Sequencing of the Malaysian H9N2 viruses
The genome sequences of the two Malaysian H9N2 viruses isolated from different types of chicken flocks in different years and states of Peninsular Malaysia were characterized in this study. The complete coding sequences of all 8 genes from the two H9N2 isolates UPM994/2018 and UPM2033/2019 have been deposited in NCBI under accession numbers OK331348 to OK331355 and OK331356 to OK331363, respectively. The sequencing results were used for phylogenetic analysis and molecular characterization in comparison to previously isolated and characterized H9N2 viruses.
Phylogenetic and pairwise sequence comparison analysis of H9N2 virus sequences
Phylogenetic analysis and pairwise sequence comparison analysis based on the individual genes of the studied H9N2 viruses were performed.
NS gene – Both the H9N2 viruses, UPM994/2018 and UPM2033/2019 NS genes, exhibited a 98.1% homology with each other. The results showed that both isolates and the majority of previously isolated H9N2 viruses contain amino acid ‘DRLRR’ at positions 34–38 of the RNA binding site. Malaysian H9N2 NS genes are most likely derived from A/chicken/Shanghai/F/1998 (SH/98) ancestral sequences with a high identity of 98%, with NS genes of H9N2 viruses isolated from Japan, Taiwan and Indonesia and G57 lineage reference H9N2 viruses [Supplementary Table 2].
M gene – The two characterized Malaysian H9N2 M genes are identical and have a 100% homology with each other and with H9N2 viruses from Indonesia. The results also show that the Malaysian H9N2 isolates are clustered closely, with the G57-like lineage sharing a 99.4% homology [Supplementary Table 3].
NA gene – The NA genes of the two Malaysian H9N2 isolates share 99.7% homology. Based on the NCBI blast results with the consensus of the NA gene of Malaysian H9N2 isolates in this study, the NA gene sequences have a close identity score of 98% with the H9N2 viruses from Taiwan and with non-avian host viruses A/canine/Guangxi/1/2011 (H9N2) and A/equine/Guangxi/3/2011 (H9N2) (Fig. 1; [Supplementary Table 4]).
NP Gene – The studied H9N2 NP gene shared a close identity with the H9N2 viruses from Vietnam, Indonesia and Cambodia with over 97% similarity [Supplementary Table 5].
HA Gene – This study covered 11 HA genes of Malaysian H9N2 isolates deposited in the NCBI databases besides the two H9N2 viruses characterized in this study. The BLAST results of the HA gene sequences revealed that both studied H9N2 isolates have a close relationship with H9N2 viruses (Melaka/10291 − 2018, Negeri Sembilan/7896 − 2018, Negeri Sembilan/7895 − 2018, Melaka/10298 − 2018, Penang/8232) previously isolated from different states in Peninsular Malaysia, with an identity above 95% followed by more than 97% identity of the Indonesian HA encoded gene sequences (Fig. 2; [Supplementary Table 6]).
PA gene –Both the Malaysian H9N2 PA genes displayed a 99.2% homology to each other (Fig. 3; [Supplementary Table 7]).
PB1 gene – The Malaysian H9N2 PB1 genes share a 97.6% homology. Phylogenetically, the PB1 gene of the Malaysian H9N2 isolates shares a cluster with Korean sequences, which have an identity of approximately 97% and a close genetic relationship with A/Chicken/Korea/38349-p96323/96 (H9N2) lineage that is a representative prototypic virus with 91% identity and 6% less genetic distance than other reference progeny viruses (Fig. 4; [Supplementary Tables 8 & 10]). On the other hand, the studied Malaysian H9N2 isolates independently clustered, at a high genetic distance, with the H9N2 isolates from Taiwanese, Indonesian, and Japanese viruses. PB1 genes of Malaysian isolates also have a close genetic similarity of 93% with H9N2 viruses – A/swine/Korea/S452/2004 (H9N2) isolated from pigs in Korea.
PB2 gene – The PB2 gene sequences of the two Malaysian H9N2 viruses share a 98.8% identity. Furthermore, the Malaysian PB2 gene exhibits more than 98% identity with Indonesian and Japanese H9N2 viruses. PB2 genes have the lowest genetic distance of all the genes, with G57 genotype viruses and their ancestor genes originating from a G1 lineage virus with 94% identity (Fig. 5; [Supplementary Tables 9 & 10]).
