Whole-genome sequencing analysis of the Chikungunya virus during 2021 outbreak in Malaysia

doi:10.21203/rs.3.rs-4877663/v1

Background

Chikungunya cases was reported to be on the rise in Malaysia from 2019 to 2021. Although potential endemicity was described previously, genotype shift during 2008 outbreak originating from the 2004 Indian Ocean Islands outbreak presents the probability of current CHIKV spread from neighboring countries. This is due to the prevalence of the new IOL sub-lineage which consists of E1-226A wildtype or reverted strains that are circulating in the Indian subcontinent before spreading to neighboring Thailand during 2018-2019 outbreak.

Method

In this study, samples received mostly from the Tangkak, Johor were analyzed. A total 56 CHIKV positive serum samples received in 2021 by Institute of Medical Research Malaysia (IMR), were collected based on sample selection criteria. Selected samples were subjected to total RNA extraction, whole-genome sequencing as well as bioinformatic analysis such as phylogenetic, variant and mutation analysis.

Results

Based on the genomic and phylogenetic analysis, the CHIKV samples from 2021 outbreak were of ECSA-IOL genotype. Genome similarity analysis also revealed that these CHIKVs are highly similar to 2018-2019 outbreak strain from Thailand. In comparison to the 2008 outbreak CHIKV isolate, the current CHIKVs lacked the E1-A226V mutation and harbored the new E1-K211E/E2-V264A sub-linage mutation. Since the E1-K211E/E2-V264A mutation facilitates adaptation to Ae. aegypti as opposed to the E1-A226V mutation which improves adaptation to Ae. albopictus, the emergence 2021 CHIKV outbreak in Malaysia can be postulated due to vector shift. Interestingly, a novel nsP3-T441A/V mutation detected in this study, may also play a role in virus transmission, pathogenicity, fitness and vector adaptation

Conclusion

In summary, the current CHIKV outbreak are strains originated from the Indian subcontinent through Thailand which may have capitalized on vector shifting by adapting to Ae. aegypti. The presence of novel nsP3-T441A/V mutation may also contribute to the spread of this virus across peninsular Malaysia.

CHIKV

Chikungunya

Malaysia

Whole-genome

Sequencing

Chikungunya is a viral-mediated disease with clinical manifestations such as mild febrile illness and arthralgia from acute infection to a more severe polyarthralgia, polyarthritis as well as rheumatism in chronic cases [1]. The Chikungunya virus (CHIKV) is an arbovirus (arthropod-borne virus) transmitted by Aedes species mosquito vector through both sylvatic (Aedes taylori, Aedes furcifer, Aedes fulgen) and urban (Aedes aegypti and Aedes Albopictus) infection cycle [2]. The sylvatic cycle involved the enzootic maintenance of CHIKV among non-human primates such as African green monkeys, baboons and guenons that are reported as potential reservoirs for virus amplification [2]. The urban cycle involves virus transmission in human by Ae. aegypti or Ae. Albopictus infected with CHIKV that replicates and spreads to its salivary glands before being transmitted into human [3]. The virus incubates in human from one to twelve days and consequently result in viremia persisting for up to ten days [4-6]. Viremia occurs due to CHIKV replication in the dermal fibroblast before migrating to lymph node and circulating in the blood by infecting macrophages. The virus migrates to joint, muscles and bones resulting in viral persistence which leads to continuous inflammation and chronic CHIKV-induced disease. Apart from the common clinical manifestation such as arthritis, arthralgia, fever and myalagia, Chikungunya fever (CHIKF) could also result in neurological, cardiovascular, renal complications as well as death [7].

