{"paper_id":"2a4c7a5d-47d4-494d-a022-7f365a7e7760","body_text":"Genetic diversity and phylogenetic analyses of Asian lineage of Zika virus whole genome sequencing derived from Culex quinquefasciatus mosquitoes and urine of patients during the epidemic in Thailand | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Genetic diversity and phylogenetic analyses of Asian lineage of Zika virus whole genome sequencing derived from Culex quinquefasciatus mosquitoes and urine of patients during the epidemic in Thailand Atchara Phumee, Suwalak Chitcharoen, Nataya Sutthanont, Proawpilart Intayot, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3354895/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 27 Oct, 2023 Read the published version in Scientific Reports → Version 1 posted 9 You are reading this latest preprint version Abstract Zika virus (ZIKV) is a mosquito-borne flavivirus that is emerging and re-emerging. Since 2010, sporadic ZIKV cases have been reported annually in Thailand, with over 1,000 confirmed positive cases in 2016. High-throughput sequencing technologies, such as whole genome sequencing (WGS), have recently enabled the rapid sequencing of pathogen genomes. Therefore, this study used multiplex amplicon sequencing by the Illumina Miseq to describe the ZIKV WGS. Six ZIKV WGS were obtained from three samples of field-caught Culex quinquefasciatus mosquitoes and three urine samples collected from human in three different provinces of Thailand. Additionally, one ZIKV isolate was successfully isolated from a female Cx. quinquefasciatus . Based on WGS analysis, the timing of the 2020 outbreak correlates with the acquisition of five amino acid changes in the Asian lineage ZIKV strains from Thailand 2006, Cambodia 2010 and 2019, and Philippines 2012, including C-T106A, prM-V1A, E-V473M, NS1-A188V, and NS5-M872V. This mutation was found in all seven WGS, which was previously reported to be associated with significantly higher mortality rates. Moreover, phylogenetic analysis indicated that the seven ZIKV sequences belonged to the Asian lineage. The genomic region of the E gene showed the highest nucleotide diversity (0.7–1.3%). The data can be applied to develop molecular tools to better understand the virus's patterns and evolution. Furthermore, it could indicate potential targets for development of more effective to control and prevent the zika outbreak. Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Zika virus (ZIKV) is an emerging RNA flavivirus transmitted by mosquitoes and closely related to yellow fever virus (YFV), Japanese encephalitis (JEV), dengue virus (DENV), and West Nile virus (WNV) 1 . The ZIKV genome is approximately 10.8 kilobases in length and consists of a positive-sense, single-stranded RNA molecule 2 . The genome organization of flaviviruses consists of 5′-C-prM-E-NS1-NS2a-NS2b-NS3-NS4a-2K-NS4b-NS5-3′ 3 , 4 . The structural components of the genome are comprised of the capsid protein (C), precursor membrane protein (Pr/M), and envelope protein (E); in addition, the seven non-structural proteins (NS) are critical for genome replication 5 . Phylogenetic analyses have played a crucial role in elucidating the evolutionary dynamics and dissemination patterns of ZIKV. Two major lineages, identified as the African and Asian lineages, have been defined based on the first finding in the specific geographic regions 2 , 6 . The analysis of genetic diversity among ZIKV lineages and across the genome can provide valuable insights into the genotype of the virus currently in circulation 7 . Several investigations have reported nucleotide (nt) mutations and amino acid (aa) changes throughout the entire diversity of ZIKV 8 – 10 . The presence of specific mutations in all sequences of a specific clade in the phylogeny may have importance for the control of disease, especially when that clade is associated with increased pathogenicity. However, the causal relationship between a mutation and a phenotype must always be verified using reverse genetics. Recently, ZIKV has rapidly spread globally and has been associated with human diseases such as microcephaly and other birth defects in neonates and Guillain-Barré syndrome in adults 11 , 12 . The main transmission route of ZIKV is through several mosquito species such as Aedes mosquitoes 13 . However, ZIKV can also be transmitted by other non-mosquito vector 14 including perinatal transmission from mother to fetus 15 , blood transfusion 16 and sexual transmission 17 . Between 2015 and 2017, ZIKV rapidly spread to 51 countries and territories in Thailand 18 . ZIKV has been circulating in Thailand for a long time and has spread significantly 19 . Previous studies have demonstrated the presence of ZIKV RNA in Aedes aegypti , Culex quinquefasciatus , and Armigeres subalbatus mosquitoes collected from the homes of actively infected ZIKV patients in Thailand 20 , 21 . However, the severity of the diseases caused by ZIKV, and its rapid spread require an immediate understanding of this virus. For ZIKV diagnosis, the recommended gold standard is molecular-based detection of ZIKV RNA in samples such as reverse transcription PCR (RT-PCR), real time RT-PCR and nested RT-PCR assay for using several alternative specimen types. Recently, high-throughput sequencing technologies, whole genome sequencing (WGS) provide the possibility to rapidly obtain the full sequence of pathogen genomes such as viruses. Therefore, our objective was to characterize ZIKV genetic diversity and phylogenetic analyses of the positive ZIKV samples by WGS using multiplex amplicon sequencing-based next-generation sequencing (NGS) on an Illumina sequencing system (MiSeq). A scale-up of ZIKV whole-genome sequencing provides a valuable prospect for understanding the genetic diversity, evolutionary dynamics, and transmission patterns of ZIKV. The information obtained from this study can be applied to further develop molecular tools and the nucleotide sequence data can also provide fundamental data for the application of molecular techniques for future development of more effective region-specific diagnostics, prevention, and control strategies of ZIKV in Thailand. Results ZIKV detection and isolation Hemi-nested RT-PCR (hn-RT-PCR) was used to detect the presence of ZIKV in three human urine samples and three Cx. quinquefasciatus mosquitoes. These mosquito samples were collected from areas around houses of confirmed ZIKV infected patients in Bangkok, Chanthaburi, and Nakhon Ratchasima. The supernatants of all hn-RT-PCR -positive mosquito samples were used to isolate ZIKV by inoculation onto C6/36 cells. At 7 days post-infection (dpi), cytopathic effects (CPE) can be observed in one out of three mosquitoes (Fig. 1 A). The most prominent morphological characteristics of CPE include a random-packed cell structure, damaged cell membranes, and lysis, while no morphological changes as a normal monolayer were observed in the C6/36 cell control by light microscopy (Fig. 1 B). In addition, we performed a ZIKV-specific immunofluorescence assay (IFA) staining to determine the location of the ZIKV antigen. The confirmation of the presence of ZIKV in mosquito isolation was achieved through staining (Fig. 1 C), whereas the presence of ZIKV antigen was not observed in uninfected C6/36 cell (Fig. 1 D). Genomic diversity of complete genome sequence of ZIKV The primer-trimming, alignment, variant calling, and consensus generation showed that the alignment of seven samples was approximately 10,270 nucleotides (nt) in length. The ZIKV genome encodes for three structural proteins: C (365 nt), Pr/M (502 nt), E (1,511 nt), and seven non-structural proteins: NS1 (1,055 nt), NS2A (677 nt), NS2B (389 nt), NS3 (1,850 nt), NS4A (380 nt), 2K (68 nt), NS4B (752 nt), and NS5 (2,708 nt). The complete genome sequences of ZIKV have been deposited in GenBank under the accession number OR535163-OR535169. Sequence analysis of ZIKV revealed that the seven genome sequences investigated the genetic diversity and molecular evolution related among the ZIKV isolated from the serum of patients in Thailand during the outbreak of ZIKV in 2006–2017, Cambodia in 2010 and 2019 (accession no. MH158236.1 and ON209935.1), and Philippines in 2012 (accession no. KU681082.1) (Fig. 2 A). The evaluation of nucleotide variation was conducted for each sample, in which the average count and proportion of minority