.
24. Richard M, de Graaf M , Herfst S: Avian influenza A viruses: from zoonosis to pandemic. Future Virol. 2014 May 1; 9(5): 513–24. PubMed Abstract | Free Full Text eng, Publisher Full Text |
25. Zhang Y, Xu C, Zhang H, et al.: Targeting Hemagglutinin: Approaches for Broad Protection against the Influenza A Virus. Viruses. 2019 Apr 30; 11(5). Epub 20190430. eng. PubMed Abstract | Publisher Full Text | Free Full Text
26. Shirvani E, Paldurai A, Varghese BP, et al.: Contributions of HA1 and HA2 Subunits of Highly Pathogenic Avian Influenza Virus in Induction of Neutralizing Antibodies and Protection in Chickens. Front. Microbiol. 2020; 11: 1085. Epub 20200605. eng. PubMed Abstract | Publisher Full Text | Free Full Text
27. Zhang J, Ma K, Li B, et al.: A risk marker of tribasic hemagglutinin cleavage site in influenza A (H9N2) virus. Commun Biol. 2021 Jan 15; 4(1): 71. Epub 20210115. eng. PubMed Abstract | Publisher Full Text | Free Full Text
28. Baron J, Tarnow C, Mayoli-Nüssle D, et al.: Matriptase, HAT, and TMPRSS2 activate the hemagglutinin of H9N2 influenza A viruses. J. Virol. 2013 Feb; 87(3): 1811–20. Epub 20121128. eng. PubMed Abstract | Publisher Full Text | Free Full Text
29. Bóna M, Kiss I, Dénes L, et al.: Tissue Tropism of H9N2 Low-Pathogenic Avian Influenza Virus in Broiler Chickens by Immunohistochemistry. Animals (Basel). 2023 Mar 14; 13(6). Epub 20230314. eng. PubMed Abstract | Publisher Full Text | Free Full Text
30. Teng Q, Xu D, Shen W, et al.: A Single Mutation at Position 190 in Hemagglutinin Enhances Binding Affinity for Human Type Sialic Acid Receptor and Replication of H9N2 Avian Influenza Virus in Mice. J. Virol. 2016 Nov 1; 90(21): 9806–25.Epub 20161014. eng. PubMed Abstract | Publisher Full Text | Free Full Text
31. Shen HQ, Yan ZQ, Zeng FG, et al.: Isolation and phylogenetic analysis of hemagglutinin gene of H9N2 influenza viruses from chickens in South China from 2012 to 2013. J. Vet. Sci. 2015; 16(3): 317–24. Epub 20150130. eng. PubMed Abstract | Publisher Full Text | Free Full Text
32. Sun H, Lin J, Liu Z, et al.: Genetic, Molecular, and Pathogenic Characterization of the H9N2 Avian Influenza Viruses Currently Circulating in South China. Viruses. 2019 Nov 8; 11(11). Epub 20191108. eng. PubMed Abstract | Publisher Full Text | Free Full Text
33. Sealy JE, Yaqub T, Peacock TP, et al.: Association of Increased Receptor-Binding Avidity of Influenza A(H9N2) Viruses with Escape from Antibody-Based Immunity and Enhanced Zoonotic Potential. Emerg. Infect. Dis. 2018 Jan; 25(1): 63–72. eng. PubMed Abstract | Publisher Full Text | Free Full Text
34. Belser JA, Sun X, Brock N, et al.: Genetically and Antigenically Divergent Influenza A(H9N2) Viruses Exhibit Differential Replication and Transmission Phenotypes in Mammalian Models. J. Virol. 2020 Aug 17; 94(17). Epub 20200817. eng. PubMed Abstract | Publisher Full Text | Free Full Text
35. Song Y, Zhang Y, Chen L, et al.: Genetic Characteristics and Pathogenicity Analysis in Chickens and Mice of Three H9N2 Avian Influenza Viruses. Viruses. 2019 Dec 6; 11(12). Epub 20191206. eng. PubMed Abstract | Publisher Full Text | Free Full Text
36. Rehman S, Rantam FA, Batool K, et al.: Emerging threats and vaccination strategies of H9N2 viruses in poultry in Indonesia: A review. F1000Res. 2022; 11: 548. Epub 20220519. eng. PubMed Abstract | Publisher Full Text | Free Full Text
37. Peacock T, Reddy K, James J, et al.: Antigenic mapping of an H9N2 avian influenza virus reveals two discrete antigenic sites and a novel mechanism of immune escape. Sci. Rep. 2016 Jan 7; 6: 18745. Epub 20160107. eng. PubMed Abstract | Publisher Full Text | Free Full Text
38. Kaverin NV, Rudneva IA, Ilyushina NA, et al.: Structural differences among hemagglutinins of influenza A virus subtypes are reflected in their antigenic architecture: analysis of H9 escape mutants. J. Virol. 2004 Jan; 78(1): 240–9. eng. PubMed Abstract | Publisher Full Text | Free Full Text
39. Deshpande KL, Fried VA, Ando M, et al.: Glycosylation affects cleavage of an H5N2 influenza virus hemagglutinin and regulates virulence. Proc. Natl. Acad. Sci. USA. 1987 Jan; 84(1): 36–40. eng. PubMed Abstract | Publisher Full Text | Free Full Text
40. Hu Z, Peng F, Xiong Z, et al.: Genetic and Molecular Characterization of H9N2 Avian Influenza Viruses Isolated from Live Poultry Markets in Hubei Province, Central China, 2013-2017. Virol. Sin. 2021 Apr; 36(2): 291–9. Epub 2020/09/15. eng. PubMed Abstract | Publisher Full Text | Free Full Text
41. Carnaccini S, Perez DR: H9 Influenza Viruses: An Emerging Challenge. Cold Spring Harb. Perspect. Med. 2020 Jun 1; 10(6). Epub 2019/12/25. eng. PubMed Abstract | Publisher Full Text | Free Full Text
42. El Mellouli F, Mouahid M, Fusaro A, et al.: Spatiotemporal Dynamics, Evolutionary History and Zoonotic Potential of Moroccan H9N2 Avian Influenza Viruses from 2016 to 2021. Viruses. 2022 Mar 1; 14(3). Epub 2022/03/27. eng. PubMed Abstract | Publisher Full Text | Free Full Text
43. Jonas M, Sahesti A, Murwijati T, et al.: Identification of avian influenza virus subtype H9N2 in chicken farms in Indonesia. Prev. Vet. Med. 2018 Nov 1; 159: 99–105. Epub 2018/10/14. eng. PubMed Abstract | Publisher Full Text
44. van der Kolk JH : Role for migratory domestic poultry and/or wild birds in the global spread of avian influenza? Vet. Q. 2019 Dec; 39(1): 161–7. eng. PubMed Abstract | Publisher Full Text | Free Full Text
45. Peacock TP, James J, Sealy JE, et al.: A Global Perspective on H9N2 Avian Influenza Virus. Viruses. 2019; 11(7). PubMed Abstract | Publisher Full Text | Free Full Text
46. Noor E, Haraldsdóttir HS, Milo R, et al.: Consistent estimation of Gibbs energy using component contributions. PLoS Comput. Biol. 2013; 9(7): e1003098. Epub 20130711. eng. PubMed Abstract | Publisher Full Text | Free Full Text
47. Golas SM, Nguyen AN, Rietman EA, et al.: Gibbs free energy of protein-protein interactions correlates with ATP production in cancer cells. J. Biol. Phys. 2019 Dec; 45(4): 423–30. Epub 20191216. eng. PubMed Abstract | Publisher Full Text | Free Full Text
48. McGuire SH, Rietman EA, Siegelmann H, et al.: Gibbs free energy as a measure of complexity correlates with time within C. elegans embryonic development. J. Biol. Phys. 2017 Dec; 43(4): 551–63. Epub 20170919. eng. PubMed Abstract | Publisher Full Text | Free Full Text
49. Bulusu G, Desiraju G: Strong and Weak Hydrogen Bonds in Protein–Ligand Recognition. J. Indian Inst. Sci. 2019 11/14; 100: 31–41. Publisher Full Text
50. Chen D, Oezguen N, Urvil P, et al.: Regulation of protein-ligand binding affinity by hydrogen bond pairing. Sci. Adv. 2016 Mar; 2(3): e1501240. Epub 20160325. eng. PubMed Abstract | Publisher Full Text | Free Full Text
51. Lins L, Brasseur R: The hydrophobic effect in protein folding. FASEB journal: official publication of the Federation of American Societies for. Exp. Biol. 1995 05/01; 9: 535–40. Publisher Full Text
52. Bogunia M, Makowski M: Influence of Ionic Strength on Hydrophobic Interactions in Water: Dependence on Solute Size and Shape. J. Phys. Chem. B. 2020 2020/11/19; 124(46): 10326–36. PubMed Abstract | Publisher Full Text | Free Full Text