The CHIKV is an Alphavirus (Togaviridae family) which harbors an 11.8 kb single-stranded, non-segmented, positive (sense) -strand genomic RNA. The genomic RNA is 5′-end 7-methylguanosine capped and polyadenylated at the 3′-end, similar to messenger RNA (mRNA) which enables direct cytoplasmic translation upon viral entry [8]. It consists of two open reading frames (ORF) encoding four non-structural proteins (nsP1-4) and five structural proteins (C, E3, E2, 6K and E1) respectively [9]. The 3’ end ORF also encodes truncated polyprotein comprised of C, E3, E2, C-terminal 6K fused with a TF peptide [10, 11]. The capsid (C) structural proteins are arranged as an icosahedron enclosing the viral genome. The resulting nucleocapsid is enveloped by host cell plasma membrane containing cholesterol, sphingolipid as well as viral E1 and E2 glycoproteins. Both viral glycoproteins forms heterodimers that are arranged as trimeric spikes that mediates CHIKV cell infection by receptor binding (E2) and viral-host membrane fusion (E1) [9, 12]. The 6K is a small hydrophobic protein that attaches E1-E2 polyprotein in order to facilitate envelope processing, membrane permeabilization, virus assembly and budding. Frameshift mutation of sub-genomic RNA also results in truncated TF protein production which is incorporated into viral particles, indicating its potential involvement in viral assembly [10, 11]. The four non-structural proteins, nsPs, form replication complex (RC) which initiates viral genomic RNA replication and transcription. The nsP1 functions as the initiator of negative strand RNA synthesis and capping whilst nsP2 functions as RNA helicase, polyprotein protease and recognizes the sub-genomic RNA promoter. The nsP3 is an RNA helicase and also an accessory protein involve in interacting with host cell proteins required for optimal viral replication. The nsP4 is the RNA-dependent RNA polymerase (RdRP) and acts as the catalytic subunit of CHIKV replicase [13].

CHIKV is classified into three main genotypes which are East/Central/South African (ECSA), West African (WA) and Asian strains (A) [14, 15]. Initially, CHIKV primarily circulates in Africa (South Africa, Democratic Republic of Congo, Uganda) and Asia (Indonesia, Thailand and India) with sporadic outbreaks being recorded in neighboring countries such as Kenya, Malaysia and Singapore [1]. However, the 2004 Indian Ocean Islands (IOL) outbreak expanded the CHIKV spread to Europe as well as South America [13, 16-18]. Besides, significant urban outbreaks in Indian subcontinent and South-east Asian countries were also reported including Malaysia which resulted in genotype shift from Asian to ECSA genotype [18, 19]. Currently, the ECSA-IOL sub lineage is the prevailing genotype circulating worldwide and re-emerging in South-East Asian countries such as Malaysia from the Indian subcontinent through neighboring country, Thailand [20]. Since 2019, increased Chikungunya cases have been reported as disease burden in Malaysia, where the last recorded major outbreak was in the year 2008 (Figure 1) [21-23]. This includes south Thailand-bordering states, Kedah and Kelantan, spreading to urban northern states, Pulau Pinang and Perak where the highest cases were recorded in the year 2020. By 2021, urban central state Selangor, the Federal territories as well as urban southern state, Johor showed increased cases which indicates the spread of the Chikungunya virus (CHIKV) across the Peninsular Malaysia (Table 1) [21-23]. In this study, we analyzed CHIKV samples mainly from Tangkak, Johor, where the last major outbreak was in 2008, to explore the potential for emergence and transmission by conducting whole genome sequencing and characterization.

Table 1 Chikungunya cases recorded in Malaysian states from 2019 to 2021 based Malaysian Ministry of Health Press Statement on Dengue, Zika and Chikungunya fever situation from 2019 to 2021 [21-23]. The report showed, a part from Thailand-bordering states, Kedah and Kelantan, most Chikungunya cases were recorded in urban states as indicated by asterisks (*).

State/Year	2019	2020	2021
Perlis	5	1	3
Kedah	60	91	140
Pulau Pinang*	5	1175	77
Perak*	635	1064	180
Selangor*	184	17	339
Kuala Lumpur*	1	6	490
Negeri Sembilan*	8	0	22
Melaka*	11	50	29
Johor*	0	33	97
Pahang	1	2	0
Terengganu	0	0	0
Kelantan	74	161	55
Sarawak	4	0	1
Sabah	1	1	0
Labuan	1	0	0
Total	990	2601	1433

Sample collection

In 2021, Chikungunya suspected cases primarily from Johor were sent to the Institute for Medical Research, IMR for qPCR diagnostics. In this study, clinical sample selection criteria for sequencing involves low Ct of less than 25 with sufficient volume (~500 µl). Based on these criteria, a total of 56 samples (IMR01-21 to IMR56-21) were selected for this sequencing study which includes 53 samples from Tangkak, Johor, one samples from Segamat, Johor and 2 samples from Serdang, Selangor. These archived samples have been approved by the Medical Research Ethics Committee (MREC) (Reference: 22-01162-PDE (2)) to be used for the current research objective.