variants were determined in relation to the reference sequence from Brazil (accession no. NC035889.1). The nucleotide variation averaged per gene within the ZIKV whole genome from Asia and non-Asia sequencing. In general, the study demonstrated that the seven samples had a relatively low level of nucleotide variation among the three different regions of ZIKV infection. The E region showed the most nucleotide diversity, ranging from 0.7–1.3%. The non-structural proteins of NS1, NS2B, NS3, NS4A, NS4B, and the NS5 determined low nucleotide diversity of approximately 0.2–0.3%, whereas NS2A region showed a higher nucleotide diversity than the other NS regions (0.5%). The percentage of nucleotide variation revealed the presence of minor variations in non-Asia ZIKV belonging to the Asian lineage of ZIKV. (Fig. 2 B). For the amino acids study, several amino acid changes were observed in all seven ZIKV genome sequences. Most of these changes were previously reported in ZIKV isolates from Thailand, Cambodia, and Philippines including C-T106A, prM-V1A, E-V473M, NS1-A188V, and NS5-M872V (Fig. 3 ). However, the five mutations were not observed in ZIKV isolates collected in Thailand during the 2013–2017 epidemic, whereas these mutations were detected in Thailand in 2006, Cambodia in 2010 and 2019, and Philippines in 2012. Phylogenetic analysis based on the complete genome sequence of ZIKV To determine the genetic relationship among the ZIKV isolates obtained during this study and other ZIKV strains collected from various geographical locations, a phylogenetic tree was constructed using complete genome sequences. A total of seven genome sequences were subjected to alignment analysis with 224 full-length ZIKV isolates derived from 29 distinct countries across the world and have been sampled between 2000 and 2023, as established in the GenBank database. The phylogenetic analysis revealed that the seven ZIKV isolates presented belonging within the Asian lineage and showed a close relationship to ZIKV strains obtained from human blood samples in Thailand in 2017–2019, as well as from human saliva sample in Cambodia in 2019. (Fig. 4 A and 4 B). For a clear understanding of clustered within close geographical proximity to neighboring regions, we reconstructed another phylogenetic tree for evolutionary distances using the neighbor-joining method and a maximum composite likelihood algorithm. The ZIKV genomes sequenced in this study are found in two separate clusters within a clade, together with genomes from Thailand in 2017 and Cambodia in 2019. However, in our genomic analyses, the non-Asia sequences formed a monophyletic clade distinct from the Asia sequences (Fig. 4 C). In addition, the phylogenetic tree analysis demonstrated that all ZIKV sequences obtained in this study from mosquitoes and humans in different regions showed no discernible differentiation. Discussion Zika virus infection has historically been a neglected viral infection in Africa, the Americas, Asia, and the Pacific, probably due to its prevalence of silent infections and relatively mild clinical manifestations 22 , 23 . ZIKV is continuously circulating in many Southeast Asian countries such as Thailand 24 , Cambodia 25 , 26 , Indonesia 27 , Philippines 28 , India 29 , Singapore 30 , Japan 31 , and Vietnam 32 , 33 . This current study reports the detection and isolation of ZIKV in field-caught mosquitoes and urine of patients from Thailand, during part of the 2020 epidemic. ZIKV was found in both male and female of Cx. quinquefasciatus and human urine. This is the first report of whole genome sequences of ZIKV obtained from Cx. quinquefasciatus from Thailand. Seven ZIKV sequences in this study demonstrated good coverage with > 80%. To better understand the evolutionary relationships between these sequences, we constructed the phylogenetic relationships between the many ZIKV strains using the ZIKV genomes. The evolutionary history of these ZIKV isolates showed strong clustering with ZIKV genomes obtained from other human ZIKV in Thailand during the outbreak of ZIKV in 2006–2017, Cambodia in 2010 and 2019, and Philippines in 2012. It is most likely that ZIKV spread among neighboring countries in Southeast Asia at the beginning, such as Cambodian, which shares a border in the East of Thailand. Our study demonstrated five mutations in the seven ZIKV genomes, including C-T106A, prM-V1A, E-V473M, NS1-A188V, and NS5-M872V. In 2019, a novel ZIKV isolate from Cambodia in 2019 (CAM/2019) revealed that 12 amino acid mutations were compared with Cambodia in 2010 (CAM/2010), including C-T106A, prM‐V123A, prM‐N130S, prM‐M151L, E‐V763M, NS1‐A982V, NS2A‐A1204T, NS2A‐P1274L, NS2B‐A1477T, NS5‐V2878A, NS5‐M3392V, and NS5‐V3403M, several of which were found to be associated with neurovirulence 34 . Furthermore, the CAM/2019 virus showed an NS2A-A1204T mutation that demonstrated the most closely related to the Thai isolate SI-BKK02 (accession no. MF996804.1) collected from Thailand in 2017, which was obtained from a case of microcephaly 35 . The reports of amino acid changes in Asian lineage ZIKV strains from 2010 to 2013 were C-T106A, prM-V1A, prM-S17N, E-V473M, NS1-A188V, NS5-M114V, and NS5-M872V 36 – 39 . Yu, et al. (2021) revealed that the C-T106A mutation increased infectivity and accelerated through the spread of ZIKV in both mosquitoes and rodents, resulting in increased transmissibility between vectors and hosts 40 . The mutation NS1-A188V was observed to have a slight effect on the infectivity of the ZIKV in laboratory Ae. aegypti mosquitoes. Additionally, this mutation was found to inhibit the production of type I interferon (IFN) in mammals 36 , 37 . Shan, et al. (2020) suggested that the mutants C-T106A, E-V473M, and NS5-M872V have been associated with significantly higher mortality rates. In particular, the E-V473M mutation has been found to enhance the replication of the ZIKV, resulting in enhanced neurovirulence, increased transmission from mother to fetus during pregnancy, and the presence of viremia in both mouse and nonhuman primate models. 41 . In this study, we conducted a comparative analysis of the genetic diversity offered by ZIKV mutants containing distinct stable substitutions that emerged within the Asian ZIKV lineage. Genetic variation can provide crucial insights into ZIKV biology and pathogenesis, as well as providing potential functional mutations in the virus. However, the potential relevance of these mutations in this study remains uncertain. The study is limited by a relatively small sample size, which includes only of seven complete genomes collected from different geographical regions from Thailand. Further investigation is required to assess the transmissibility of the mutant strains of the ZIKV within the mosquitoes using a mosquito-mouse transmission model. The present study not only offers a scientific basis for understanding the evolution of the Zika virus (ZIKV) in Southeast Asia but also illustrates the importance of continued monitoring in future outbreaks of ZIKV, in addition to focus on diagnostic challenges, surveillance, control and prevention strategies of ZIKV to stop the rapid spread of the epidemic in the future. Methods Sample collection Adult mosquitoes were collected both inside and outside the homes of ZIKV-infected patients during the outbreaks in Bangkok, Chanthaburi, and Nakhon Ratchasima in 2020, using a backpack aspirator (Bioquip, CA, USA). All mosquitos were classified according to their sex and species using morphological identification. Urine of ZIKV-infected patients were collected. The individual mosquitoes and human samples were then tested for ZIKV infection. ZIKV RNA detection by hn-RT-PCR Individual mosquitoes were placed in 1.5 ml microcentrifuge tubes with 200 µl of 1X phosphate buffered saline (PBS) and were ground and centrifuged at 13,000× g for 10 min. Then, 200 µl of the supernatant was transferred to a sterile 1.5 ml microcentrifuge tubes mixed with 200 µl of 2X minimum essential medium Eagle (MEM) (HyClone, USA) containing 2% heat-inactivated FBS (Gibco, USA), 2% penicillin (100 U/ml), and streptomycin (100 µg/ml) (Sigma-Aldrich, USA) and stored at − 80°C for further virus isolation attempts. The mosquito carcass/ or human urine sample was mixed with 300 µl of lysis buffer and processed for viral RNA extraction using an Invisorb Spin Virus RNA Mini Kit (STRATEC Molecular GmbH, Germany). The mosquito/or human urine RNA samples were amplified for detection of ZIKV at NS5 gene using hn-RT-PCR (Thai Patent No. 2001004011, 2020) 20 . The positive control was constructed and used the lower band of a synthetic positive control plasmid. The amplified PCR products were separated on a 1.5% agarose gel, stained with ethidium bromide, and visualized using Quantity One Quantification Analysis Software Version 4.5.2, Gel Doc EQ System (Bio-Rad, USA). Virus isolation and propagation The supernatant from ZIKV-PCR positive samples was passed through 0.2 µm syringe filters and seeded onto monolayers of Ae. albopictus C6/36 cells in 12-well plates were gently mixed at room temperature for 1 hour. The supernatant was removed and replaced with 2 ml of MEM containing 1% heat-inactivated FBS (Gibco, USA), penicillin (100 U/ml), and streptomycin (100 µg/ml) (Sigma-Aldrich, USA). The plates were incubated at 28°C, 5% CO 2 . CPE was monitored daily for 6–7 days. The hn-RT-PCR amplification of CPE-positive culture supernatant on day 7 confirmed ZIKV infection. The positive isolates were propagated in C6/36 cells cultured in MEM with 10% heat-inactivated FBS (Gibco, USA) at 28°C and 5% CO 2 for three more passages. Harvested supernatant was kept at -80°C until use. Immunofluorescence assay (IFA) At 0, 3, 5, and 7 days post-infection (dpi), infected cells grown on glass coverslips were harvested and rinsed with 1X PBS (3x5 min), fixed with 4% formaldehyde for 15 min at room temperature, and then incubated for 15 min in 0.1% of Triton X-100 in 1X PBS for 15 min at room temperature. After washing with 1X PBS (3x5 min), the fixed cells were incubated with a 1:500 primary rabbit-Zika virus NS1 protein antibody (GeneTex, USA), rinsed with 1X PBS (3x5 min), and incubated with 1:10000 Goat Anti-Rabbit IgG H&L (Alexa Fluor 488) (Abcam, UK) at room temperature for 1 hour as the secondary antibody and then washed with 1xPBS (3x5 min). Finally, a drop of Prolong gold antifade (Invitrogen, USA) was added on each slide and topped with a cover slide. The non-infected C6/36 cells are used as a negative control in the experiment. All slides were examined under a fluorescence microscope (Nikon, Japan). WGS using multiplex amplicon sequencing-based NGS RNA from positive samples were synthesized for cDNA using Superscript IV enzyme (Invitrogen, USA) followed by ZIKV multiplex PCR of 2 primer pools, long-amplicon 35‐plex PCR primer panel with ~ 11,000‐bp amplicon lengths for full‐genome sequencing of ZIKV 42 . The PCR products from 2 primer pools were purified and end-repaired using KAPA Hyper prep kit (Kapa Biosystems, USA). The index adaptor was then added to the end of the fragments and PCR was performed. The library DNA was checked for quality and quantity using QIAxcel screen gel (Qiagen, USA) and Qubit high sensitivity DNA kit (Thermo Fisher Scientific, USA), respectively. The concentration of each library DNA was calculated. The pooled DNA library was then loaded into the MiSeq reagent kit V3 (Illumina, USA) and sequenced using MiSeq machine (Illumina, USA). Phylogenetic tree construction The genome sequences were assembled using multiple alignments using fast Fourier transform (MAFFT) version 7 ( https://mafft.cbrc.jp/alignment/server/ ) 43 . Based on the complete genome sequence of ZIKV from this study and reference strains obtained from the GenBank database The phylogenetic trees were constructed using the neighbor-joining (NJ) method and maximum composite likelihood model, based on the general time reversible (GTR) model in MEGA 11 software 44 . Bootstrap values were estimated for 1000 replicates. The phylogenetic trees were edited using Interactive Tree Of Life (iTOL) v6.3 45 . Variation and mutation in the genome are confirmed by manually checking the alignment with ZIKV reference genome from Brazil (accession no. NC035889.1) in Unipro UGENE v47.0 46 . Declarations Acknowledgements This Research is supported by Thailand Science research and Innovation Fund Chulalongkorn University (Grant No. CU_FRB65_hea (20)_027_30_08) and Health Systems Research Institute (Grant No. 64-156). Competing interests The authors declare no competing interests. Data availability statement The data that support the findings of this study are available from the corresponding author on reasonable request. The ZIKV- sequences are available in GenBank, accession number OR535163-OR535169. Ethics declarations The study was approved by the animal research ethics committee of Chulalongkorn University and adhered to the Animal Care and Use Protocol (CU-ACUP). The Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand (COA No. 025/2564) approved this study, which abided by the Animals for Scientific Purposes Act and all relevant institutional policies and regulations regarding animal care and use at Chulalongkorn University. 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Liu, Y., et al. Evolutionary enhancement of Zika virus infectivity in Aedes aegypti mosquitoes. Nature . 545 (7655), 482-486 (2017). https://doi.org/10.1038/nature22365. Yuan, L., et al. A single mutation in the prM protein of Zika virus contributes to fetal microcephaly. Science . 358 (6365), 933-936 (2017). https://doi.org/10.1126/science.aam7120. Jaeger, A. S., et al. Zika viruses of African and Asian lineages cause fetal harm in a mouse model of vertical transmission. PLoS. Negl. Trop. Dis . 13 (4), e0007343 (2019). https://doi.org/10.1371/journal.pntd.0007343. Yu, X., et al. A mutation-mediated evolutionary adaptation of Zika virus in mosquito and mammalian host. Proc. Natl. Acad. Sci. U S A . 118 (42), e2113015118 (2021). https://doi.org/10.1073/pnas.2113015118. Shan, C., et al. A Zika virus envelope mutation preceding the 2015 epidemic enhances virulence and fitness for transmission. Proc. Natl. Acad. Sci. U S A . 117 (33), 20190-20197 (2020). https://doi.org/10.1073/pnas.2005722117. Quick, J., et al. Multiplex PCR method for MinION and Illumina sequencing of Zika and other virus genomes directly from clinical samples. Nat. Protoc . 12 (6), 1261-1276 (2017). https://doi.org/10.1038/nprot.2017.066. Katoh, K. & Standley, D. M. MAFFT multiple sequence alignment software version 7: improvements in performance and usability. Mol. Biol. Evol. 30 (4), 772-780 (2013). https://doi.org/10.1093/molbev/mst010. Tamura, K., Stecher, G. & Kumar, S. MEGA11: Molecular Evolutionary Genetics Analysis Version 11. Mol. Biol. Evol. 38 (7), 3022-3027 (2021). https://doi.org/10.1093/molbev/msab120. Letunic, I. & Bork, P. Interactive Tree Of Life (iTOL) v5: an online tool for phylogenetic tree display and annotation. Nucleic. Acids. Res. 49 (W1), W293-W296 (2021). https://doi.org/10.1093/nar/gkab301. Okonechnikov, K., Golosova, O., Fursov, M. & UGENE team. Unipro UGENE: a unified bioinformatics toolkit. Bioinformatics . 28 (8), 1166-1167 (2012). https://doi.org/10.1093/bioinformatics/bts091. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Published Journal Publication published 27 Oct, 2023 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Major revision 06 Oct, 2023 Reviews received at journal 03 Oct, 2023 Reviews received at journal 03 Oct, 2023 Reviewers agreed at journal 22 Sep, 2023 Reviewers invited by journal 22 Sep, 2023 Editor assigned by journal 22 Sep, 2023 Editor invited by journal 19 Sep, 2023 Submission checks completed at journal 19 Sep, 2023 First submitted to journal 14 Sep, 2023 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {\"props\":{\"pageProps\":{\"initialData\":{\"identity\":\"rs-3354895\",\"acceptedTermsAndConditions\":true,\"allowDirectSubmit\":false,\"archivedVersions\":[],\"articleType\":\"Article\",\"associatedPublications\":[],\"authors\":[{\"id\":234603262,\"identity\":\"a1d921c1-09e5-451c-a5fa-f880818d8fc4\",\"order_by\":0,\"name\":\"Atchara Phumee\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Walailak University\",\"correspondingAuthor\":false,\"submittingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Atchara\",\"middleName\":\"\",\"lastName\":\"Phumee\",\"suffix\":\"\"},{\"id\":234603264,\"identity\":\"fed714c6-e4cd-4eed-af2a-b3020204f0cb\",\"order_by\":1,\"name\":\"Suwalak Chitcharoen\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Khon Kaen