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Author details Author details

¹ Master of Animal Biomedical Sciences, School of Veterinary and Biomedical, IPB University, Bogor, West Java, 16680, Indonesia
² Department of Microbiology, Faculty of Medicine, University of Indonesia, Jakarta, Jakarta, 10320, Indonesia
³ Division of Medical Microbiology, School of Veterinary Medicine and Biomedical Sciences, IPB University, Bogor, West Java, 16680, Indonesia
⁴ Animal Health Diagnostic Unit, PT. Medika Satwa Laboratoris, Bogor, West Java, 16166, Indonesia
⁵ Division of Central Laboratory and Disease Research Center, Technology and Research Development, Central Proteina Prima (CP Prima) Inc., Tangerang, Banten, 15560, Indonesia
⁶ Department of Medical Pharmacy, Faculty of Medicine, University of Indonesia, Jakarta, Jakarta, 10430, Indonesia

Muhammad Ade Putra
Roles: Conceptualization, Formal Analysis, Methodology, Project Administration, Supervision, Visualization, Writing – Original Draft Preparation, Writing – Review & Editing

Amin Soebandrio
Roles: Conceptualization, Investigation, Supervision, Validation, Writing – Original Draft Preparation, Writing – Review & Editing

I Wayan Teguh Wibawan
Roles: Conceptualization, Investigation, Methodology, Validation

Christian Marco Hadi Nugroho Nugroho
Roles: Conceptualization, Data Curation, Formal Analysis, Visualization, Writing – Original Draft Preparation, Writing – Review & Editing

Ryan Septa Kurnia
Roles: Data Curation, Formal Analysis, Methodology, Resources, Supervision, Validation, Visualization

Otto Sahat Martua Silaen
Roles: Project Administration

Rifky Rizkiantino
Roles: Formal Analysis, Methodology, Resources, Software, Validation

Agustin Indrawati
Roles: Investigation, Methodology, Supervision, Validation, Writing – Original Draft Preparation

Okti Nadia Poetri
Roles: Conceptualization, Data Curation, Formal Analysis, Project Administration, Supervision, Writing – Original Draft Preparation, Writing – Review & Editing

Desak Gede Budi Krisnamurti
Roles: Conceptualization, Formal Analysis, Funding Acquisition, Methodology, Project Administration, Supervision, Writing – Review & Editing

Competing interests

No competing interests were disclosed.

Grant information

This study was funded by Universitas Indonesia through a PUTI Grant with contract number NKB-366/UN2.RST/HKP.05.00/2023.
The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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Putra MA, Soebandrio A, Wibawan IWT et al. Analyzing Molecular Traits of H9N2 Avian Influenza Virus Isolated from a Same Poultry Farm in West Java Province, Indonesia, in 2017 and 2023 [version 2; peer review: 1 approved, 2 approved with reservations]. F1000Research 2024, 13:571 (https://doi.org/10.12688/f1000research.150975.2)

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Albert Perlas, Helmholtz AI, Helmholtz Zentrum München, Helmholtz AI, 85764, Spain; Helmholtz Pioneer Campus, Helmholtz Zentrum Muenchen, Neuherberg, 85764, Germany

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https://doi.org/10.5256/f1000research.171635.r333988

The authors compared the molecular characteristics of a subtype H9N2 avian influenza virus strain isolated in 2023 with another strain isolated in 2017 from the same farm in Indonesia. They sequenced the HA segment of the 2023 strain after RT-PCR ... Continue reading

The authors compared the molecular characteristics of a subtype H9N2 avian influenza virus strain isolated in 2023 with another strain isolated in 2017 from the same farm in Indonesia. They sequenced the HA segment of the 2023 strain after RT-PCR amplification using Sanger sequencing. Based on these sequencing results, the authors performed phylogenetic analysis, molecular homology comparisons, and molecular docking analysis. The results revealed 11 amino acid residue differences between the two strains, confirmed that both strains belong to the H9.4.2.5 subclade, and suggested, through molecular docking, a preference of the 2023 strain for Neu5AC2-6Gal, which is more commonly found in mammalian species. Overall, the manuscript provides relevant information on the concerning H9N2 avian influenza subtype. However, the text is not consistently clear or well-organized, and improvements in these areas would enhance the quality of the work.

Comments

The authors should clarify the criteria used to include other H9N2 strains in the phylogenetic analysis, aside from the two isolates from the same farm in West Java

I agree with Reviewer 1 that the authors should refer to the 'recent strain' by its correct influenza strain name, adhering to the proper nomenclature.

Figure 1 could be moved to the supplementary material as it does not add critical information to the manuscript.

Additionally, the authors should clarify how the percentage of similarity between strains, as shown in Table 2, was calculated.