Genotype identification of Malaysian H9N2 isolates
Based on the genome sequence analysis, the two studied Malaysian H9N2 virus (UPM994/2018 and UPM2033/2019) backbones of PA, NS and NP genes originated from SF/98-like lineages, and the M and PB2 genes have a G1-like backbone. Furthermore, HA and NA originated from BJ/94 (Y280) and G9 lineages, respectively. Besides, all these seven internal gene cassettes are similar to G57 viruses except for the PB1 gene originating from the Korean lineage. Therefore, the studied Malaysian isolates UPM994/2018 and UPM2033/2019 are novel reassortants from G57 lineages. Moreover, the Malaysian H9N2 virus that was isolated from ducks in 2001 (Duck/2001), the virus NS, M, NP and PA genes originated from Korean lineages, PB1 and PB2 genes have an SF/98 origin, while the HA and the NA gene have a BJ/94- and G9-like origin, respectively [Supplementary Table 11]. The Malaysian UPM994/2018 and UPM2033/2019 H9N2 isolates also closely relate to lineages from neighboring countries, such as Indonesia, Taiwan, Japan and Cambodia.
Molecular analysis
The genome sequences of the Malaysian H9N2 isolates were evaluated based on the amino acid sequence variations associated with various biological properties of the virus as reported by previous studies.
NS gene – Both Malaysian H9N2 isolates have an NS gene with 217 amino acids with 13 deletions of amino acids and contain the conserved 214-LSTK-217 domain in the C terminal of the PDZ ligand. A similar PDZ ligand was noted in other AIVs (Table 2). However, the previously NCBI-deposited H9N2 (Duck/2001) from Malaysia consists of 230 full-length amino acids.
M gene – The M2 peptide of both Malaysian UPM isolates possesses a drug resistance marker at 31N, a mutation associated with resistance to the antiviral property of amantadine and rimantadine [24].
A gene – Deletion in the stalk region of the NA gene was not observed in UPM994/2018 and UPM2033/2019. The nucleotide sequences of the NA genes of both Malaysian H9N2 isolates were compared with previously reported drug resistance mutation markers. Both Malaysian H9N2 isolates have several amino acid substitutions, such as V116, E119, Q136, R152, D198, I222, S246, T247, H274, E276, E277, R292 and K432 in the receptor-binding pocket which is associated with zanamivir and oseltamivir antiviral drug susceptibility. NA genes of both Malaysian isolates have nine predicted glycosylation sites (GLS) at 44NST, 61NVT, 69NNT, 86NWS, 146NGT, 200NAT, 234NGT, 306NMT and 402NSS positions. However, the previously isolated Malaysian H9N2 from ducks (Duck/2001) possesses seven predicted GLS.
HA gene – In this study, UPM994/2018 and UPM2033/2019 H9N2 Malaysian isolates consist of dibasic HA1/HA2 that connect peptides at the 333-PSRSSR*GLF-341 position (Table 1). The cleavage sites were also compared with the H9N2 viruses recently deposited by researchers from VRI, Ipoh, Perak in Malaysia. The HA genes of the studied H9N2 isolates as well as Melaka/10291 − 2018, Perak/9745 − 2018, Penang/9541 − 2018 and Negeri Sembilan/7895/2018, which are H9N2 isolates from different states in Malaysia, showed the presence of a dibasic cleavage site motif RSSR. However, the previously characterized Malaysian H9N2, A/chicken/Perak/2061 − 2015/2015 and A/chicken/Penang/3174 − 2017/2017 showed 333- PARSKR*GLF-341 (Table 1) that is commonly detected in Korean-like H9N2, while the Duck/2021 isolates showed the presence of 333-PAISNR*GLF-341.