University\",\"correspondingAuthor\":false,\"submittingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Suwalak\",\"middleName\":\"\",\"lastName\":\"Chitcharoen\",\"suffix\":\"\"},{\"id\":234603267,\"identity\":\"fec1a2ba-c8f3-45c0-8a5e-9d6c20dcfeb6\",\"order_by\":2,\"name\":\"Nataya Sutthanont\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Mahidol University\",\"correspondingAuthor\":false,\"submittingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Nataya\",\"middleName\":\"\",\"lastName\":\"Sutthanont\",\"suffix\":\"\"},{\"id\":234603269,\"identity\":\"fe32158c-a65b-4c0f-80ab-fd1b02dedc4a\",\"order_by\":3,\"name\":\"Proawpilart Intayot\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"The Government Pharmaceutical Organization\",\"correspondingAuthor\":false,\"submittingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Proawpilart\",\"middleName\":\"\",\"lastName\":\"Intayot\",\"suffix\":\"\"},{\"id\":234603270,\"identity\":\"0e5ee02d-2e3b-4222-bf72-58d3b099079d\",\"order_by\":4,\"name\":\"Supaporn Wacharapluesadee\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"King Chulalongkorn Memorial Hospital, Chulalongkorn University\",\"correspondingAuthor\":false,\"submittingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Supaporn\",\"middleName\":\"\",\"lastName\":\"Wacharapluesadee\",\"suffix\":\"\"},{\"id\":234603271,\"identity\":\"aa97116e-b9a4-441b-9f0d-26144e5dccc6\",\"order_by\":5,\"name\":\"Padet Siriyasatien\",\"email\":\"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA20lEQVRIiWNgGAWjYHACAxAhxyDBwHgAIsBDhBagUmOgFgaIFjYitSQ2EK3FnP3wxscfGOrS+2f3GBz4wWAnzyDfewCvFsuetGKDAwyHc2fcOWNwsIch2bCBjS8Bv6sO5JhJHGA4kLtBIsfgAA8DcwLQYQb4tZx/Y/7jANBhBkAtB/8w1BOh5UaOGdDbzAkgLYd5GA4T1mI541mxxBmDw4YzbqQVHJYxOG7YxpaDX4s5f/LGDxUVdfL8M5I3PnxTUS3Pz3yGgMOQSAiDDa96ZMWjYBSMglEwCnACAB7pQY5iZuuhAAAAAElFTkSuQmCC\",\"orcid\":\"\",\"institution\":\"Chulalongkorn University\",\"correspondingAuthor\":true,\"submittingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Padet\",\"middleName\":\"\",\"lastName\":\"Siriyasatien\",\"suffix\":\"\"}],\"badges\":[],\"createdAt\":\"2023-09-14 09:59:26\",\"currentVersionCode\":1,\"declarations\":\"\",\"doi\":\"10.21203/rs.3.rs-3354895/v1\",\"doiUrl\":\"https://doi.org/10.21203/rs.3.rs-3354895/v1\",\"draftVersion\":[],\"editorialEvents\":[{\"content\":\"https://doi.org/10.1038/s41598-023-45814-9\",\"type\":\"published\",\"date\":\"2023-10-27T15:01:55+00:00\"}],\"editorialNote\":\"\",\"failedWorkflow\":false,\"files\":[{\"id\":43663652,\"identity\":\"488e14dc-9a5c-45d2-90ca-86b610620cb5\",\"added_by\":\"auto\",\"created_at\":\"2023-09-25 23:34:18\",\"extension\":\"png\",\"order_by\":1,\"title\":\"Figure 1\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":3900371,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eThe isolation of ZIKV from \\u003cem\\u003eCx. quinquefasciatus\\u003c/em\\u003ein C6/36 cells. The CPE in infected cells at 7 dpi (A) and uninfected cells (B) (100x magnification). The immunofluorescence assay (IFA) was used to detect ZIKV antigens in C6/36 cells at 7 dpi (C) and uninfected cells (D) (400x magnification); Green fluorescent color indicates viral replication; DAPI stains cell nuclei (blue fluorescent color). Photos taken by Atchara Phumee, First author.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Figure1.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-3354895/v1/aabfe7360e5d7c856d266544.png\"},{\"id\":43662255,\"identity\":\"25c9ed13-0acf-4256-a314-ffbcae3c8038\",\"added_by\":\"auto\",\"created_at\":\"2023-09-25 23:26:18\",\"extension\":\"png\",\"order_by\":2,\"title\":\"Figure 2\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":720478,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eAnalysis of nucleotide variation in each sample based on the number of consensus nucleotides (A) and the percentage of average nucleotide variation per gene in the study, Asia, and non-Asia ZIKV sequencings (B). All figures were modified from free software under public domain or a free license.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Figure2.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-3354895/v1/087cb88dfcc49b99bbd2c55d.png\"},{\"id\":43663651,\"identity\":\"3381b694-f51e-40e4-b0ad-c052b237ff6e\",\"added_by\":\"auto\",\"created_at\":\"2023-09-25 23:34:18\",\"extension\":\"png\",\"order_by\":3,\"title\":\"Figure 3\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":615810,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eThe five mutation-mediated evolutionary adaptation of ZIKV isolates belonged to the Asian lineage. All figures were modified from free software under public domain or a free license.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Figure3.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-3354895/v1/f49c6d13b4be6b7d67650e42.png\"},{\"id\":43662256,\"identity\":\"ed5f2b86-0915-4d78-ba49-909b88e73930\",\"added_by\":\"auto\",\"created_at\":\"2023-09-25 23:26:18\",\"extension\":\"png\",\"order_by\":4,\"title\":\"Figure 4\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":1402641,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003ePhylogenetic analysis of 224 ZIKV complete nucleotide sequences isolates obtained from the NCBI Virus database worldwide in 2000-2023 (A), expansion clade of tree containing ZIKV sequence in this study (B), and the maximum likelihood (ML) phylogenetic based on ZIKV isolates from Asia and non-Asia regions (C). The red and blue colors indicated the mosquito and human samples, respectively. The green color denotes the ZIKV non-Asia isolates. All figures were modified from free software under public domain or a free license.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Figure4.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-3354895/v1/348923887372e4c309ce7c91.png\"},{\"id\":45453806,\"identity\":\"bcd28f7f-b509-4147-91bb-fcbecfed323f\",\"added_by\":\"auto\",\"created_at\":\"2023-10-30 15:07:03\",\"extension\":\"pdf\",\"order_by\":0,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"manuscript-pdf\",\"size\":3255018,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"manuscript.pdf\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-3354895/v1/144a2dca-0bb1-4411-964a-fbf26a693103.pdf\"}],\"financialInterests\":\"No competing interests reported.\",\"formattedTitle\":\"Genetic diversity and phylogenetic analyses of Asian lineage of Zika virus whole genome sequencing derived from Culex quinquefasciatus mosquitoes and urine of patients during the epidemic in Thailand\",\"fulltext\":[{\"header\":\"Introduction\",\"content\":\"\\u003cp\\u003eZika virus (ZIKV) is an emerging RNA flavivirus transmitted by mosquitoes and closely related to yellow fever virus (YFV), Japanese encephalitis (JEV), dengue virus (DENV), and West Nile virus (WNV)\\u003csup\\u003e\\u003cspan citationid=\\\"CR1\\\" class=\\\"CitationRef\\\"\\u003e1\\u003c/span\\u003e\\u003c/sup\\u003e. The ZIKV genome is approximately 10.8 kilobases in length and consists of a positive-sense, single-stranded RNA molecule\\u003csup\\u003e\\u003cspan citationid=\\\"CR2\\\" class=\\\"CitationRef\\\"\\u003e2\\u003c/span\\u003e\\u003c/sup\\u003e. The genome organization of flaviviruses consists of 5\\u0026prime;-C-prM-E-NS1-NS2a-NS2b-NS3-NS4a-2K-NS4b-NS5-3\\u0026prime;\\u003csup\\u003e\\u003cspan citationid=\\\"CR3\\\" class=\\\"CitationRef\\\"\\u003e3\\u003c/span\\u003e,\\u003cspan citationid=\\\"CR4\\\" class=\\\"CitationRef\\\"\\u003e4\\u003c/span\\u003e\\u003c/sup\\u003e. The structural components of the genome are comprised of the capsid protein (C), precursor membrane protein (Pr/M), and envelope protein (E); in addition, the seven non-structural proteins (NS) are critical for genome replication\\u003csup\\u003e\\u003cspan citationid=\\\"CR5\\\" class=\\\"CitationRef\\\"\\u003e5\\u003c/span\\u003e\\u003c/sup\\u003e. Phylogenetic analyses have played a crucial role in elucidating the evolutionary dynamics and dissemination patterns of ZIKV. Two major lineages, identified as the African and Asian lineages, have been defined based on the first finding in the specific geographic regions\\u003csup\\u003e\\u003cspan citationid=\\\"CR2\\\" class=\\\"CitationRef\\\"\\u003e2\\u003c/span\\u003e,\\u003cspan citationid=\\\"CR6\\\" class=\\\"CitationRef\\\"\\u003e6\\u003c/span\\u003e\\u003c/sup\\u003e. The analysis of genetic diversity among ZIKV lineages and across the genome can provide valuable insights into the genotype of the virus currently in circulation\\u003csup\\u003e\\u003cspan