The discussion should address the potential role of other viral segments, beyond HA, in the adaptation of avian influenza to vaccination. The focus solely on HA sequencing represents a significant limitation of this work, which should be clearly acknowledged.

It would be valuable to discuss the role of low-frequency variants in adaptation to vaccination, not just the consensus sequence data. This aspect is missing due to the sequencing technique employed and should be highlighted as a limitation.

Another major limitation of the study is that it relies on samples from only one farm. To effectively analyze the adaptation potential of H9N2 strains to vaccination, a broader sampling strategy that includes multiple farms from the region would be necessary. If expanding the sample set is not feasible, this limitation should be explicitly discussed.

The discussion section is currently written in a somewhat confusing manner. Some content belongs in the results section, while other parts would be more appropriate in the introduction. The discussion should be rewritten to reduce its length and focus on the main findings of the manuscript:
- Both strains belong to the same clade.
- Specific amino acid mutations, particularly in antigenic site.
- The preference of the 2023 strain for Neu5Ac2-6Gal.

Is the work clearly and accurately presented and does it cite the current literature?

Partly
Is the study design appropriate and is the work technically sound?

Yes
Are sufficient details of methods and analysis provided to allow replication by others?

Yes
If applicable, is the statistical analysis and its interpretation appropriate?

Not applicable
Are all the source data underlying the results available to ensure full reproducibility?

Yes
Are the conclusions drawn adequately supported by the results?

Yes

Competing Interests: No competing interests were disclosed.

Reviewer Expertise: virology, avian influenza, genomics, animal health

I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.

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Reviewer Report 09 Oct 2024

Mark Zanin, The University of Hong Kong, Hong Kong SAR, China

Hui Lei, Centre for Immunology & Infection, Hong Kong, Hong Kong

Approved

https://doi.org/10.5256/f1000research.171635.r323384

The authors have addressed the ... Continue reading

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VERSION 1

PUBLISHED 04 Jun 2024

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Reviewer Report 21 Aug 2024

Mark Zanin, The University of Hong Kong, Hong Kong SAR, China

Hui Lei, Centre for Immunology & Infection, Hong Kong, Hong Kong

Approved with Reservations

https://doi.org/10.5256/f1000research.165593.r303175

The authors conducted this study to compare the molecular characteristics of avian influenza virus subtype H9N2 isolated from West Java in 2017 and 2023. They collected samples including brain, trachea, and oviduct pooled from deceased chickens and inoculated them into ... Continue reading

The authors conducted this study to compare the molecular characteristics of avian influenza virus subtype H9N2 isolated from West Java in 2017 and 2023. They collected samples including brain, trachea, and oviduct pooled from deceased chickens and inoculated them into specific pathogen-free embryonated chicken eggs for virus isolation. RNA extraction and RT-qPCR were used to confirm the presence of H9 and N2 genes in the samples. Sequences were obtained through RT-PCR and sanger sequencing from positive samples and the similarity of the recent isolates sequences were compared to reference isolates in Genbank through phylogenetic trees. They also compared the interactions of the recent isolates with the sialic acids Neu5Ac2-3Gal and Neu5Ac2-6Gal to that of the isolate A/Layer/Indonesia/WestJava-04/2017 (H9N2). They found that the recent virus exhibited 11 amino acid residue differences from A/Layer/Indonesia/WestJava-04/2017 (H9N2). Phylogenetically, the recent virus is also of the h9.4.2.5 subclade. The recent isolate had amino acid ‘N’ at position 183 at antigenic site II, which was different from A/Layer/Indonesia/WestJava-04/2017 (H9N2). Molecular docking analysis revealed a preference of HA1 from the 2017 virus for Neu5Ac2-3Gal, while the 2023 virus displayed a tendency to predominantly bind with Neu5Ac2-6Gal.
These findings help to gain insights into the evolution of avian influenza virus subtype H9N2 in the region, necessary for understanding the potential risks associated with them. Overall the manuscript is well writted and well presented.

MAJOR COMMENTS;

No figure legends are included.
The binding ability to sialic acids of the 2017 virus and 2023 virus were analyzed through molecular docking instead of direct experiment data. Conducting a receptor binding assay such as using analogues of different sialic acids will be more convincing. This should be noted as a limitation or a consideration in the discussion.
Please use correct influenza strain name nomenclature, i.e., A/Layer/Indonesia/WestJava-04/2017 (H9N2).
The authors should refer to the ‘recent isolate’ by its correct influenza strain name using correct nomenclature in the abstract and introduction after calling it the recent isolate as this is more informative.

MINOR COMMENTS;
Abstract
“Homology comparison and phylogenetic tree analysis, utilizing the neighbor-joining tree method, assessed the recent isolate’s similarity to reference isolates from GenBank.” Suggest revision to; “Homology comparison and phylogenetic tree analysis, utilizing the neighbor-joining tree method, were applied to assess the recent isolate’s similarity to reference isolates from GenBank.”

“The recent virus exhibited 11 amino acid residue differences compared to the A/Layer/Indonesia/WestJava-04/2017 isolate.”. please specify which gene is being referred to, or if this is the whole genome.
Introduction
There are 11 different subtypes of NA, please amend the second line of the introduction.
“facilitates the easy spread and mutation of AIVs”. Recommend removing ‘easy’ from this sentence.
Methods
“After five years of vaccination with a homologous vaccine at the farm, a decrease in production and bird mortality occurred in February 2023.” The authors may wish to consider publication of these potentially interesting data in another manuscript if they haven’t already. In this manuscript, information RE the vaccine used and the administration regimens should be included. This could be included in the introduction or results.
“Samples consisting of brain, trachea, and oviduct organs pooled from deceased chickens” More information would be helpful, perhaps in the results and/or as supplementary information.

How many chickens?
How many samples were tested and by which method?
How did the chickens die? Were they symptomatic of influenza or other illnesses?
Define the meaning of ‘pooled’ – were all organs from each chicken combined together in one sample or were different organs of the same type polled?
How large is the farm in terms of numbers of chickens?

