Characterization of two novel Negevirus strains and analysis of global Negevirus distribution and host diversity

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Furthermore, mosquitoes carry a wide range of insect-specific viruses (ISVs), and studying these ISVs is important due to their potential influence on mosquito behavior and the transmission of mosquito-borne viruses. In this study, we report the first isolation of two Negevirus strains from Aedes and Culex mosquitoes in Xinjiang, China: Dezidougou virus (DEZV, isolate XJ-ALT23-420-01) and Negev-like virus (NEGLV, isolate XJ-JH20-91-01). Phylogenetically, the nucleotide sequence of DEZV exhibits high similarity (98.96%) with the DEZV isolate 8345 from Germany but shows lower similarity (< 91.91%) with other DEZV strains. In contrast, the NEGLV isolate XJ-JH20-91-01 displays significant divergence from other Nege virus (NEGV) or NEGLV, sharing only 79.39% nucleotide similarity with the most closely related strain, NEGV BeAr805514. Both DEZV/XJ-ALT23-420-01 and NEGLV/XJ-JH20-91-01 replicated rapidly in mosquito cell lines (C6/36 and Aag2), reaching viral loads of up to 10 9–10 copies/mL, causing significant cytopathic effect (CPE) in these cell lines but failing to replicate in vertebrate cells. In addition, analysis of the location and potential hosts of all published Negevirus members indicated their wide distribution and diverse host range. These findings extend the current spectrum of Negevirus group and provide deeper insights into its geographical distribution and host diversity. Dezidougou virus Negev-like virus Negevirus insect specific virus Aedes vexans Culex pipiens Xinjiang Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 1. Introduction There are two major groups of viruses that circulated in mosquito: mosquito-borne viruses (MBV) and mosquito-specific viruses (MSV) (Moonen et al., 2023 ). MBV, a subset of arbovirus, can infect both mosquito and vertebrate, whereas MSV belong to the insect-specific virus (ISV) group and replicate exclusively in mosquito (Stollar and Thomas, 1975 ). The first discovered ISV was Cell-fusing agent virus (CFAV), which was identified by Stollar and colleagues in 1975 from Aedes aegypti cell lines. With advances in high-throughput sequencing and the implementation of mosquito virome surveys, an increasing number of ISVs have since been discovered. ISVs are widely distributed across diverse viral taxonomic groups, such as Flaviviridae (Anakha et al., 2023 ; Espinoza-Gómez et al., 2011 ; Roiz et al., 2012 ), Bunyaviridae (Auguste et al., 2014 ), Togaviridae (Bennouna et al., 2019 ), Mesoniviridae (Lauber et al., 2012 ; Thuy et al., 2013 ), Reoviridae (Hermanns et al., 2014 ; Langat et al., 2023 ), Rhabdoviridae (Vasilakis et al., 2014 ), and new taxons such as Negevirus (Bishop et al., 2020 ; da Silva Ribeiro et al., 2022 ; Ribeiro et al., 2024 ; Vasilakis et al., 2013 ; Zhao et al., 2019 ). Researchs have demonstrated that the presence of MSVs can influence the replication and proliferation of MBVs. For example, Nhumirim virus (NHUV), isolated from mosquitoes in Brazil, significantly suppresses the amplification of West Nile virus (WNV), Japanese encephalitis virus (JEV), and St. Louis encephalitis virus (SLEV) when co-infecting C6/36 cells. The inhibitory effect was most pronounced against WNV and SLEV, with peak viral titers reduced by 10 6 PFU/mL and 10 4 PFU/mL, respectively (Kenney et al., 2014 ). These findings suggest that studying MSV-MBV interactions may offer novel strategies for controlling spreading of MBV. However, the mechanisms by which MSVs modulate MBV replication, dissemination, and transmission remain unclear. Negevirus is a new taxon group of non-segmented, single-stranded positive-sense RNA viruses with wide geographic distribution. The genome size for Negevirus is around 9–10 kb, containing three open reading frames (ORFs), flanked by 5′ and 3′ untranslated regions (UTRs), and terminates in a 3′ poly (A) tail. The virus particles are ellipsoidal or spherical in shape, with a diameter of 45–55 nm (Vasilakis et al., 2013 ). The genus Negevirus has been classified into two distinct clades: Nelorpivirus and Sandewavirus (Kallies et al., 2014 ) Based on genomic organization and phylogenetic analysis, which are distantly related to some plant viruses, such as Cileviruses , Higrevirus and Blunervirus . While primarily detected in mosquitoes, they have also been identified in sandflies, bees, and other arthropods. In this study, during mosquito and virus surveillance conducted in Xinjiang, China, we isolated one strain of Dezidougou virus (DEZV) from Aedes vexans and one strain of Negev-like virus (NEGLV) from Culex pipiens . The genome sequencing, viral purification, growth characteristics, and phylogenetic relationships of these two Negevirus strains were analyzed. Additionally, the global distribution patterns and host diversity of all known Negevirus members were investigated. 