citationid=\\\"CR7\\\" class=\\\"CitationRef\\\"\\u003e7\\u003c/span\\u003e\\u003c/sup\\u003e. Several investigations have reported nucleotide (nt) mutations and amino acid (aa) changes throughout the entire diversity of ZIKV\\u003csup\\u003e\\u003cspan additionalcitationids=\\\"CR9\\\" citationid=\\\"CR8\\\" class=\\\"CitationRef\\\"\\u003e8\\u003c/span\\u003e\\u0026ndash;\\u003cspan citationid=\\\"CR10\\\" class=\\\"CitationRef\\\"\\u003e10\\u003c/span\\u003e\\u003c/sup\\u003e. The presence of specific mutations in all sequences of a specific clade in the phylogeny may have importance for the control of disease, especially when that clade is associated with increased pathogenicity. However, the causal relationship between a mutation and a phenotype must always be verified using reverse genetics. Recently, ZIKV has rapidly spread globally and has been associated with human diseases such as microcephaly and other birth defects in neonates and Guillain-Barr\\u0026eacute; syndrome in adults\\u003csup\\u003e\\u003cspan citationid=\\\"CR11\\\" class=\\\"CitationRef\\\"\\u003e11\\u003c/span\\u003e,\\u003cspan citationid=\\\"CR12\\\" class=\\\"CitationRef\\\"\\u003e12\\u003c/span\\u003e\\u003c/sup\\u003e. The main transmission route of ZIKV is through several mosquito species such as \\u003cem\\u003eAedes\\u003c/em\\u003e mosquitoes\\u003csup\\u003e\\u003cspan citationid=\\\"CR13\\\" class=\\\"CitationRef\\\"\\u003e13\\u003c/span\\u003e\\u003c/sup\\u003e. However, ZIKV can also be transmitted by other non-mosquito vector\\u003csup\\u003e\\u003cspan citationid=\\\"CR14\\\" class=\\\"CitationRef\\\"\\u003e14\\u003c/span\\u003e\\u003c/sup\\u003e including perinatal transmission from mother to fetus\\u003csup\\u003e\\u003cspan citationid=\\\"CR15\\\" class=\\\"CitationRef\\\"\\u003e15\\u003c/span\\u003e\\u003c/sup\\u003e, blood transfusion\\u003csup\\u003e\\u003cspan citationid=\\\"CR16\\\" class=\\\"CitationRef\\\"\\u003e16\\u003c/span\\u003e\\u003c/sup\\u003e and sexual transmission\\u003csup\\u003e\\u003cspan citationid=\\\"CR17\\\" class=\\\"CitationRef\\\"\\u003e17\\u003c/span\\u003e\\u003c/sup\\u003e. Between 2015 and 2017, ZIKV rapidly spread to 51 countries and territories in Thailand\\u003csup\\u003e\\u003cspan citationid=\\\"CR18\\\" class=\\\"CitationRef\\\"\\u003e18\\u003c/span\\u003e\\u003c/sup\\u003e. ZIKV has been circulating in Thailand for a long time and has spread significantly\\u003csup\\u003e\\u003cspan citationid=\\\"CR19\\\" class=\\\"CitationRef\\\"\\u003e19\\u003c/span\\u003e\\u003c/sup\\u003e. Previous studies have demonstrated the presence of ZIKV RNA in \\u003cem\\u003eAedes aegypti\\u003c/em\\u003e, \\u003cem\\u003eCulex quinquefasciatus\\u003c/em\\u003e, and \\u003cem\\u003eArmigeres subalbatus\\u003c/em\\u003e mosquitoes collected from the homes of actively infected ZIKV patients in Thailand\\u003csup\\u003e\\u003cspan citationid=\\\"CR20\\\" class=\\\"CitationRef\\\"\\u003e20\\u003c/span\\u003e,\\u003cspan citationid=\\\"CR21\\\" class=\\\"CitationRef\\\"\\u003e21\\u003c/span\\u003e\\u003c/sup\\u003e. However, the severity of the diseases caused by ZIKV, and its rapid spread require an immediate understanding of this virus. For ZIKV diagnosis, the recommended gold standard is molecular-based detection of ZIKV RNA in samples such as reverse transcription PCR (RT-PCR), real time RT-PCR and nested RT-PCR assay for using several alternative specimen types. Recently, high-throughput sequencing technologies, whole genome sequencing (WGS) provide the possibility to rapidly obtain the full sequence of pathogen genomes such as viruses. Therefore, our objective was to characterize ZIKV genetic diversity and phylogenetic analyses of the positive ZIKV samples by WGS using multiplex amplicon sequencing-based next-generation sequencing (NGS) on an Illumina sequencing system (MiSeq). A scale-up of ZIKV whole-genome sequencing provides a valuable prospect for understanding the genetic diversity, evolutionary dynamics, and transmission patterns of ZIKV. The information obtained from this study can be applied to further develop molecular tools and the nucleotide sequence data can also provide fundamental data for the application of molecular techniques for future development of more effective region-specific diagnostics, prevention, and control strategies of ZIKV in Thailand.\\u003c/p\\u003e\"},{\"header\":\"Results\",\"content\":\"\\u003cdiv id=\\\"Sec3\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eZIKV detection and isolation\\u003c/h2\\u003e \\u003cp\\u003eHemi-nested RT-PCR (hn-RT-PCR) was used to detect the presence of ZIKV in three human urine samples and three \\u003cem\\u003eCx. quinquefasciatus\\u003c/em\\u003e mosquitoes. These mosquito samples were collected from areas around houses of confirmed ZIKV infected patients in Bangkok, Chanthaburi, and Nakhon Ratchasima. The supernatants of all hn-RT-PCR -positive mosquito samples were used to isolate ZIKV by inoculation onto C6/36 cells. At 7 days post-infection (dpi), cytopathic effects (CPE) can be observed in one out of three mosquitoes (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003eA). The most prominent morphological characteristics of CPE include a random-packed cell structure, damaged cell membranes, and lysis, while no morphological changes as a normal monolayer were observed in the C6/36 cell control by light microscopy (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003eB). In addition, we performed a ZIKV-specific immunofluorescence assay (IFA) staining to determine the location of the ZIKV antigen. The confirmation of the presence of ZIKV in mosquito isolation was achieved through staining (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003eC), whereas the presence of ZIKV antigen was not observed in uninfected C6/36 cell (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003eD).\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec4\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eGenomic diversity of complete genome sequence of ZIKV\\u003c/h2\\u003e \\u003cp\\u003eThe primer-trimming, alignment, variant calling, and consensus generation showed that the alignment of seven samples was approximately 10,270 nucleotides (nt) in length. The ZIKV genome encodes for three structural proteins: C (365 nt), Pr/M (502 nt), E (1,511 nt), and seven non-structural proteins: NS1 (1,055 nt), NS2A (677 nt), NS2B (389 nt), NS3 (1,850 nt), NS4A (380 nt), 2K (68 nt), NS4B (752 nt), and NS5 (2,708 nt). The complete genome sequences of ZIKV have been deposited in GenBank under the accession number OR535163-OR535169. Sequence analysis of ZIKV revealed that the seven genome sequences investigated the genetic diversity and molecular evolution related among the ZIKV isolated from the serum of patients in Thailand during the outbreak of ZIKV in 2006\\u0026ndash;2017, Cambodia in 2010 and 2019 (accession no. MH158236.1 and ON209935.1), and Philippines in 2012 (accession no. KU681082.1) (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eA). The evaluation of nucleotide variation was conducted for each sample, in which the average count and proportion of minority variants were determined in relation to the reference sequence from Brazil (accession no. NC035889.1). The nucleotide variation averaged per gene within the ZIKV whole genome from Asia and non-Asia sequencing. In general, the study demonstrated that the seven samples had a relatively low level of nucleotide variation among the three different regions of ZIKV infection. The E region showed the most nucleotide diversity, ranging from 0.7\\u0026ndash;1.3%. The non-structural proteins of NS1, NS2B, NS3, NS4A, NS4B, and the NS5 determined low nucleotide diversity of approximately 0.2\\u0026ndash;0.3%, whereas NS2A region showed a higher nucleotide diversity than the other NS regions (0.5%). The percentage of nucleotide variation revealed the presence of minor variations in non-Asia ZIKV belonging to the Asian lineage of ZIKV. (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eB). For the amino acids study, several amino acid changes were observed in all seven ZIKV genome sequences. Most of these changes were previously reported in ZIKV isolates from Thailand, Cambodia, and Philippines including C-T106A, prM-V1A, E-V473M, NS1-A188V, and NS5-M872V (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003e). However, the five mutations were not observed in ZIKV isolates collected in Thailand during the 2013\\u0026ndash;2017 epidemic, whereas these mutations were detected in Thailand in 2006, Cambodia in 2010 and 2019, and Philippines in 2012.