“Tissue was combined with sterile phosphate-buffered saline from ThermoFisher (28348) pH 7.4 containing antibiotics (200 g/ml penicillin and 100 g/ml streptomycin) and then centrifuged at 1,000 g and 4 °C for fifteen minutes. The remaining supernatant was filtered through a 0.45 m membrane filter and injected intra-allantoically into 9-day-old SPF ECEs.” Recommend modification to read; “Tissue was combined with sterile phosphate-buffered saline (pH 7.4) from ThermoFisher (28348) containing antibiotics (200 g/ml penicillin and 100 g/ml streptomycin) and then centrifuged at 1,000 g and 4 °C for fifteen minutes. The remaining supernatant was filtered through a 0.45 μm membrane filter and injected intra-allantoically into 9-day-old SPF eggs.”
How many samples were used to inoculate eggs?
“The allantoic fluid that was collected was further extracted using a total RNA mini kit reagent from Geneaid®, Taiwan (RPD050).” Recommend modification to read; “RNA was further extracted from the allantoic fluid that was collected using a total RNA mini kit reagent from Geneaid®, Taiwan (RPD050).”
Results
“A positive result was determined when the Ct value was less than 40 and displayed an amplification curve resembling that of the positive control. Conversely, a negative result was determined if the Ct value was 45 or higher, and no amplification curve resembling the positive control was observed. The results with a Ct value between 40 and 45 were classified as indeterminate or dubious.” Perhaps this is too repetitive of the same information in the methods section?
“The amino acid sequence at the cleavage site of the recent isolate is PSRSSR↓GLF, while the receptor binding site has the motif PWTNTLY (Table 1). Additionally, at position 217 on the left side of the receptor binding site (RBS), the recent isolate contained the amino acid M (methionine). It should be noted that this amino acid sequence is identical to A/Layer/Indonesia/WestJava-04/2017, which originated from the same farm.” A bit more explanation of the significance of these would be useful.
Discussion
“In conclusion, this study investigated the molecular characteristics of avian influenza virus subtype H9N2 in Indonesia, focusing on a farm in West Java province from 2017 to 2023.” Is this correct? Was continued surveillance performed on this farm during this time period? If so, then these data should be included (such as numbers of samples collected, method of detection of influenza etc). Otherwise this sentence should be revised as it is a bit misleading.

Is the work clearly and accurately presented and does it cite the current literature?

Yes
Is the study design appropriate and is the work technically sound?

Yes
Are sufficient details of methods and analysis provided to allow replication by others?

Yes
If applicable, is the statistical analysis and its interpretation appropriate?

Not applicable
Are all the source data underlying the results available to ensure full reproducibility?

No source data required
Are the conclusions drawn adequately supported by the results?

Partly

Competing Interests: No competing interests were disclosed.

Reviewer Expertise: Influenza virus zoonosis and interspecies transmission

We confirm that we have read this submission and believe that we have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however we have significant reservations, as outlined above.

CITE

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17

Reviewer Report 14 Aug 2024

Amany Adel, Animal Health Research Institute, Agriculture Research Center (ARC), Giza, Egypt

Approved with Reservations

https://doi.org/10.5256/f1000research.165593.r303169

Thanks for giving me the chance to review this paper.
I think it is an important study that supposed to present updated situation for H9N2 viruses in Indonesia, and I believe that this kind of study is very important ... Continue reading

Thanks for giving me the chance to review this paper.
I think it is an important study that supposed to present updated situation for H9N2 viruses in Indonesia, and I believe that this kind of study is very important and expressive to give an updated vision for the viruses around the world.
Unfortunately, the study is not well developed and not well written, I think it needs to be more improved in writing and structuring.
If you please take these notes in your consideration:
1- The paper needs to be re-written well, the discussion is too long and needs to be more concise and targeted.
2- The number of the collected samples are not clarified in the paper, also the epidemiological data is very leaked and does not represent the situation in Indonesia.
3- I have a confusion in the classification of H9 in your study; your classification is not compatible with classification in the study of Silvia Carnaccini and Daniel R. Perez, 2020 (H9 Influenza Viruses: An Emerging Challenge), although you cited this paper in your study. please take a look in this study again and give explanation for this conflict.

Best wishes.

Is the work clearly and accurately presented and does it cite the current literature?

Partly
Is the study design appropriate and is the work technically sound?

Partly
Are sufficient details of methods and analysis provided to allow replication by others?

Partly
If applicable, is the statistical analysis and its interpretation appropriate?

Not applicable
Are all the source data underlying the results available to ensure full reproducibility?

Yes
Are the conclusions drawn adequately supported by the results?

Partly

Competing Interests: No competing interests were disclosed.

Reviewer Expertise: virology, avian influenza, molecular diagnosis

I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.

CITE

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Version 3

VERSION 3 PUBLISHED 04 Jun 2024

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Version 3 (revision) 05 Mar 25
Version 2 (revision) 13 Sep 24		read	read
Version 1 04 Jun 24	read	read

Amany Adel, Animal Health Research Institute, Agriculture Research Center (ARC), Giza, Egypt
Mark Zanin, The University of Hong Kong, Hong Kong SAR, China

Hui Lei, Centre for Immunology & Infection, Hong Kong, Hong Kong
Albert Perlas, Helmholtz Zentrum München, Helmholtz AI, Spain; Helmholtz Zentrum Muenchen, Neuherberg, Germany

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Reviewer Report

8 Views

27 Nov 2024 | for Version 2

Albert Perlas, Helmholtz AI, Helmholtz Zentrum München, Helmholtz AI, 85764, Spain; Helmholtz Pioneer Campus, Helmholtz Zentrum Muenchen, Neuherberg, 85764, Germany

8 Views Cite this report Responses(0)

Approved With Reservations

The authors compared the molecular characteristics of a subtype H9N2 avian influenza virus strain isolated in 2023 with another strain isolated in 2017 from the same farm in Indonesia. They sequenced the HA segment of the 2023 strain after RT-PCR amplification using Sanger sequencing. Based on these sequencing results, the authors performed phylogenetic analysis, molecular homology comparisons, and molecular docking analysis. The results revealed 11 amino acid residue differences between the two strains, confirmed that both strains belong to the H9.4.2.5 subclade, and suggested, through molecular docking, a preference of the 2023 strain for Neu5AC2-6Gal, which is more commonly found in mammalian species. Overall, the manuscript provides relevant information on the concerning H9N2 avian influenza subtype. However, the text is not consistently clear or well-organized, and improvements in these areas would enhance the quality of the work.

Comments

The authors should clarify the criteria used to include other H9N2 strains in the phylogenetic analysis, aside from the two isolates from the same farm in West Java

I agree with Reviewer 1 that the authors should refer to the 'recent strain' by its correct influenza strain name, adhering to the proper nomenclature.

Figure 1 could be moved to the supplementary material as it does not add critical information to the manuscript.

Additionally, the authors should clarify how the percentage of similarity between strains, as shown in Table 2, was calculated.

The discussion should address the potential role of other viral segments, beyond HA, in the adaptation of avian influenza to vaccination. The focus solely on HA sequencing represents a significant limitation of this work, which should be clearly acknowledged.

It would be valuable to discuss the role of low-frequency variants in adaptation to vaccination, not just the consensus sequence data. This aspect is missing due to the sequencing technique employed and should be highlighted as a limitation.

Another major limitation of the study is that it relies on samples from only one farm. To effectively analyze the adaptation potential of H9N2 strains to vaccination, a broader sampling strategy that includes multiple farms from the region would be necessary. If expanding the sample set is not feasible, this limitation should be explicitly discussed.