Table 1
Key amino acids residue analysis of important sites of HA molecules of Malaysian H9N2 isolates with reference H9N2 viruses
Virus
|
Cleavage sites
|
Left-edge of receptor-binding pocket
|
Receptor binding sites
|
Right-edge of receptor-binding pocket
|
333–341
|
146–150
|
109
|
161
|
163
|
191
|
198
|
202
|
203
|
232–237
|
A/chicken/UPM/994/2018 (H9N2)
|
PSRSSR-GLF
|
GTSKA
|
Y
|
W
|
T
|
N
|
V
|
L
|
Y
|
NGLMGR
|
A/chicken/UPM/2033/2019 (H9N2)
|
PSRSSR-GLF
|
GTSKA
|
Y
|
W
|
T
|
N
|
V
|
L
|
Y
|
NGLMGR
|
A/chicken/quail/Hong Kong/G1/97 (H9N2)
|
PSRSSR-GLF
|
GTSKA
|
Y
|
W
|
T
|
H
|
A
|
L
|
Y
|
NGLIGR
|
A/duck/Hong Kong/Y280/97 (H9N2)
|
PAKSSR-GLF
|
GTSKS
|
Y
|
W
|
T
|
H
|
A
|
L
|
Y
|
NGLIGR
|
A/chicken/Hong Kong/G9/97 (H9N2)
|
PSRSSR-GLF
|
GISRA
|
Y
|
W
|
T
|
H
|
E
|
L
|
Y
|
NDLQGR
|
A/chicken/Melaka/10291 − 2018/2018 (H9N2)
|
PSRSSR-GLF
|
GTSRA
|
Y
|
W
|
T
|
N
|
E
|
L
|
Y
|
NGLMGR
|
A/chicken/Perak/2061 − 2015/2015 (H9N2)
|
PARSKR-GLF
|
GTSKA
|
Y
|
W
|
T
|
H
|
E
|
L
|
Y
|
NGQQGR
|
A/chicken/Perak/9745 − 2018/2018 (H9N2)
|
PSRSSR-GLF
|
GTSKA
|
Y
|
W
|
T
|
N
|
E
|
L
|
Y
|
NGLMGR
|
A/chicken/Negeri Sembilan/7896/2018 (H9N2)
|
PSRSSR-GLF
|
GTSRA
|
Y
|
W
|
T
|
N
|
E
|
L
|
Y
|
NGLMGR
|
A/chicken/Negeri Sembilan/7895/2018 (H9N2)
|
PSRSSR-GLF
|
GTSKA
|
Y
|
W
|
T
|
N
|
A
|
L
|
Y
|
NGLMGR
|
A/chicken/Melaka/10298 − 2018/2018 (H9N2)
|
PSRSSR-GLF
|
GTSKA
|
Y
|
W
|
T
|
N
|
E
|
L
|
Y
|
NGLMGR
|
A/chicken/Penang/9541 − 2018/2018 (H9N2)
|
PSRSSR-GLF
|
GTSKA
|
Y
|
W
|
T
|
N
|
A
|
L
|
Y
|
NGLMGR
|
A/chicken/Penang/8232 − 2018/2018 (H9N2)
|
PSRSSR-GLF
|
GTSKA
|
Y
|
W
|
T
|
N
|
A
|
L
|
Y
|
NGLMGR
|
A/chicken/Penang/3174 − 2017/2017 (H9N2)
|
PARSKR-GLF
|
GTSKA
|
Y
|
W
|
T
|
H
|
E
|
L
|
Y
|
NGQQGR
|
A/duck/2001(H9N2)
|
PAISNR-GLF
|
GTSKA
|
Y
|
W
|
T
|
H
|
E
|
L
|
Y
|
NGQQGR
|
Malaysian H9N2 viruses showed conservation of residues at Y109, W161, T163, Y203 and G234 positions in the receptor-binding pocket of the HA gene. However, the Malaysian isolates have a unique point mutation at position V198. The left edge of the receptor-binding pocket of both Malaysian isolates has a 146-GTSKA-150 amino acid motif, and the right edge of the receptor-binding pocket consists of a 232-NGLMGR-237 motif (Table 1).
The present analysis of HA protein sequences shows that H9N2 UPM isolates have eight potential GLS at position 29 (NSTE), 82 (NPSC), 141 (NVSY), 298 (NTTL), 305 (NVSR), 313 (NCSR), 492 (NGTY) and 551 (NGSC).
In this study, we analyzed the potential of the antibody escapes mutant of the Malaysian H9N2 isolates based on the key amino acid residues of the HA molecules found in previous studies. The Malaysian H9N2 isolates possess amino acid mutations N166, N167, N201 and D207 located at the head of the HA molecules, which may associate with antibody escape mutant viruses. UPM994/2018 and UPM2033/2019 Malaysian H9N2 isolates possess well-known mutations at position L226 and other signature mutations at T155, V190, N183, M227 and W153 in the head region of the HA gene, suggesting an increased affinity for binding to mammalian host cells (Table 2).