\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec5\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003ePhylogenetic analysis based on the complete genome sequence of ZIKV\\u003c/h2\\u003e \\u003cp\\u003eTo determine the genetic relationship among the ZIKV isolates obtained during this study and other ZIKV strains collected from various geographical locations, a phylogenetic tree was constructed using complete genome sequences. A total of seven genome sequences were subjected to alignment analysis with 224 full-length ZIKV isolates derived from 29 distinct countries across the world and have been sampled between 2000 and 2023, as established in the GenBank database. The phylogenetic analysis revealed that the seven ZIKV isolates presented belonging within the Asian lineage and showed a close relationship to ZIKV strains obtained from human blood samples in Thailand in 2017\\u0026ndash;2019, as well as from human saliva sample in Cambodia in 2019. (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003eA and \\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003eB). For a clear understanding of clustered within close geographical proximity to neighboring regions, we reconstructed another phylogenetic tree for evolutionary distances using the neighbor-joining method and a maximum composite likelihood algorithm. The ZIKV genomes sequenced in this study are found in two separate clusters within a clade, together with genomes from Thailand in 2017 and Cambodia in 2019. However, in our genomic analyses, the non-Asia sequences formed a monophyletic clade distinct from the Asia sequences (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003eC). In addition, the phylogenetic tree analysis demonstrated that all ZIKV sequences obtained in this study from mosquitoes and humans in different regions showed no discernible differentiation.\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003c/div\\u003e\"},{\"header\":\"Discussion\",\"content\":\"\\u003cp\\u003eZika virus infection has historically been a neglected viral infection in Africa, the Americas, Asia, and the Pacific, probably due to its prevalence of silent infections and relatively mild clinical manifestations\\u003csup\\u003e\\u003cspan citationid=\\\"CR22\\\" class=\\\"CitationRef\\\"\\u003e22\\u003c/span\\u003e,\\u003cspan citationid=\\\"CR23\\\" class=\\\"CitationRef\\\"\\u003e23\\u003c/span\\u003e\\u003c/sup\\u003e. ZIKV is continuously circulating in many Southeast Asian countries such as Thailand\\u003csup\\u003e\\u003cspan citationid=\\\"CR24\\\" class=\\\"CitationRef\\\"\\u003e24\\u003c/span\\u003e\\u003c/sup\\u003e, Cambodia\\u003csup\\u003e\\u003cspan citationid=\\\"CR25\\\" class=\\\"CitationRef\\\"\\u003e25\\u003c/span\\u003e,\\u003cspan citationid=\\\"CR26\\\" class=\\\"CitationRef\\\"\\u003e26\\u003c/span\\u003e\\u003c/sup\\u003e, Indonesia\\u003csup\\u003e\\u003cspan citationid=\\\"CR27\\\" class=\\\"CitationRef\\\"\\u003e27\\u003c/span\\u003e\\u003c/sup\\u003e, Philippines\\u003csup\\u003e\\u003cspan citationid=\\\"CR28\\\" class=\\\"CitationRef\\\"\\u003e28\\u003c/span\\u003e\\u003c/sup\\u003e, India\\u003csup\\u003e\\u003cspan citationid=\\\"CR29\\\" class=\\\"CitationRef\\\"\\u003e29\\u003c/span\\u003e\\u003c/sup\\u003e, Singapore\\u003csup\\u003e\\u003cspan citationid=\\\"CR30\\\" class=\\\"CitationRef\\\"\\u003e30\\u003c/span\\u003e\\u003c/sup\\u003e, Japan\\u003csup\\u003e\\u003cspan citationid=\\\"CR31\\\" class=\\\"CitationRef\\\"\\u003e31\\u003c/span\\u003e\\u003c/sup\\u003e, and Vietnam\\u003csup\\u003e\\u003cspan citationid=\\\"CR32\\\" class=\\\"CitationRef\\\"\\u003e32\\u003c/span\\u003e,\\u003cspan citationid=\\\"CR33\\\" class=\\\"CitationRef\\\"\\u003e33\\u003c/span\\u003e\\u003c/sup\\u003e. This current study reports the detection and isolation of ZIKV in field-caught mosquitoes and urine of patients from Thailand, during part of the 2020 epidemic. ZIKV was found in both male and female of \\u003cem\\u003eCx. quinquefasciatus\\u003c/em\\u003e and human urine. This is the first report of whole genome sequences of ZIKV obtained from \\u003cem\\u003eCx. quinquefasciatus\\u003c/em\\u003e from Thailand. Seven ZIKV sequences in this study demonstrated good coverage with \\u0026gt;\\u0026thinsp;80%. To better understand the evolutionary relationships between these sequences, we constructed the phylogenetic relationships between the many ZIKV strains using the ZIKV genomes. The evolutionary history of these ZIKV isolates showed strong clustering with ZIKV genomes obtained from other human ZIKV in Thailand during the outbreak of ZIKV in 2006\\u0026ndash;2017, Cambodia in 2010 and 2019, and Philippines in 2012. It is most likely that ZIKV spread among neighboring countries in Southeast Asia at the beginning, such as Cambodian, which shares a border in the East of Thailand. Our study demonstrated five mutations in the seven ZIKV genomes, including C-T106A, prM-V1A, E-V473M, NS1-A188V, and NS5-M872V. In 2019, a novel ZIKV isolate from Cambodia in 2019 (CAM/2019) revealed that 12 amino acid mutations were compared with Cambodia in 2010 (CAM/2010), including C-T106A, prM‐V123A, prM‐N130S, prM‐M151L, E‐V763M, NS1‐A982V, NS2A‐A1204T, NS2A‐P1274L, NS2B‐A1477T, NS5‐V2878A, NS5‐M3392V, and NS5‐V3403M, several of which were found to be associated with neurovirulence\\u003csup\\u003e\\u003cspan citationid=\\\"CR34\\\" class=\\\"CitationRef\\\"\\u003e34\\u003c/span\\u003e\\u003c/sup\\u003e. Furthermore, the CAM/2019 virus showed an NS2A-A1204T mutation that demonstrated the most closely related to the Thai isolate SI-BKK02 (accession no. MF996804.1) collected from Thailand in 2017, which was obtained from a case of microcephaly\\u003csup\\u003e\\u003cspan citationid=\\\"CR35\\\" class=\\\"CitationRef\\\"\\u003e35\\u003c/span\\u003e\\u003c/sup\\u003e. The reports of amino acid changes in Asian lineage ZIKV strains from 2010 to 2013 were C-T106A, prM-V1A, prM-S17N, E-V473M, NS1-A188V, NS5-M114V, and NS5-M872V\\u003csup\\u003e\\u003cspan additionalcitationids=\\\"CR37 CR38\\\" citationid=\\\"CR36\\\" class=\\\"CitationRef\\\"\\u003e36\\u003c/span\\u003e\\u0026ndash;\\u003cspan citationid=\\\"CR39\\\" class=\\\"CitationRef\\\"\\u003e39\\u003c/span\\u003e\\u003c/sup\\u003e. Yu, et al. (2021) revealed that the C-T106A mutation increased infectivity and accelerated through the spread of ZIKV in both mosquitoes and rodents, resulting in increased transmissibility between vectors and hosts\\u003csup\\u003e\\u003cspan citationid=\\\"CR40\\\" class=\\\"CitationRef\\\"\\u003e40\\u003c/span\\u003e\\u003c/sup\\u003e. The mutation NS1-A188V was observed to have a slight effect on the infectivity of the ZIKV in laboratory \\u003cem\\u003eAe. aegypti\\u003c/em\\u003e mosquitoes. Additionally, this mutation was found to inhibit the production of type I interferon (IFN) in mammals\\u003csup\\u003e\\u003cspan citationid=\\\"CR36\\\" class=\\\"CitationRef\\\"\\u003e36\\u003c/span\\u003e,\\u003cspan citationid=\\\"CR37\\\" class=\\\"CitationRef\\\"\\u003e37\\u003c/span\\u003e\\u003c/sup\\u003e. Shan, et al. (2020) suggested that the mutants C-T106A, E-V473M, and NS5-M872V have been associated with significantly higher mortality rates. In particular, the E-V473M mutation has been found to enhance the replication of the ZIKV, resulting in enhanced neurovirulence, increased transmission from mother to fetus during pregnancy, and the presence of viremia in both mouse and nonhuman primate models.\\u003csup\\u003e\\u003cspan citationid=\\\"CR41\\\" class=\\\"CitationRef\\\"\\u003e41\\u003c/span\\u003e\\u003c/sup\\u003e. In this study, we conducted a comparative analysis of the genetic diversity offered by ZIKV mutants containing distinct stable substitutions that emerged within the Asian ZIKV lineage. Genetic variation can provide crucial insights into ZIKV biology and pathogenesis, as well as providing potential functional mutations in the virus. However, the potential relevance of these mutations in this study remains uncertain. The study is limited by a relatively small sample size, which includes only of seven complete genomes collected from different geographical regions from Thailand. Further investigation is required to assess the transmissibility of the mutant strains of the ZIKV within the mosquitoes using a mosquito-mouse transmission model. The present study not only offers a scientific basis for understanding the evolution of the Zika virus (ZIKV) in Southeast Asia but also illustrates the importance of continued monitoring in future outbreaks of ZIKV, in addition to focus on diagnostic challenges, surveillance, control and prevention strategies of ZIKV to stop the rapid spread of the epidemic in the future.