The discussion section is currently written in a somewhat confusing manner. Some content belongs in the results section, while other parts would be more appropriate in the introduction. The discussion should be rewritten to reduce its length and focus on the main findings of the manuscript:
- Both strains belong to the same clade.
- Specific amino acid mutations, particularly in antigenic site.
- The preference of the 2023 strain for Neu5Ac2-6Gal.

Is the work clearly and accurately presented and does it cite the current literature?

Partly
Is the study design appropriate and is the work technically sound?

Yes
Are sufficient details of methods and analysis provided to allow replication by others?

Yes
If applicable, is the statistical analysis and its interpretation appropriate?

Not applicable
Are all the source data underlying the results available to ensure full reproducibility?

Yes
Are the conclusions drawn adequately supported by the results?

Yes

Competing Interests

No competing interests were disclosed.

Reviewer Expertise

virology, avian influenza, genomics, animal health

I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.

Respond to this report

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Reviewer Report

5 Views

09 Oct 2024 | for Version 2

Mark Zanin, The University of Hong Kong, Hong Kong SAR, China

Hui Lei, Centre for Immunology & Infection, Hong Kong, Hong Kong

5 Views Cite this report Responses(0)

Approved

The authors have addressed the comments raised in the review.

Competing Interests

No competing interests were disclosed.

Reviewer Expertise

Influenza virus zoonosis and interspecies transmission

We confirm that we have read this submission and believe that we have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.

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Reviewer Report

10 Views

21 Aug 2024 | for Version 1

Mark Zanin, The University of Hong Kong, Hong Kong SAR, China

Hui Lei, Centre for Immunology & Infection, Hong Kong, Hong Kong

10 Views Cite this report Responses(0)

Approved With Reservations

The authors conducted this study to compare the molecular characteristics of avian influenza virus subtype H9N2 isolated from West Java in 2017 and 2023. They collected samples including brain, trachea, and oviduct pooled from deceased chickens and inoculated them into specific pathogen-free embryonated chicken eggs for virus isolation. RNA extraction and RT-qPCR were used to confirm the presence of H9 and N2 genes in the samples. Sequences were obtained through RT-PCR and sanger sequencing from positive samples and the similarity of the recent isolates sequences were compared to reference isolates in Genbank through phylogenetic trees. They also compared the interactions of the recent isolates with the sialic acids Neu5Ac2-3Gal and Neu5Ac2-6Gal to that of the isolate A/Layer/Indonesia/WestJava-04/2017 (H9N2). They found that the recent virus exhibited 11 amino acid residue differences from A/Layer/Indonesia/WestJava-04/2017 (H9N2). Phylogenetically, the recent virus is also of the h9.4.2.5 subclade. The recent isolate had amino acid ‘N’ at position 183 at antigenic site II, which was different from A/Layer/Indonesia/WestJava-04/2017 (H9N2). Molecular docking analysis revealed a preference of HA1 from the 2017 virus for Neu5Ac2-3Gal, while the 2023 virus displayed a tendency to predominantly bind with Neu5Ac2-6Gal.
These findings help to gain insights into the evolution of avian influenza virus subtype H9N2 in the region, necessary for understanding the potential risks associated with them. Overall the manuscript is well writted and well presented.

MAJOR COMMENTS;

No figure legends are included.
The binding ability to sialic acids of the 2017 virus and 2023 virus were analyzed through molecular docking instead of direct experiment data. Conducting a receptor binding assay such as using analogues of different sialic acids will be more convincing. This should be noted as a limitation or a consideration in the discussion.
Please use correct influenza strain name nomenclature, i.e., A/Layer/Indonesia/WestJava-04/2017 (H9N2).
The authors should refer to the ‘recent isolate’ by its correct influenza strain name using correct nomenclature in the abstract and introduction after calling it the recent isolate as this is more informative.

MINOR COMMENTS;
Abstract
“Homology comparison and phylogenetic tree analysis, utilizing the neighbor-joining tree method, assessed the recent isolate’s similarity to reference isolates from GenBank.” Suggest revision to; “Homology comparison and phylogenetic tree analysis, utilizing the neighbor-joining tree method, were applied to assess the recent isolate’s similarity to reference isolates from GenBank.”

“The recent virus exhibited 11 amino acid residue differences compared to the A/Layer/Indonesia/WestJava-04/2017 isolate.”. please specify which gene is being referred to, or if this is the whole genome.
Introduction
There are 11 different subtypes of NA, please amend the second line of the introduction.
“facilitates the easy spread and mutation of AIVs”. Recommend removing ‘easy’ from this sentence.
Methods
“After five years of vaccination with a homologous vaccine at the farm, a decrease in production and bird mortality occurred in February 2023.” The authors may wish to consider publication of these potentially interesting data in another manuscript if they haven’t already. In this manuscript, information RE the vaccine used and the administration regimens should be included. This could be included in the introduction or results.
“Samples consisting of brain, trachea, and oviduct organs pooled from deceased chickens” More information would be helpful, perhaps in the results and/or as supplementary information.

How many chickens?
How many samples were tested and by which method?
How did the chickens die? Were they symptomatic of influenza or other illnesses?
Define the meaning of ‘pooled’ – were all organs from each chicken combined together in one sample or were different organs of the same type polled?
How large is the farm in terms of numbers of chickens?

“Tissue was combined with sterile phosphate-buffered saline from ThermoFisher (28348) pH 7.4 containing antibiotics (200 g/ml penicillin and 100 g/ml streptomycin) and then centrifuged at 1,000 g and 4 °C for fifteen minutes. The remaining supernatant was filtered through a 0.45 m membrane filter and injected intra-allantoically into 9-day-old SPF ECEs.” Recommend modification to read; “Tissue was combined with sterile phosphate-buffered saline (pH 7.4) from ThermoFisher (28348) containing antibiotics (200 g/ml penicillin and 100 g/ml streptomycin) and then centrifuged at 1,000 g and 4 °C for fifteen minutes. The remaining supernatant was filtered through a 0.45 μm membrane filter and injected intra-allantoically into 9-day-old SPF eggs.”
How many samples were used to inoculate eggs?
“The allantoic fluid that was collected was further extracted using a total RNA mini kit reagent from Geneaid®, Taiwan (RPD050).” Recommend modification to read; “RNA was further extracted from the allantoic fluid that was collected using a total RNA mini kit reagent from Geneaid®, Taiwan (RPD050).”
Results
“A positive result was determined when the Ct value was less than 40 and displayed an amplification curve resembling that of the positive control. Conversely, a negative result was determined if the Ct value was 45 or higher, and no amplification curve resembling the positive control was observed. The results with a Ct value between 40 and 45 were classified as indeterminate or dubious.” Perhaps this is too repetitive of the same information in the methods section?
“The amino acid sequence at the cleavage site of the recent isolate is PSRSSR↓GLF, while the receptor binding site has the motif PWTNTLY (Table 1). Additionally, at position 217 on the left side of the receptor binding site (RBS), the recent isolate contained the amino acid M (methionine). It should be noted that this amino acid sequence is identical to A/Layer/Indonesia/WestJava-04/2017, which originated from the same farm.” A bit more explanation of the significance of these would be useful.
Discussion
“In conclusion, this study investigated the molecular characteristics of avian influenza virus subtype H9N2 in Indonesia, focusing on a farm in West Java province from 2017 to 2023.” Is this correct? Was continued surveillance performed on this farm during this time period? If so, then these data should be included (such as numbers of samples collected, method of detection of influenza etc). Otherwise this sentence should be revised as it is a bit misleading.