Table 2
Summary of whole genome mutation analysis markers of Malaysian H9N2 viruses
Gene
|
Phenotypic character
|
UPM994/2018 and UPM2033/2019 H9N2
|
Duck/2001 H9N2
|
PB2
|
Mammalian adaptation
|
V292, D309, G477, V495, V588, A661, I616, K702
|
D309, G477,V495, V588, A661, I616, K702
|
Enhanced virulence markers
|
V504, V588
|
V504, V588
|
PB1
|
Increased polymerase activity in mice
|
G662
|
G662
|
Enhanced virulence markers
|
G22
|
G22
|
Increased polymerase activity in H5N1
|
H436Y, R207K, M677T
|
H436Y, R207K
|
Mammalian adaptation
|
T39, K207, R386, A448, L472, M523
|
T39, K207, R386, A448, L472, M523
|
Full-length second peptide (PB1-F2)
|
90 AA
|
90 AA
|
PA
|
PA-X length
|
252 AA
|
252 AA
|
Mammalian adaptation
|
D160, S277, Q278, S291, N409, T515, L549
|
D160, S277, Q278, S291, N409, T515, L549
|
Enhanced virulence markers
|
V127, L550, R356
|
V127, L550, R356
|
NP
|
Mammalian adaptation
|
N52, Q231, D275, R422, T433, D255, D480
|
Q231, D275, R422, T433, D255, D480
|
HA
|
Mammalian adaptation
|
L226, T155, V190, N183, M227, W153
|
T155, W153
|
Airborne transmission
|
H183, A189, E190, L226
|
E190
|
Escape mutants
|
N167, D196, T147, N201, D207
|
N167, D196, T147, D207
|
Cleavage site motif
|
PSRSSR-GLF
|
PAISNR-GLF
|
Glycosylation site
|
8 (NPSC, NVSY, NSTE, NTTL, NVSR, NCSK, NGTY, NGSC)
|
7 (NPSC, NVTV, NRTF, NTTL, NVSK, NGTY, NGSC)
|
Mutation associated with addition or deletion of new glycosylation site
|
S218, S315
|
Not detected
|
NA
|
Mammalian adaptation
|
N356
|
Not detected
|
Airborne transmission
|
S370, K313, G381
|
S370, K313
|
Antivirus drug-resistant markers
|
Not detected
|
Not detected
|
Deletion of stalk length
|
Not detected
|
Not detected
|
M
|
Antivirus drug resistance (amantadine & rimantadine)
|
N31
|
Not detected
|
Mammalian adaptation
|
V28, I55
|
Not detected
|
NS
|
NS1 length
|
217 AA
|
230 AA
|
AA deletion at 80–84 positions
|
Not detected
|
Not detected
|
Enhanced virulence markers
|
G184
|
G184
|
C Terminus PDZ
|
LSTK
|
ESEV
|
PA gene – The PA genes of the Malaysian H9N2 isolates contain amino acid substitutions, which are associated with mammalian receptor binding affinity at position D160, S277, Q278, S291, N409, T515 and L549 as well as pathogenicity-enhanced markers at position V127, L550 and R356. The PA genes of both Malaysian isolates also harbour airborne transmission markers at L672. The Malaysian H9N2 isolates of this study also encode for full-length 252 amino acids in the coding region of PA-X gene. Furthermore, UPM2033/2019 isolate consists of L108, C305, P357 and G364 amino acid polymorphism in the PA gene. However, other closely similar viruses, including UPM994/2018, have I108, Y305, T357 and S364 (Table 2).
PB1 gene – The PB1 genes of both Malaysian isolates, virulence-enhanced markers, mammalian adaptation markers and mutations were analyzed. Both Malaysian isolates have 667V, whereas other similar viruses have 667I. Both studied viruses show two amino acid substitution markers at positions P13 and G662 associated with enhanced polymerase activity as reported by previous studies. In addition, the PB1 genes of both Malaysian H9N2 isolates have virulence-enhanced markers related to amino acid mutations at R207K, H436Y and M677T (Table 2).
Both Malaysian H9N2 isolates have multiple mammalian adaptation mutation positions at 39T, 207K, 386R, 448A, 472L and 523M. In addition, the amino acid variations at 68I, 69Q and 70G positions are associated with enhanced virulence markers. These two viruses also have a full-length (90 aa) PB1-F2 encoded secondary peptide.
PB2 gene – Both Malaysian H9N2 isolates display mammalian adaptation markers at V292, D309, G477, V495, V588, A661, I616, and K702 positions and increased virulence mutation at V504 and V588 positions of PB2 (Table 2).