\\u003c/p\\u003e\"},{\"header\":\"Methods\",\"content\":\"\\u003cdiv id=\\\"Sec8\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eSample collection\\u003c/h2\\u003e \\u003cp\\u003eAdult mosquitoes were collected both inside and outside the homes of ZIKV-infected patients during the outbreaks in Bangkok, Chanthaburi, and Nakhon Ratchasima in 2020, using a backpack aspirator (Bioquip, CA, USA). All mosquitos were classified according to their sex and species using morphological identification. Urine of ZIKV-infected patients were collected. The individual mosquitoes and human samples were then tested for ZIKV infection.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec9\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eZIKV RNA detection by hn-RT-PCR\\u003c/h2\\u003e \\u003cp\\u003eIndividual mosquitoes were placed in 1.5 ml microcentrifuge tubes with 200 \\u0026micro;l of 1X phosphate buffered saline (PBS) and were ground and centrifuged at 13,000\\u0026times; g for 10 min. Then, 200 \\u0026micro;l of the supernatant was transferred to a sterile 1.5 ml microcentrifuge tubes mixed with 200 \\u0026micro;l of 2X minimum essential medium Eagle (MEM) (HyClone, USA) containing 2% heat-inactivated FBS (Gibco, USA), 2% penicillin (100 U/ml), and streptomycin (100 \\u0026micro;g/ml) (Sigma-Aldrich, USA) and stored at \\u0026minus;\\u0026thinsp;80\\u0026deg;C for further virus isolation attempts. The mosquito carcass/ or human urine sample was mixed with 300 \\u0026micro;l of lysis buffer and processed for viral RNA extraction using an Invisorb Spin Virus RNA Mini Kit (STRATEC Molecular GmbH, Germany). The mosquito/or human urine RNA samples were amplified for detection of ZIKV at NS5 gene using hn-RT-PCR (Thai Patent No. 2001004011, 2020)\\u003csup\\u003e\\u003cspan citationid=\\\"CR20\\\" class=\\\"CitationRef\\\"\\u003e20\\u003c/span\\u003e\\u003c/sup\\u003e. The positive control was constructed and used the lower band of a synthetic positive control plasmid. The amplified PCR products were separated on a 1.5% agarose gel, stained with ethidium bromide, and visualized using Quantity One Quantification Analysis Software Version 4.5.2, Gel Doc EQ System (Bio-Rad, USA).\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec10\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eVirus isolation and propagation\\u003c/h2\\u003e \\u003cp\\u003eThe supernatant from ZIKV-PCR positive samples was passed through 0.2 \\u0026micro;m syringe filters and seeded onto monolayers of \\u003cem\\u003eAe. albopictus\\u003c/em\\u003e C6/36 cells in 12-well plates were gently mixed at room temperature for 1 hour. The supernatant was removed and replaced with 2 ml of MEM containing 1% heat-inactivated FBS (Gibco, USA), penicillin (100 U/ml), and streptomycin (100 \\u0026micro;g/ml) (Sigma-Aldrich, USA). The plates were incubated at 28\\u0026deg;C, 5% CO\\u003csub\\u003e2\\u003c/sub\\u003e. CPE was monitored daily for 6\\u0026ndash;7 days. The hn-RT-PCR amplification of CPE-positive culture supernatant on day 7 confirmed ZIKV infection. The positive isolates were propagated in C6/36 cells cultured in MEM with 10% heat-inactivated FBS (Gibco, USA) at 28\\u0026deg;C and 5% CO\\u003csub\\u003e2\\u003c/sub\\u003e for three more passages. Harvested supernatant was kept at -80\\u0026deg;C until use.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec11\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eImmunofluorescence assay (IFA)\\u003c/h2\\u003e \\u003cp\\u003eAt 0, 3, 5, and 7 days post-infection (dpi), infected cells grown on glass coverslips were harvested and rinsed with 1X PBS (3x5 min), fixed with 4% formaldehyde for 15 min at room temperature, and then incubated for 15 min in 0.1% of Triton X-100 in 1X PBS for 15 min at room temperature. After washing with 1X PBS (3x5 min), the fixed cells were incubated with a 1:500 primary rabbit-Zika virus NS1 protein antibody (GeneTex, USA), rinsed with 1X PBS (3x5 min), and incubated with 1:10000 Goat Anti-Rabbit IgG H\\u0026amp;L (Alexa Fluor 488) (Abcam, UK) at room temperature for 1 hour as the secondary antibody and then washed with 1xPBS (3x5 min). Finally, a drop of Prolong gold antifade (Invitrogen, USA) was added on each slide and topped with a cover slide. The non-infected C6/36 cells are used as a negative control in the experiment. All slides were examined under a fluorescence microscope (Nikon, Japan).\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec12\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eWGS using multiplex amplicon sequencing-based NGS\\u003c/h2\\u003e \\u003cp\\u003eRNA from positive samples were synthesized for cDNA using Superscript IV enzyme (Invitrogen, USA) followed by ZIKV multiplex PCR of 2 primer pools, long-amplicon 35‐plex PCR primer panel with ~\\u0026thinsp;11,000‐bp amplicon lengths for full‐genome sequencing of ZIKV\\u003csup\\u003e\\u003cspan citationid=\\\"CR42\\\" class=\\\"CitationRef\\\"\\u003e42\\u003c/span\\u003e\\u003c/sup\\u003e. The PCR products from 2 primer pools were purified and end-repaired using KAPA Hyper prep kit (Kapa Biosystems, USA). The index adaptor was then added to the end of the fragments and PCR was performed. The library DNA was checked for quality and quantity using QIAxcel screen gel (Qiagen, USA) and Qubit high sensitivity DNA kit (Thermo Fisher Scientific, USA), respectively. The concentration of each library DNA was calculated. The pooled DNA library was then loaded into the MiSeq reagent kit V3 (Illumina, USA) and sequenced using MiSeq machine (Illumina, USA).\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec13\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003ePhylogenetic tree construction\\u003c/h2\\u003e \\u003cp\\u003eThe genome sequences were assembled using multiple alignments using fast Fourier transform (MAFFT) version 7 (\\u003cspan class=\\\"ExternalRef\\\"\\u003e\\u003cspan class=\\\"RefSource\\\"\\u003ehttps://mafft.cbrc.jp/alignment/server/\\u003c/span\\u003e\\u003cspan address=\\\"https://mafft.cbrc.jp/alignment/server/\\\" targettype=\\\"URL\\\" class=\\\"RefTarget\\\"\\u003e\\u003c/span\\u003e\\u003c/span\\u003e)\\u003csup\\u003e43\\u003c/sup\\u003e. Based on the complete genome sequence of ZIKV from this study and reference strains obtained from the GenBank database The phylogenetic trees were constructed using the neighbor-joining (NJ) method and maximum composite likelihood model, based on the general time reversible (GTR) model in MEGA 11 software\\u003csup\\u003e\\u003cspan citationid=\\\"CR44\\\" class=\\\"CitationRef\\\"\\u003e44\\u003c/span\\u003e\\u003c/sup\\u003e. Bootstrap values were estimated for 1000 replicates. The phylogenetic trees were edited using Interactive Tree Of Life (iTOL) v6.3\\u003csup\\u003e45\\u003c/sup\\u003e. Variation and mutation in the genome are confirmed by manually checking the alignment with ZIKV reference genome from Brazil (accession no. NC035889.1) in Unipro UGENE v47.0\\u003csup\\u003e46\\u003c/sup\\u003e.\\u003c/p\\u003e \\u003c/div\\u003e\"},{\"header\":\"Declarations\",\"content\":\"\\u003cp\\u003e\\u003cstrong\\u003eAcknowledgements\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThis Research is supported by Thailand Science research\\u0026nbsp;and Innovation Fund Chulalongkorn University (Grant\\u0026nbsp;No.\\u0026nbsp;CU_FRB65_hea (20)_027_30_08)\\u0026nbsp;and Health Systems Research Institute (Grant No. 64-156).\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eCompeting interests\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThe authors declare no competing interests.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eData availability statement\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThe data that support the findings of this study are available from the corresponding author on reasonable request. The ZIKV- sequences are available in GenBank, accession number OR535163-OR535169.