Is the work clearly and accurately presented and does it cite the current literature?

Yes
Is the study design appropriate and is the work technically sound?

Yes
Are sufficient details of methods and analysis provided to allow replication by others?

Yes
If applicable, is the statistical analysis and its interpretation appropriate?

Not applicable
Are all the source data underlying the results available to ensure full reproducibility?

No source data required
Are the conclusions drawn adequately supported by the results?

Partly

Competing Interests

No competing interests were disclosed.

Reviewer Expertise

Influenza virus zoonosis and interspecies transmission

We confirm that we have read this submission and believe that we have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however we have significant reservations, as outlined above.

Respond to this report

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Reviewer Report

17 Views

14 Aug 2024 | for Version 1

Amany Adel, Animal Health Research Institute, Agriculture Research Center (ARC), Giza, Egypt

17 Views Cite this report Responses(0)

Approved With Reservations

Thanks for giving me the chance to review this paper.
I think it is an important study that supposed to present updated situation for H9N2 viruses in Indonesia, and I believe that this kind of study is very important and expressive to give an updated vision for the viruses around the world.
Unfortunately, the study is not well developed and not well written, I think it needs to be more improved in writing and structuring.
If you please take these notes in your consideration:
1- The paper needs to be re-written well, the discussion is too long and needs to be more concise and targeted.
2- The number of the collected samples are not clarified in the paper, also the epidemiological data is very leaked and does not represent the situation in Indonesia.
3- I have a confusion in the classification of H9 in your study; your classification is not compatible with classification in the study of Silvia Carnaccini and Daniel R. Perez, 2020 (H9 Influenza Viruses: An Emerging Challenge), although you cited this paper in your study. please take a look in this study again and give explanation for this conflict.

Best wishes.

Is the work clearly and accurately presented and does it cite the current literature?

Partly
Is the study design appropriate and is the work technically sound?

Partly
Are sufficient details of methods and analysis provided to allow replication by others?

Partly
If applicable, is the statistical analysis and its interpretation appropriate?

Not applicable
Are all the source data underlying the results available to ensure full reproducibility?

Yes
Are the conclusions drawn adequately supported by the results?

Partly

Competing Interests

No competing interests were disclosed.

Reviewer Expertise

virology, avian influenza, molecular diagnosis

I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.

Respond to this report

Responses (0)

[1] 1. Balkhy H, Al-Hajjar S: Avian influenza: are our feathers ruffled? Ann. Saudi Med. 2006 May-Jun; 26(3): 175–82. eng. PubMed Abstract | Publisher Full Text | Free Full Text

[2] 2. Nugroho C, Pawitan J: Production of Antibody for Direct Fluorescence Antibody Assay against Avian Influenza H9N2. Hosts and Viruses. 2020; 7(28): 7. Publisher Full Text

[3] 3. Kanaujia R, Bora I, Ratho RK, et al.: Avian influenza revisited: concerns and constraints. Virusdisease. 2022 Dec; 33(4): 456–65. Epub 20221028. eng. PubMed Abstract | Publisher Full Text | Free Full Text

[4] 4. Shao W, Li X, Goraya MU, et al.: Evolution of Influenza A Virus by Mutation and Re-Assortment. Int. J. Mol. Sci. 2017 Aug 7; 18(8). Epub 20170807. eng. PubMed Abstract | Publisher Full Text | Free Full Text

[5] 5. Lee DH, Song CS: H9N2 avian influenza virus in Korea: evolution and vaccination. Clin Exp. Vaccine Res. 2013 Jan; 2(1): 26–33. Epub 20130115. eng. PubMed Abstract | Publisher Full Text | Free Full Text

[6] 6. Gao X, Wang N, Chen Y, et al.: Sequence characteristics and phylogenetic analysis of H9N2 subtype avian influenza A viruses detected from poultry and the environment in China, 2018. PeerJ. 2021; 9: e12512. Epub 20211220. eng. PubMed Abstract | Publisher Full Text | Free Full Text

[7] 7. Li C, Yu K, Tian G, et al.: Evolution of H9N2 influenza viruses from domestic poultry in Mainland China. Virology. 2005 2005/09/15/; 340(1): 70–83. Publisher Full Text

[8] 8. Bouvier NM, Palese P: The biology of influenza viruses. Vaccine. 2008 Sep 12; 26 Suppl 4(Suppl 4): D49–53. eng. PubMed Abstract | Publisher Full Text | Free Full Text

[9] 9. Sriwilaijaroen N, Suzuki Y: Molecular basis of the structure and function of H1 hemagglutinin of influenza virus. Proc. Jpn. Acad. Ser. B Phys. Biol. Sci. 2012; 88(6): 226–49. Epub 2012/06/26. eng. PubMed Abstract | Publisher Full Text | Free Full Text

[10] 10. Neumann G: H5N1 influenza virulence, pathogenicity and transmissibility: what do we know? Future Virol. 2015; 10(8): 971–80. eng. PubMed Abstract | Publisher Full Text | Free Full Text

[11] 11. Taubenberger JK: Influenza virus hemagglutinin cleavage into HA1, HA2: no laughing matter. Proc. Natl. Acad. Sci. USA. 1998 Aug 18; 95(17): 9713–5. eng. PubMed Abstract | Publisher Full Text | Free Full Text

[12] 12. Xu H, Qian J, Song Y, et al.: The adaptability of H9N2 avian influenza A virus to humans: A comparative docking simulation study. Biochem. Biophys. Res. Commun. 2020 2020/09/03/; 529(4): 963–9. PubMed Abstract | Publisher Full Text

[13] 13. Gu M, Xu L, Wang X, et al.: Current situation of H9N2 subtype avian influenza in China. Vet. Res. 2017 12/01; 48: 49. PubMed Abstract | Publisher Full Text | Free Full Text

[14] 14. Bonfante F, Mazzetto E, Zanardello C, et al.: A G1-lineage H9N2 virus with oviduct tropism causes chronic pathological changes in the infundibulum and a long-lasting drop in egg production. Vet. Res. 2018 Aug 29; 49(1): 83. Epub 20180829. eng. PubMed Abstract | Publisher Full Text | Free Full Text