2. Method 2.1 Cells The C6/36 and Aag2 (mosquito cell lines), VeroE6, BHK-21, SW13, and PK15 (vertebrate cell lines) were used in this study. C6/36 and Aag2 cells were maintained in CO₂ incubator at 28°C in Roswell Park Memorial Institute (RPMI) 1640 medium containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (PS). BHK-21, VeroE6, SW13, and PK15 cells were cultured in CO₂ incubator at 37°C in Dulbecco’s minimal essential medium (DMEM) containing 10% FBS and 1% penicillin-streptomycin. 2.2 Mosquito collection and preparation From June 2020 to July 2023, mosquito collections were conducted in Xinjiang Uygur Autonomous Region, China, focusing on the Ebinur Lake and Takeshiken Port area. Mosquitoes were captured using CO₂-baited traps and subsequently morphology identification was conducted. Approximately 50–60 mosquitoes were pooled per sample and homogenized in RPMI 1640 medium supplemented with 2% penicillin-streptomycin. The homogenate was centrifuged at 4°C and 15,000 g for 30 min, then the debris in the bottom was discarded and centrifuged again at 15,000 g for 10 min to get the supernatant. 2.3 Virus isolation and purification Cells were seeded into 24-well plates (C6/36: 2×10 5 cells/well; VeroE6 and BHK-21: 1.6×10 5 cells/well). The supernatant acquired in section 2.2 was used to inoculate cell monolayers in 24-well plates. After a 1-hour incubation at 28°C (C6/36) or 37°C (VeroE6 and BHK-21), the inoculum was removed and replaced with maintenance medium (RPMI 1640 for C6/36 and DMEM for mammalian cells) containing 2% FBS. Cells were observed daily for 7 days to examine cytopathic effect (CPE), with all procedures replicated three times. To acquire pure virus stocks, the supernatant in the well showing CPE was harvested, diluted, and subjected to plaque purification on C6/36 cells. Virus-specific PCR primers (STable 1) were designed for viral identification. 2.4 Virus sequencing Supernatant from infected cells were harvested for viral RNA extraction using an automated nucleic acid extraction system (NanoMagBio, S-48 and NMG0966-16). RNA sequencing libraries were developed by Institutional Center for Shared Technologies and Facilities of Wuhan Institute of Virology, CAS. Sequencing was performed at Center for Instrumental Analysis and Metrology of Wuhan Institute of Virology, CAS, using the Illumina (Illumina Novaseq) platform. The resulting sequences were assembled de novo using Trinity v2.15.2 (Grabherr et al., 2011 ) and subjected to sequence comparison by Blast-n. Overlapping primers (STable 2 and STable 3) were designed according to the assembled contigs using Primalscheme (Quick et al., 2017 ) ( https://primalscheme.com/ ). Purified viral RNA was reverse transcribed and amplified by PCR, followed by purification of PCR products using the E.Z.N.A.® Gel Extraction Kit (Omega Bio-Tek, D2500-02). Missing terminal sequences were obtained using the HiScript-TS 5'/3' RACE Kit (Vazyme, RA101) according to the manufacturer's protocol. The complete viral genome was obtained through sequence splicing and assembly using DNASTAR Lasergene SeqMan Pro v7.1. 2.5 Viral morphological characteristics The virus was amplified using C6/36 cell lines. After 2 days of cultivation, the supernatant from samples exhibiting CPE was collected and purified by sucrose density gradient centrifugation (Beckman Coulter SW32, 4°C, 32,000 rpm for 3 hours). The supernatant was then discarded, and the precipitates were resuspended in 400 µL phosphate-buffered saline (PBS). Prior to ultracentrifugation, a sucrose gradient solution was prepared, and the viral supernatant was carefully layered onto it. Ultracentrifugation was performed at 4°C for 4 hours using a Beckman MLS50 rotor at 45,000 rpm. After centrifugation, the position of the viral band in the centrifuge tube was observed and recorded. A 1 mL syringe was then inserted into the band to aspirate the target viral band. Excess sucrose was removed using an Amicon Ultra-0.5 Centrifugal Filter