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eEthics declarations\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThe study was approved by the animal research ethics committee of Chulalongkorn University and adhered to the Animal Care and Use Protocol (CU-ACUP). The Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand (COA No. 025/2564) approved this study, which abided by the Animals for Scientific Purposes Act and all relevant institutional policies and regulations regarding animal care and use at Chulalongkorn University. The use of hazardous agents was only initiated after approval from the institutional animal care and use committee (IACUC), Institutional Biosafety Committee (IBC), and/or Environmental Health and Safety Department. The use of human blood was approved by the Institutional Review Board of the Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand (COE No. 016/2017), and the study was conducted in compliance with the international guidelines for human research protection as stated in the Declaration of Helsinki, The Belmont Report, the Council for International Organizations of Medical Sciences (CIOMS) guidelines and the International Conference on Harmonization in Good Clinical Practice (ICH-GCP). Informed consent was obtained from all participants. All experimental protocols requiring biosafety were approved by Institutional Biosafety Committees (IBC) of the Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand (MDCUIBC002/2017).\\u003c/p\\u003e\"},{\"header\":\"References\",\"content\":\"\\u003col\\u003e\\n\\u003cli\\u003eHayes E. B. Zika virus outside Africa. \\u003cem\\u003eEmerg. Infect. 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Unipro UGENE: a unified bioinformatics toolkit. \\u003cem\\u003eBioinformatics\\u003c/em\\u003e. \\u003cstrong\\u003e28\\u003c/strong\\u003e(8), 1166-1167 (2012). https://doi.org/10.1093/bioinformatics/bts091.\\u003c/li\\u003e\\n\\u003c/ol\\u003e\"}],\"fulltextSource\":\"\",\"fullText\":\"\",\"funders\":[],\"hasAdminPriorityOnWorkflow\":false,\"hasManuscriptDocX\":true,\"hasOptedInToPreprint\":true,\"hasPassedJournalQc\":\"\",\"hasAnyPriority\":false,\"hideJournal\":false,\"highlight\":\"\",\"institution\":\"\",\"isAcceptedByJournal\":true,\"isAuthorSuppliedPdf\":false,\"isDeskRejected\":\"\",\"isHiddenFromSearch\":false,\"isInQc\":false,\"isInWorkflow\":false,\"isPdf\":false,\"isPdfUpToDate\":true,\"isWithdrawnOrRetracted\":false,\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"scientific-reports\",\"isNatureJournal\":false,\"hasQc\":true,\"allowDirectSubmit\":false,\"externalIdentity\":\"scirep\",\"sideBox\":\"Learn more about [Scientific Reports](http://www.nature.com/srep/)\",\"snPcode\":\"\",\"submissionUrl\":\"\",\"title\":\"Scientific Reports\",\"twitterHandle\":\"\",\"acdcEnabled\":true,\"dfaEnabled\":true,\"editorialSystem\":\"stoa\",\"reportingPortfolio\":\"Scientific Reports\",\"inReviewEnabled\":true,\"inReviewRevisionsEnabled\":true},\"keywords\":\"\",\"lastPublishedDoi\":\"10.21203/rs.3.rs-3354895/v1\",\"lastPublishedDoiUrl\":\"https://doi.org/10.21203/rs.3.rs-3354895/v1\",\"license\":{\"name\":\"CC BY 4.0\",\"url\":\"https://creativecommons.org/licenses/by/4.0/\"},\"manuscriptAbstract\":\"\\u003cp\\u003eZika virus (ZIKV) is a mosquito-borne flavivirus that is emerging and re-emerging. Since 2010, sporadic ZIKV cases have been reported annually in Thailand, with over 1,000 confirmed positive cases in 2016. High-throughput sequencing technologies, such as whole genome sequencing (WGS), have recently enabled the rapid sequencing of pathogen genomes. Therefore, this study used multiplex amplicon sequencing by the Illumina Miseq to describe the ZIKV WGS. Six ZIKV WGS were obtained from three samples of field-caught \\u003cem\\u003eCulex quinquefasciatus\\u003c/em\\u003e mosquitoes and three urine samples collected from human in three different provinces of Thailand. Additionally, one ZIKV isolate was successfully isolated from a female \\u003cem\\u003eCx. quinquefasciatus\\u003c/em\\u003e. Based on WGS analysis, the timing of the 2020 outbreak correlates with the acquisition of five amino acid changes in the Asian lineage ZIKV strains from Thailand 2006, Cambodia 2010 and 2019, and Philippines 2012, including C-T106A, prM-V1A, E-V473M, NS1-A188V, and NS5-M872V. This mutation was found in all seven WGS, which was previously reported to be associated with significantly higher mortality rates. Moreover, phylogenetic analysis indicated that the seven ZIKV sequences belonged to the Asian lineage. The genomic region of the E gene showed the highest nucleotide diversity (0.7\\u0026ndash;1.3%). The data can be applied to develop molecular tools to better understand the virus's patterns and evolution. Furthermore, it could indicate potential targets for development of more effective to control and prevent the zika outbreak.\\u003c/p\\u003e\",\"manuscriptTitle\":\"Genetic diversity and phylogenetic analyses of Asian lineage of Zika virus whole genome sequencing derived from Culex quinquefasciatus mosquitoes and urine of patients during the epidemic in Thailand\",\"msid\":\"\",\"msnumber\":\"\",\"nonDraftVersions\":[{\"code\":1,\"date\":\"2023-09-25 23:26:13\",\"doi\":\"10.21203/rs.3.rs-3354895/v1\",\"editorialEvents\":[{\"type\":\"communityComments\",\"content\":0},{\"type\":\"decision\",\"content\":\"Major revision\",\"date\":\"2023-10-06T12:28:03+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"editorInvitedReview\",\"content\":\"\",\"date\":\"2023-10-03T11:19:02+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"editorInvitedReview\",\"content\":\"\",\"date\":\"2023-10-03T06:46:56+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"reviewerAgreed\",\"content\":\"d58fd42f-63ec-4a28-9a5b-8cfdda71d52c\",\"date\":\"2023-09-22T14:55:54+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"reviewersInvited\",\"content\":\"\",\"date\":\"2023-09-22T14:44:08+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"editorAssigned\",\"content\":\"\",\"date\":\"2023-09-22T14:36:36+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"editorInvited\",\"content\":\"\",\"date\":\"2023-09-19T07:29:56+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"checksComplete\",\"content\":\"\",\"date\":\"2023-09-19T07:23:16+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"submitted\",\"content\":\"Scientific Reports\",\"date\":\"2023-09-14T09:50:38+00:00\",\"index\":\"\",\"fulltext\":\"\"}],\"status\":\"published\",\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"scientific-reports\",\"isNatureJournal\":false,\"hasQc\":true,\"allowDirectSubmit\":false,\"externalIdentity\":\"scirep\",\"sideBox\":\"Learn more about [Scientific Reports](http://www.nature.com/srep/)\",\"snPcode\":\"\",\"submissionUrl\":\"\",\"title\":\"Scientific Reports\",\"twitterHandle\":\"\",\"acdcEnabled\":true,\"dfaEnabled\":true,\"editorialSystem\":\"stoa\",\"reportingPortfolio\":\"Scientific Reports\",\"inReviewEnabled\":true,\"inReviewRevisionsEnabled\":true}}],\"origin\":\"\",\"ownerIdentity\":\"50482d5c-f662-470d-86b0-73655b8806f0\",\"owner\":[],\"postedDate\":\"September 25th, 2023\",\"published\":true,\"recentEditorialEvents\":[],\"rejectedJournal\":[],\"revision\":\"\",\"amendment\":\"\",\"status\":\"published-in-journal\",\"subjectAreas\":[],\"tags\":[],\"updatedAt\":\"2023-10-30T15:04:00+00:00\",\"versionOfRecord\":{\"articleIdentity\":\"rs-3354895\",\"link\":\"https://doi.org/10.1038/s41598-023-45814-9\",\"journal\":{\"identity\":\"scientific-reports\",\"isVorOnly\":false,\"title\":\"Scientific Reports\"},\"publishedOn\":\"2023-10-27 15:01:55\",\"publishedOnDateReadable\":\"October 27th, 2023\"},\"versionCreatedAt\":\"2023-09-25 23:26:13\",\"video\":\"\",\"vorDoi\":\"10.1038/s41598-023-45814-9\",\"vorDoiUrl\":\"https://doi.org/10.1038/s41598-023-45814-9\",\"workflowStages\":[]},\"version\":\"v1\",\"identity\":\"rs-3354895\",\"journalConfig\":\"researchsquare\"},\"__N_SSP\":true},\"page\":\"/article/[identity]/[[...version]]\",\"query\":{\"redirect\":\"/article/rs-3354895\",\"identity\":\"rs-3354895\",\"version\":[\"v1\"]},\"buildId\":\"WrCJVZZCHTDjtuVLN7oU0\",\"isFallback\":false,\"isExperimentalCompile\":false,\"dynamicIds\":[84888],\"gssp\":true,\"scriptLoader\":[]}","source_license":"CC-BY-4.0","license_restricted":false}