Total RNA extraction and purification

The total RNA extraction of selected CHIKV positive serum samples were conducted using TRIzol™ LS Reagent (Invitrogen, USA) according to manufacturer’s protocol. A volume of 250 µl of serum was mixed with 750 µl of TRIzol™ LS Reagent and homogenized through resuspension. The mixture was incubated for 5 minutes at room temperature before adding 200 µl of chloroform and followed by further 2-3 minutes of room temperature incubation. The lysis suspension was centrifuged at 12 000 x g at 4^oC for 15 minutes. The upper colourless aqueous phase (~700 µl) was transferred into new tube and added with equal volume of 70% ethanol followed by mixing with vortex. The suspension was then be subjected to RNA purification using the PureLink™ RNA Mini Kit (Invitrogen, USA) according to manufacturer’s protocol. A volume of ~700 µl of suspension was be transferred to a spin cartridge with attached collection tube. Once centrifuged and washed with Washing Buffer I, on column DNase treatment was performed using PureLink® DNase (Invitrogen, USA). DNase treated column was washed with Wash Buffer II and eluted by adding 30 µl of RNase-Free Water. The total RNA yield was quantified using Qubit™ RNA HS Assay Kit (Invitrogen, USA) according to manufacturer’s protocol

cDNA Conversion

The total RNA of individual samples were converted into double-stranded, (ds) cDNA using Maxima H Minus Double-Stranded cDNA Synthesis Kit (Thermo Scientific, USA) in reference to manufacturer’s protocol. Once first and second strand synthesis completed, the final reaction mixture was subjected to residual RNA removal. The resulting ds cDNA was purified using GeneJET PCR Purification Kit (Thermo Scientific, USA) according to manufacturer’s protocol. The yield of purified ds cDNA was analysed by Qubit™ dsDNA HS Assay Kit (Invitrogen, USA) and through qPCR using in-house primer/probe.

Library Preparation

The ds cDNAs subjected to library preparation by using Illumina DNA Prep (Illumina, USA) kit. A volume of 30 µl (100-500 ng) of cDNA fragments was used for the library preparation by first, tagging the cDNA fragments with adapter sequencing through Bead-Linked Transposomes (BLT). The tagmented cDNAs fragments were added with unique indexes from Illumina DNA/RNA UD Indexes Sets C kit (Illumina, USA) for each individual samples through limited-cycle PCR. Subsequently library clean-up was performed on the short-read libraries according to manufacturer’s protocol before conducting denaturation and dilution in order to obtain normalized concentration of (20 pM) of single stranded DNA library. The prepared libraries were sequenced using the Illumina MiSeq sequencing platform.

Bioinformatic Analysis

Raw reads obtained from MiSeq sequencer were assembled by Geneious prime Assembler (Geneious prime version 2023.1.1) by using Thailand CHIKV isolate full genome sequence (Accession number: MN 974211 .1) as reference sequence, at 100 times depth coverage. The resulting consensus full genome sequence were characterized for untranslated regions, ORFs and viral genes based on the aforementioned reference sequence. The Geneious prime software was also utilized for SNPs and variant calling for each individual samples as well as similarity analysis among the sequenced samples. Additionally, mutation comparison between the current CHIKV consensus sequence with the 2008 outbreak CHIKV isolate sequence (Accession number: KT324226.1) was performed by using GISAID/EpiArbo/ChikSurver database. Six selected complete genomic sequences obtained in this study were deposited into NCBI database.

Phylogenetic analysis was conducted by constructing maximum likelihood tree (ML tree) using 53 reference sequences from all genotypes. All complete coding sequences (CDS) were aligned by MEGA 11 software before proceeding to construct ML tree for genotyping of the sequenced genomes. Bayesian phylogenetic was also performed, in order to explore the evolutionary expansion of the ECSA-IOL lineage to the current investigated genomic sequences. The time-scaled, maximum clade credibility (MCC) tree was constructed as previously described [20], by using 147 ECSA-IOL reference sequences along with sequences obtained in this study. All CDS were aligned using Geneious prime software before being subjected to Markov Chain Monte Carlo (MCMC) sampling by BEAST software to estimate posterior distribution parameters such as divergence time, substitution rates and tree topology. The input file parameters include best-fit substitution model (GTR + F + I +G4) with relaxed uncorrelated longnormal clock to construct Bayesian Skygrid coalescent tree. The MCMC sampling was performed at 60,000,000 generations each with sampling for every 6000 generations at three different occasions. The output file was analysed by Tracer v1.7.1, to determine the convergence and effective samples size (ESS), which attributes to the MCMC chain reaching stable distribution. The resulting tree topology was annotated by TreeAnnotater and visualized by FigTree software.