Unit (50 kDa), following the manufacturer’s protocol, and this step was repeated five times. The purified viruses were loaded onto Formvar carbon-coated copper grid and negatively stained with 1% uranyl acetate. Morphological analysis was conducted using a Thermo Scientific Talos L120C transmission electron microscope operated at 120 kV. 2.6 Plaque and TCID50 assay Virus titration was performed as described by Vasilakis’ team (Vasilakis et al., 2013 ). Viral progeny formed plaques on C6/36 cell monolayers in 24-well plates. The viral supernatant was serially diluted 10-fold in culture medium. For each well, 100 µL of virus dilution in different fold was added, and the plates were incubated at 28°C for 1 hour, gently rocked every 15 minutes. Afterward, the supernatant was discarded and replaced with 100 µL of a medium consisting of a 1:1 mixture of 2% tragacanth suspension and 2 × RPMI with 5% FBS, 2% tryptose phosphate broth (TPB), and 2% penicillin-streptomycin. The cells were incubated at 28°C for 2 days to allow plaque formation. After incubation, the overlay was discarded, and the cells were fixed with 10% formaldehyde for 1 hour. Staining was done overnight at room temperature with 2% crystal violet and plaques were counted and recorded after removing excess stain under running water. Viral titers were additionally quantified using the 50% tissue culture infectious dose (TCID50) endpoint dilution method. A monolayer of C6/36 cells was spread in a 96-well plate. The viral supernatant was serially diluted 10-fold from 1×10⁻¹ to 1×10⁻¹¹, repeated eight wells per gradient. For each column, 100 µl of the viral supernatant was added and incubated for 1 hour. The plate was gently shaken every 15 min, then the supernatant was discarded, and the plate was replaced with 200 µL RPMI (2% FBS and 2% PS) medium for incubation at 28°C. The cell growth status was monitored daily and quantified for each well. 2.7 Virus grow kinetics The growth kinetics of DEZV/XJ-ALT23-420-01 and NEGLV/XJ-JH20-91-01 were determined in two mosquito cell lines: C6/36 ( Aedes albopictus ) and Aag2 ( Aedes aegypti ), and four vertebrate cell lines: VeroE6 (monkey), BHK-21 (baby hamster), SW13 (human), and PK-15 (pig). Briefly, cells were seeded into T-25 flasks (10 5–6 cells/per flask). After cultivation for 24h, the cells were incubated with virus at a ratio of viral RNA copies to cell number (R/C) from 0.001 to 1000. After incubation for 1 h, the inoculation was removed, washed three times with PBS, and replaced by fresh RPMI-1640 medium containing 2% FBS. Supernatant from the infected cell cultures were collected daily and stored at 80 ºC for viral RNA detection. The RNA template for qRT-PCR standard curve of NEGV and DEZV were produced by Takara In Vitro Transcription T7 kit and the designed primers (STable 4). 2.8 Phylogenetic analysis, geographical distribution, and host diversity of Negevirus First, RNA-dependent RNA polymerase (RdRp) sequences from both isolates were predicted using NCBI's Conserved Domain Search ( https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi ). Subsequently, a maximum likelihood phylogenetic tree was constructed using the complete RdRp coding regions of: (1) selected Negevirus isolates, and (2) representative members of two phylogenetically related plant virus families ( Kitaviridae and Virgaviridae ). Phylogenetic analysis was performed in PhyloSuite v1.2.3 (Zhang et al., 2020 ) with 1,000 standard bootstrap replicates. Trees were visualized and edited using Normal tree visualization software (Xie et al., 2023 ). All publicly available Negevirus related sequences (as of January 17, 2025) were retrieved from NCBI Virus, with associated host and geographical metadata. Sankey diagrams for distribution and host diversity analyses were generated using Origin 2024b. 3. Results 3.1 Virus isolation and morphology A total of three C6/36 wells inoculated with Aedes vexans homogenate tested positive for DEZV, while thirteen wells inoculated with Culex pipiens sample tested positive for NEGLV. Subsequently, one DEZV strain (XJ23-420-01) and one NEGLV strain (XJ-JH20-91-01) were successfully isolated. Both DEZV/XJ23-420-01) and NEGLV/XJ-JH20-91-01) induced prominent CPE in C6/36 cells at 2 days post-inoculation (Fig. 1 A). In NEGLV infected C6/36 monolayers, plaques displayed distinct edges and were readily quantifiable. In contrast, DEZV infected cells produced heterogeneous plaques with fuzzy, indistinct boundaries (Fig. 1 B), making plaque-based titer determination unreliable. Therefore, titer for DEZV was quantified using the TCID assay. In C6/36 cells, the titer of DEZV can reach 10 7.9 TCID 50 /mL (equal to 1.4× 10 7 PFU/mL) (STable 5) (Spearman, 1908; Kärber, 1931) (Lei et al., 2021 ), and NEGLV was 10 6 PFU/mL. In addition, electron microscopy revealed that both purified DEZV and NEGLV particles were exhibited elliptical shape (40–60 nm in diameter), with envelope (Fig. 1 C). 