[15] 15. Iqbal M, Yaqub T, Mukhtar N, et al.: Infectivity and transmissibility of H9N2 avian influenza virus in chickens and wild terrestrial birds. Vet. Res. 2013 Oct 17; 44(1): 100. Epub 20131017. eng. PubMed Abstract | Publisher Full Text | Free Full Text

[16] 16. Nugroho CMH, Silaen OSM, Kurnia RS, et al.: Isolation and molecular characterization of the hemagglutinin gene of H9N2 avian influenza viruses from poultry in Java, Indonesia. J. Adv. Vet. Anim. Res. 2021 Sep; 8(3): 423–34. Epub 2021/11/02. eng. PubMed Abstract | Publisher Full Text | Free Full Text

[17] 17. Kurnia RS, Tarigan S, Nugroho CMH, et al.: Potency of bacterial sialidase Clostridium perfringens as antiviral of Newcastle disease infections using embryonated chicken egg in ovo model. Vet. World. 2022 Aug; 15(8): 1896–905. Epub 20220806. eng. PubMed Abstract | Publisher Full Text | Free Full Text

[18] 18. Chaharaein B, Omar AR, Aini I, et al.: Detection of H5, H7 and H9 subtypes of avian influenza viruses by multiplex reverse transcription-polymerase chain reaction. Microbiol. Res. 2009; 164(2): 174–9. Epub 2007/03/06. eng. PubMed Abstract | Publisher Full Text

[19] 19. Abid M, Yaqub T, Mehboob A, et al.: Characterization and Phylogenetic Analysis of Avian Influenza Virus Subtype h9n2 in Pakistan.2017 08/24.

[20] 20. Nugroho CMH, Kurnia RS, Tarigan S, et al.: Screening and purification of NanB sialidase from Pasteurella multocida with activity in hydrolyzing sialic acid Neu5Acα(2-6) Gal and Neu5Acα(2-3)Gal. Sci. Rep. 2022 Jun 8; 12(1): 9425. Epub 2022/06/09. eng. PubMed Abstract | Publisher Full Text | Free Full Text

[21] 21. Maharani NR, Susetya H, Wibowo MH: Characterization of Hemagglutinin Gene Fragment of H9N2 Avian Influenza Virus Isolated from Environmental Live Bird Market in the Greater Jakarta Area. Trop. Anim. Sci. J. 2022 05/18; 45(2): 141–153. Publisher Full Text

[22] 22. Silaen OSM, Murtini S, Pamungkas J, et al.: Isolation and molecular characterization of fowl aviadenovirus associated with inclusion body hepatitis from poultry in Banten and West Java, Indonesia. Vet. World. 2020 Sep; 13(9): 1940–6. Epub 20200922. eng. PubMed Abstract | Publisher Full Text | Free Full Text

[23] 23. Syahputra G: Docking Simulation of Curcumin and Its Analogs as Inhibitors on 12-Lipoxygenase Enzyme.2014.

[24] 24. Richard M, de Graaf M , Herfst S: Avian influenza A viruses: from zoonosis to pandemic. Future Virol. 2014 May 1; 9(5): 513–24. PubMed Abstract | Free Full Text eng, Publisher Full Text |

[25] 25. Zhang Y, Xu C, Zhang H, et al.: Targeting Hemagglutinin: Approaches for Broad Protection against the Influenza A Virus. Viruses. 2019 Apr 30; 11(5). Epub 20190430. eng. PubMed Abstract | Publisher Full Text | Free Full Text

[26] 26. Shirvani E, Paldurai A, Varghese BP, et al.: Contributions of HA1 and HA2 Subunits of Highly Pathogenic Avian Influenza Virus in Induction of Neutralizing Antibodies and Protection in Chickens. Front. Microbiol. 2020; 11: 1085. Epub 20200605. eng. PubMed Abstract | Publisher Full Text | Free Full Text

[27] 27. Zhang J, Ma K, Li B, et al.: A risk marker of tribasic hemagglutinin cleavage site in influenza A (H9N2) virus. Commun Biol. 2021 Jan 15; 4(1): 71. Epub 20210115. eng. PubMed Abstract | Publisher Full Text | Free Full Text

[28] 28. Baron J, Tarnow C, Mayoli-Nüssle D, et al.: Matriptase, HAT, and TMPRSS2 activate the hemagglutinin of H9N2 influenza A viruses. J. Virol. 2013 Feb; 87(3): 1811–20. Epub 20121128. eng. PubMed Abstract | Publisher Full Text | Free Full Text

[29] 29. Bóna M, Kiss I, Dénes L, et al.: Tissue Tropism of H9N2 Low-Pathogenic Avian Influenza Virus in Broiler Chickens by Immunohistochemistry. Animals (Basel). 2023 Mar 14; 13(6). Epub 20230314. eng. PubMed Abstract | Publisher Full Text | Free Full Text

[30] 30. Teng Q, Xu D, Shen W, et al.: A Single Mutation at Position 190 in Hemagglutinin Enhances Binding Affinity for Human Type Sialic Acid Receptor and Replication of H9N2 Avian Influenza Virus in Mice. J. Virol. 2016 Nov 1; 90(21): 9806–25.Epub 20161014. eng. PubMed Abstract | Publisher Full Text | Free Full Text

[31] 31. Shen HQ, Yan ZQ, Zeng FG, et al.: Isolation and phylogenetic analysis of hemagglutinin gene of H9N2 influenza viruses from chickens in South China from 2012 to 2013. J. Vet. Sci. 2015; 16(3): 317–24. Epub 20150130. eng. PubMed Abstract | Publisher Full Text | Free Full Text

[32] 32. Sun H, Lin J, Liu Z, et al.: Genetic, Molecular, and Pathogenic Characterization of the H9N2 Avian Influenza Viruses Currently Circulating in South China. Viruses. 2019 Nov 8; 11(11). Epub 20191108. eng. PubMed Abstract | Publisher Full Text | Free Full Text

[33] 33. Sealy JE, Yaqub T, Peacock TP, et al.: Association of Increased Receptor-Binding Avidity of Influenza A(H9N2) Viruses with Escape from Antibody-Based Immunity and Enhanced Zoonotic Potential. Emerg. Infect. Dis. 2018 Jan; 25(1): 63–72. eng. PubMed Abstract | Publisher Full Text | Free Full Text

[34] 34. Belser JA, Sun X, Brock N, et al.: Genetically and Antigenically Divergent Influenza A(H9N2) Viruses Exhibit Differential Replication and Transmission Phenotypes in Mammalian Models. J. Virol. 2020 Aug 17; 94(17). Epub 20200817. eng. PubMed Abstract | Publisher Full Text | Free Full Text

[35] 35. Song Y, Zhang Y, Chen L, et al.: Genetic Characteristics and Pathogenicity Analysis in Chickens and Mice of Three H9N2 Avian Influenza Viruses. Viruses. 2019 Dec 6; 11(12). Epub 20191206. eng. PubMed Abstract | Publisher Full Text | Free Full Text