Genome annotation and characterization

Consensus sequence for all 56 samples were successfully obtained through assembly by Geneious Assembler (Geneious Prime Version 2023.1.1) by using Thailand’s CHIKV isolate full genome sequence (Accession number: MN 974211 .1) as reference sequence. Total consensus sequence sizes for each sample ranged from 11 810 bps to 11 526 bps with varied coverage of flanking 5’-end and 3’-end untranslated regions. The full-length coding sequences (CDS) of 11 238 nucleotides, for all samples were successfully sequenced which includes two open-reading-frame (ORFs) and an intergenic region. The first ORF of 7 425 nucleotides, encodes non-structural proteins (nsP1, nsP2, nsP3 and nsP4) and the second ORF of 3 747 nucleotides encodes structural proteins (C, E3, E2, 6K and E1). The intergenic region of 66 nucleotides, consists of a regulatory sub-genomic promoter for structural protein ORF transcription. Six selected genomic sequences were submitted to NCBI GenBank database (Accession number: PP236105.1- PP236110.1). Genomic features were annotated by using Geneious Prime (version 2023.1.1) and Clone Manager 9 and an example of annotated genome (IMR24-21) is shown in Figure 2.

Phylogenetic and Mutation analysis

Maximum likelihood (ML) tree constructed with 156 reference sequences revealed the investigated virus genotype is ECSA-IOL sublinegae (Figure 3). Additionally, Maximum Clade Credibility (MCC) tree was also constructed, in order to analyse the evolution of the expanding IOL sublinage using 147 references sequence (Figure 4).

Mutation analysis in reference to strain S27 (African prototype), showed the lack of IOL sub-lineage initiating, E1-A226V mutation, as well as secondary adaptive mutation, E2-K252Q, compared to the isolate from previous major outbreak in Tangkak, Johor in 2008 (Accession number: KT324226.1) (Table 2). The presence of E1-K211E, E2-V264A and E1-I317V mutation, define these investigated viruses as part of the new E1-K211E/E2-V264A IOL sub-lineage CHIKVs (Figure 4) which comprise of wildtype or reverted E1-226A strains [20]. This indicates the investigated viruses originated from the dominant wildtype or reverted IOL strain detected in India from 2007-2010 [24-27] which expanded to northern India subcontinent during 2016-2017 outbreaks [28-30] before spreading to Thailand from Bangladesh in 2017 [31]. Besides, these characterized viruses also consist of nsp2-E145D and nsp4-S55N mutations which are defined as Indian subcontinent/Southeast Asia clade (IS clade). In addition, the presence of geographically distributed nsP2-N495S and C-K73R mutations among the investigated CHIKVs, classifies these viruses into Southeast Asia subclade of the IS clade [20]. Similar to Thailand 2018-2019 outbreak viruses, both nsp3-D372E and E2- G205S were also detected in the current investigated viruses which indicates the 2021 CHIKV E1-226A strain detected in Peninsular Malaysia is from neighboring Thailand [20].

The current circulation of E1-226A strain have been attributed to the presence of E1-K211E:E2-V264A epistatic mutation which are reported to result in higher fitness in Ae. aegypti through increased viral infectivity, dissemination and transmission [32]. Thus, in contrast to the E1-A226V strains detected in 2008 outbreak which consists of Ae. Albopictus adaptive mutations (E2-K252Q), the 2021 CHIKV E1-226A strain may have adapted to Ae. aegypti [33, 34]. This indicates a potential vector shift which would have been integral in the recent re-emergence of CHIKV in Malaysia where urban areas were mostly affected due to the high population of Ae. aegypti [35, 36]. Therefore, it is crucial to investigate Ae. aegypti and Ae. Albopictus in Malaysia CHIKV hotspots, in order to determine the possibility of CHIKV vector shift which leads to increasing local transmission.

Table 2 Amino acids differences comparison between outbreak strain and isolate from 2008-2009 outbreak (KT324226.1) by using GISAID, EpiArboTM, ChikSurver with strain S27 (African prototype) as reference. Different mutations detected in the current outbreak strains compared to KT324226.1, are bolded and underlined, while, KT324226.1 mutations that are absent in current outbreak strains are bolded with asterisks.

Protein	Regions	Malaysia 2021 outbreak strain	KT324226.1 Chikungunya virus isolate MY/09/5668
Non-structural	nsp1	T128K, L172V, E234K, T376M, M383L, I384L, T481I, Q488R, L507R	T128K, L172V, E234K, T376M, M383L, I384L, T481I, Q488R, L507R
	nsp2	S54N, H130Y, E145D, H374Y, N495S, C642Y, S643N	S54N, H374Y, *L539S, C642Y, S643N, *A793V
	nsp3	V175I, Y217H, P326S, V331A, T337I, K352E, D372E, I376T, A382T, T441A/V, L461P, S462N, P471S, R524stop	V175I, Y217H, P326S, V331A, T337I, *L340P, K352E, I376T, A382T, L461P, S462N, P471S, R524stop
	nsp4	S55N, T75A, R85G, T254A, Q500L, I514T, V555I, V604I	T75A, R82S, T254A, Q500L, I514T, V555I, V604I
Structural	C	P23S, V27I, K63R, K73R	P23S, V27I, K63R
	E3	I23T	I23T
	E2	G57K, I74M, G79E, N160T, A164T, L181M, S194G, G205S, I211T, V264A, M267R, S299N, T312M, A344T, S375T, V386A	G57K, I74M, G79E, N160T, A164T, L181M, S194G, I211T, *N218S, *K252Q, M267R, S299N, T312M, A344T, S375T, V386A
	6k	V8I, I54V	V8I, I54V
	E1	K211E, M269V, D284E, I317V, V322A	*A226V, M269V, D284E, V322A

A new mutation was detected in the outbreak virus which is threonine-to-valine mutation at position 441 within the nsp3 protein (nsp3-T441V) that may help define the current Malaysian IS subclade with estimated divergence time between 2018.17 to 2019.61 (Figure 4). Interestingly, 54 samples received from southern state, Johor (53 from Tangkak; 1 from Segamat) consist of nsp3-T441V mutation while 2 samples received from central state, Selangor harbor nsp3-T441A and nsp3-T441A/V mutation respectively. Alignment of the CHIKV nsp3 431-449 amino acid region among 49 reference sequences from all genotypes revealed the nsp3-441T is highly conserved among 48 sequences while a recently sequenced CHIKV isolate from a 2020 case study in Malaysia consist of nsp3-T441A mutation similar to that detected in current sample received from Selangor (Table 3)[37]. The CHIKV nsp3 is a multidomain protein which consists of N-terminal macro domain (1-161 a.a), Alphavirus-Unique domain (AUD) (162-320 a.a) and the C-terminal hypervariable domain (HVD) (321-550 a.a) [38]. Both macro domain and AUD plays a crucial role in virus genome replication and transcription. The C-terminal HVD involves in binding to various host proteins which contributes to viral pathogenesis [39, 40]. The nsp3-T441A/V mutation detected in the hypervariable domain region, therefore, may affect the nsp3 interaction with cellular factors that could translate into CHIKV immunomodulation and its’ pathogenicity [38]. Since alphavirus nsp3 protein is a phosphoprotein, the nsp3-T441V/A could potentially replace a threonine phosphorylation site. Whilst the exact phosphorylation site for CHIKV nsp3 protein is unknown, it was reported that unlike other alphaviruses, the lack of phosphorylation sites significantly diminished virus replication which resulted in attenuated CHIKV mutants [41]. The nsp3-T441A/V mutation is also within the region of HVD (411-519 a.a) which serves as binding site for Four and a half LIM domains protein 1 (FHL1), a host protein that promotes CHIKV infection. Replacement of all phosphorylation sites in this region to alanine were shown to significantly reduce the binding of FHL1 which also could potentially diminish virus infection and pathogenesis [42]. Although the nsp3-T441A/V may possibly contribute to an avirulent CHIKV strain, further study is needed, since the 2020 Malaysian CHIKV (Accession number: MW557661) which harbors nsp3-T441A was isolated from an infant suffering from severe Chikungunya disease with encephalopathy and pneumonia [37]. Apart from that, the nsp3-T441A/V mutation may also result in novel nsp3 interaction with cellular protein which may help in elucidating nsp3 function and its potential immunomodulatory properties in human and mosquito.