[36] 36. Rehman S, Rantam FA, Batool K, et al.: Emerging threats and vaccination strategies of H9N2 viruses in poultry in Indonesia: A review. F1000Res. 2022; 11: 548. Epub 20220519. eng. PubMed Abstract | Publisher Full Text | Free Full Text

[37] 37. Peacock T, Reddy K, James J, et al.: Antigenic mapping of an H9N2 avian influenza virus reveals two discrete antigenic sites and a novel mechanism of immune escape. Sci. Rep. 2016 Jan 7; 6: 18745. Epub 20160107. eng. PubMed Abstract | Publisher Full Text | Free Full Text

[38] 38. Kaverin NV, Rudneva IA, Ilyushina NA, et al.: Structural differences among hemagglutinins of influenza A virus subtypes are reflected in their antigenic architecture: analysis of H9 escape mutants. J. Virol. 2004 Jan; 78(1): 240–9. eng. PubMed Abstract | Publisher Full Text | Free Full Text

[39] 39. Deshpande KL, Fried VA, Ando M, et al.: Glycosylation affects cleavage of an H5N2 influenza virus hemagglutinin and regulates virulence. Proc. Natl. Acad. Sci. USA. 1987 Jan; 84(1): 36–40. eng. PubMed Abstract | Publisher Full Text | Free Full Text

[40] 40. Hu Z, Peng F, Xiong Z, et al.: Genetic and Molecular Characterization of H9N2 Avian Influenza Viruses Isolated from Live Poultry Markets in Hubei Province, Central China, 2013-2017. Virol. Sin. 2021 Apr; 36(2): 291–9. Epub 2020/09/15. eng. PubMed Abstract | Publisher Full Text | Free Full Text

[41] 41. Carnaccini S, Perez DR: H9 Influenza Viruses: An Emerging Challenge. Cold Spring Harb. Perspect. Med. 2020 Jun 1; 10(6). Epub 2019/12/25. eng. PubMed Abstract | Publisher Full Text | Free Full Text

[42] 42. El Mellouli F, Mouahid M, Fusaro A, et al.: Spatiotemporal Dynamics, Evolutionary History and Zoonotic Potential of Moroccan H9N2 Avian Influenza Viruses from 2016 to 2021. Viruses. 2022 Mar 1; 14(3). Epub 2022/03/27. eng. PubMed Abstract | Publisher Full Text | Free Full Text

[43] 43. Jonas M, Sahesti A, Murwijati T, et al.: Identification of avian influenza virus subtype H9N2 in chicken farms in Indonesia. Prev. Vet. Med. 2018 Nov 1; 159: 99–105. Epub 2018/10/14. eng. PubMed Abstract | Publisher Full Text

[44] 44. van der Kolk JH : Role for migratory domestic poultry and/or wild birds in the global spread of avian influenza? Vet. Q. 2019 Dec; 39(1): 161–7. eng. PubMed Abstract | Publisher Full Text | Free Full Text

[45] 45. Peacock TP, James J, Sealy JE, et al.: A Global Perspective on H9N2 Avian Influenza Virus. Viruses. 2019; 11(7). PubMed Abstract | Publisher Full Text | Free Full Text

[46] 46. Noor E, Haraldsdóttir HS, Milo R, et al.: Consistent estimation of Gibbs energy using component contributions. PLoS Comput. Biol. 2013; 9(7): e1003098. Epub 20130711. eng. PubMed Abstract | Publisher Full Text | Free Full Text

[47] 47. Golas SM, Nguyen AN, Rietman EA, et al.: Gibbs free energy of protein-protein interactions correlates with ATP production in cancer cells. J. Biol. Phys. 2019 Dec; 45(4): 423–30. Epub 20191216. eng. PubMed Abstract | Publisher Full Text | Free Full Text

[48] 48. McGuire SH, Rietman EA, Siegelmann H, et al.: Gibbs free energy as a measure of complexity correlates with time within C. elegans embryonic development. J. Biol. Phys. 2017 Dec; 43(4): 551–63. Epub 20170919. eng. PubMed Abstract | Publisher Full Text | Free Full Text

[49] 49. Bulusu G, Desiraju G: Strong and Weak Hydrogen Bonds in Protein–Ligand Recognition. J. Indian Inst. Sci. 2019 11/14; 100: 31–41. Publisher Full Text

[50] 50. Chen D, Oezguen N, Urvil P, et al.: Regulation of protein-ligand binding affinity by hydrogen bond pairing. Sci. Adv. 2016 Mar; 2(3): e1501240. Epub 20160325. eng. PubMed Abstract | Publisher Full Text | Free Full Text

[51] 51. Lins L, Brasseur R: The hydrophobic effect in protein folding. FASEB journal: official publication of the Federation of American Societies for. Exp. Biol. 1995 05/01; 9: 535–40. Publisher Full Text

[52] 52. Bogunia M, Makowski M: Influence of Ionic Strength on Hydrophobic Interactions in Water: Dependence on Solute Size and Shape. J. Phys. Chem. B. 2020 2020/11/19; 124(46): 10326–36. PubMed Abstract | Publisher Full Text | Free Full Text

Analyzing Molecular Traits of H9N2 Avian Influenza Virus Isolated from a Same Poultry Farm in West Java Province, Indonesia, in 2017 and 2023

Abstract

Background

Methods

Results

Conclusion

Keywords

Revised Amendments from Version 1

Introduction

Methods

Sample collection

Virus isolation

Viral RNA was identified using a reverse transcription-polymerase chain reaction (RT–PCR) assay

Sequencing

Molecular Docking of Protein HA1 (2017 and 2023 isolates) with Ligands Neu5Ac2-3Gal and Neu5Ac2-6Gal

Nucleotide sequence accession numbers

Results

H9N2 subtype AI virus detection

Figure 1. Curves of H9 (a) and N2 (b) RT–qPCR results. The sample revealed single band (c).

Amplification of the Hemagglutinin (HA) gene

Amino acid analysis

Table 1. Amino acid analysis of important sites in the HA gene of H9N2 viruses.

Homology comparison

Table 2. Nucleotide sequence similarities between the current strain and several H9N2 isolates in GenBank.

Phylogenetic tree

Figure 2. Phylogenetic tree of the hemagglutinin (HA) gene of the AIV H9N2 virus was constructed.

Docking analysis

Figure 3. Modeled protein HA1 of H9N2 predicted by the Robetta server, visualized in Discovery Studio Visualizer.

Table 3. Docking results of proteins HA12017 and HA12023 with ligands Sia3 and Sia6 selected.

Figure 4. Interactions occurring in HA12017 and HA12023 with ligand Sia3 and Sia6.

Table 4. Hydrogen bonds and interacting residues.

Discussion

Ethical statement

Data availability statement

Acknowledgment

References

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