Table 3 Alignment of CHIKV nsp3 amino acid (431-449) sequences

Virus-Accession no- country - year	nsP3 sequence
	431	441	449
CHIKV- PP236105-Malaysia-2021	RAELCPVVQEVAETRDTA
CHIKV- PP236106-Malaysia-2021	RAELCPVVQEVAETRDTA
CHIKV- PP236107-Malaysia-2021	RAELCPVVQEVAETRDTA
CHIKV- PP236110-Malaysia-2021	RAELCPVVQEVAETRDTA
CHIKV- PP236108-Malaysia-2021	RAELCPVVQEA/VAETRDTA
CHIKV- PP236109-Malaysia-2021	RAELCPVVQEAAETRDTA
CHIKV-MW557661-Malaysia-2020	RAELCPVVQEAAETRDTA
CHIKV-KM923920-Malaysia-2015	RAELCPVVQETAETRDTA
CHIKV-KT324226-Malaysia-2009	RAELCPVVQETAETRDTA
CHIKV-KX262997-Malaysia-2009	RAELCPVVQETAETRDTA
CHIKV-MF773568-Malaysia-2008	RAELCPVVQETAETRDTA
CHIKV-KX168429-Malaysia-2009	RAEQCPAVQETAETRDTA
CHIKV- EU703762-Malaysia-2006	RAEQCPAVQETAETRDTA
CHIKV-MN974211-Thailand-2018	RAELCPVVQETAETRDTA
CHIKV- KX619422-India-2014	RAELCPVVQETAETRDTA
CHIKV- MT640256-Thailand-2021	RAELCPVVQETAETRDTA
CHIKV- DQ443544-La Reunion-2006	RAELCPVVQETAETRDTA
CHIKV- FJ807898-Taiwan-2009	RAELCPVVQETAETRDTA
CHIKV- GQ428210-India-2006	RAELCPVVQETAETRDTA
CHIKV- EU244823-Italy-2007	RAELCPVVQETAETRDTA
CHIKV- EF012359-Mauritius-2007	RAELCPVVQETAETRDTA
CHIKV- KC862329- Indonesia-2010	RAELCPVVQETAETRDTA
CHIKV- MF076568-Laos-2013	RAELCPVVQETAETRDTA
CHIKV- HM045810-Thailand-1958	RAELCPAVQETAETRDTA
CHIKV- EF027140-India-1963	RAELCPAVQETAETRDTA
CHIKV- EF452493-Thailand-1962	RAELCPAVQETAETRDTA
CHIKV- HM045813-India-1963	RAELCPAVQETAETRDTA
CHIKV- HM045803-India-1963	RAELCPAVQETAETRDTA
CHIKV- EF027141-India-1973	RAELCPAVQETAETRDTA
CHIKV- HM045788- India-1973	RAELCPAVQETAETRDTA
CHIKV- HM045791-Thailand-1983	RAELCPAVQETAETRDTA
CHIKV- HM045797- Indonesia-1985	RAELCPAVQETAETRDTA
CHIKV- HM045814-Thailand-1975	RAELCPAVQETAETRDTA
CHIKV- HM045800-Philippines-1985	RAEMCPAVQETAETRDTA
CHIKV- HM045790-Philippines-1985	RAEMCPAVQETAETRDTA
CHIKV- HM045796-Thailand-1995	RAELCPAVQETAETRDTA
CHIKV- HM045802- Thailand -1995	RAELCPAVQETAETRDTA
CHIKV- HM045787-Thailand-1995	RAELCPAVQETAETRDTA
CHIKV- HM045789- Thailand -1988	RAELCPAVQETAETRDTA
CHIKV- AB455493-India-2006	RAELCPVVQETAETRDTA
CHIKV- AF369024-Tanzania-1953	RAELCPVVQETAETRDTA
CHIKV- FJ807897- Indonesia -2007	RAEQCPAVQETAETRDTA
CHIKV- KF318729 -China-2012	RAEQCPTVQETAETRDTA
CHIKV- FN295483 -Malaysia-2006	RAEQCPAVQETAETRDTA
CHIKV- KJ451623 -Micronesia-2013	RAEQCPTVQETAETRDTA
CHIKV- HE806461- New Caledonia -2011	RAEQCPAVQETAETRDTA
CHIKV- KF872195-Russia-2013	RAEQCPAVQETAETRDTA
CHIKV- FJ000068- India-2006	RAELCPVVQETAETRDTA
CHIKV- AB860301- Philippines-2013	RAEQCPTVQETAETRDTA
CHIKV- KT327165- Mexico-2014	RAEQCPTVQETAETRDTA
CHIKV- KT327167- Mexico-2014	RAEQCPTVQETAETRDTA
CHIKV- KT327164- Mexico-2014	RAEQCPTVQETAETRDTA
CHIKV- KJ451624- British Virgin Islands-2014	RAEQCPTVQETAETRDTA
CHIKV- KX262991-Saint Martin-2013	RAEQCPTVQETAETRDTA
CHIKV- MH124579 -India-2010	RAELCPVVQETAETRDTA
CHIKV- MH124583-India-2016	RAELCPVVQETAETRDTA

The current investigated CHIKVs collected during the 2021 outbreak in Malaysia was characterized to be the new E1-K211E/E2-V264A sub-linage of ECSA-IOL genotype. Since this strain were detected to be highly similar to the neighbouring Thailand outbreak virus isolate from 2018 to 2019, it can be concluded that the investigated CHIKVs originated from Thailand through the Indian subcontinent. The new E1-K211E/E2-V264A mutations which were reported to improve adaptation to Ae. aegypti, may have caused vector shift in Malaysia leading to outbreaks primarily detected in urban west coast states as well as urban southern state, Johor. The detection of novel nsp3-T441V and nsp3-T441A among CHIKV from Johor and Selangor respectively may provide additional insight on factors resulting in the spread of CHIKV in Malaysia from 2019-2021. Therefore, further study on CHIKV isolated from Ae. aegypti and Ae. albopictus vectors from local CHIKV hotspots as well as nsp3-T441A/V mutation analysis need to be conducted in order to better understand the re-emergence of CHIKV in Malaysia.

If abbreviations are used in the text, they should be defined in the text at first use, and a list of abbreviations should be provided as below:

CHIKV: Chikungunya virus

CHIKF: Chikungunya fever

IOL: Indian ocean linage

ECSA: East/Central/South Africa lineage

ECSA-IOL: East, Central and South Africa-Indian ocean linage

nsP: non-structural protein

RNA: Ribonucleic acid

mRNA: messenger RNA

ORF: Open reading frame

C: Capsid protein

E: Envelope protein

TF: transframe protein

6k: small hydrophobic, class IIA viroporins

RC: replication complex

RdRP: RNA-dependent RNA polymerase

qPCR: quantitative PCR

cDNA: complementary DNA

ds: double stranded

SNP: single nucleotide polymorphism

CDS: coding sequence

ML tree: maximum likelihood tree

MCC tree: maximum clade credibility tree

MCMC: Markov Chain Monte Carlo

ESS: effective sample size

AUD: Alphavirus-Unique domain

HVD: hypervariable domain

FHL1: Four and a half LIM domains protein 1

GTR: General Time Reversible substitution model

+F: Empirical base frequencies

+I: Invariable sites

+G4: discrete gamma heterogeneity model 4 rate categories

Ethics approval and consent to participate

All archived clinical samples utilised in this study has been approved for research usage by the Medical Research and Ethics Committee (MREC) (Reference: 22-01162-PDE (2)) of the Ministry of Health (MOH) in reference to the MOH medical research grant NMRR-22-01162-PDE/ 22-018

Consent for publication

This study utilizes archived clinical samples with randomly generated Research ID, no personal data or unique identifiers has been revealed in the study or manuscript.

Availability of data and material

Not applicable

Competing interests

The authors declare that they have no competing interests

Funding

This study is funded by the Ministry of Health Malaysia (MOH) and registered under the National Medical Research Registry (NMRR) accession number NMRR-22-01162-PDE/ 22-018

Authors’ Contribution

J.K involved in study design, conducting the experiments and manuscript writing; Z.M.Z involved in qPCR detection and quality control experiments; M.I.A, K.A.K, E.T.A, K.E and M.A.A assisted in bioinformatical analysis and manuscript editing; M.M.S.Z involved in sample collection; R.M.Z provided consultation for study design, sample collection and manuscript editing.

Acknowledgements

We would like to thank the Director General of Health Malaysia for his permission to publish this article.

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No competing interests reported.

Whole-genome sequencing analysis of the Chikungunya virus during 2021 outbreak in Malaysia

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