3.2 Viral replication in mosquito and vertebrate cell lines In both C6/36 and Aag2 cells, DEZV/XJ23-420-01 and NEGLV/XJ-JH20-91-01 infection induced extensive CPE within 2 d.p.i whether low or high doses (Fig. 2 A, 2 B, 2 D, and 2 E). Viral RNA copies in infected C6/36 cells increased rapidly by 1 d.p.i, reaching concentrations ≥ 10 8 copies/mL. In contrast, no significant CPE was observed in any vertebrate cell lines tested (Vero E6, SW13, BHK-21, PK-15) derived from human, monkey, hamster or pig, even at high infection doses (MOI = 10) (Fig. 2 C and 2 F). 3.3 Viral sequencing and genome structure The complete genomes of both viruses were obtained through next-generation sequencing (NGS) and rapid amplification of cDNA ends (RACE), and the sequences were deposited in GenBank under accession numbers PP908777 (DEZV/XJ-ALT23-420-01) and PQ493505 (NEGLV/XJ-JH20-91-01). The genome structure was predicted through NCBI Conserved Region Prediction Tool ( https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi ). DEZV/XJ-ALT23-420-01 possesses a 9,066 nt single-stranded, positive-sense RNA genome with a poly(A) tail. BLASTn analysis revealed 98.96% nucleotide identity to DEZV strain 8345 (from Germany), with high similarity. Its genome contains three open reading frames (ORFs): (1) ORF1 (nt 70-6810) encodes: FtsJ-like methyltransferase domain (nt positions 787–994), viral RNA helicase domain (nt 1316–1612), and RdRp domain (nt 1921–2202); (2) ORF2 (nt 6842–8125) contains DisA glycoprotein domain (nt 6989–8116); (3) ORF3 (nt 8233–8829) contains SP24 domain (nt 8353–8772) (Fig. 3 A). NEGLV/XJ-JH20-91-01 possesses a 9,531 nt single-stranded, positive-sense RNA genome with a poly(A) tail. BLASTn analysis revealed only 79.39% nucleotide identity to Negev virus strain BeAr805514 (from Brazilian Amazon), establishing it as a novel strain. Genomic annotation identified three open reading frames (ORFs): (1) ORF1 (nt 233–7339) encodes: viral methyltransferase (vMet; nt 187–539), AdoMet-dependent MTase superfamily domain (AdoMet; aa 914–1101), viral RNA helicase (VRhel; nt 1471–1733), and RdRp (nt 2113–2379); (2) ORF2 (nt 7373–8575) contains DisB glycoprotein domain (nt 7553–7744); (3) ORF3 (nt 8624–9250) contains SP24 domain (nt 8774–9190) (Fig. 3 B). 3.4 Phylogenetic analysis A maximum likelihood phylogenetic tree was constructed using the RdRp coding regions of representative members from Negevirus , Virgaviridae , and Kitaviridae , with branch support assessed by 1,000 bootstrap replicates (Fig. 4 ). The analysis revealed two distinct subgroup of Negevirus , Sandewavirus and Nelorpivirus . DEZVs belong to Sandewavirus subgroup and formed into two clusters: our DEZV/XJ-ALT23-420-01 and German strain 8345 (WGH58589.1) in one cluster, and FIN/PP-2018/82 (UUV42173.1), Yakutsk_2023 (XCM48930.1), FTA2-3 (QIN93579.1), and the prototype strain (ARQ15945.1) in the second cluster. NEGVs and NEGLVs belong to Nelorpivirus subgroup, and NEGLV/XJ-JH20-91-01 formed a separate branch, distinguishing from other NEGV or NEGLV strains. 3.5 Geographical distribution and host diversity of Negevirus Analysis of host diversity and geographic distribution of Negavirus group revealed global occurrence across all continents except Antarctica. Most sequences (136/168; 81%) originated from mosquitoes, with Culex species representing the highest proportion (68/168; 40.5%). Negevirus members were also detected in other invertebrates including bees, aphids, and flies, and notably in flatworms (Platyhelminthes). Piura virus was the most prevalent strain (45/168; 26.8%), detected in multiple mosquito vectors including Culex and Anopheles species (Fig. 5 ). 4. Discussion Negevirus comprises two subgroups: Sandewavirus and Nelorpivirus . DEZV, classified within Sandewavirus , has been reported in Africa (Shrivastava et al., 2017a ), Asia (Stepanyuk et al., 2025 ), and Europe (Agboli et al., 2023 ). In contrast, NEGV/NEGLV belongs to Nelorpivirus and has been detected in North America (Vasilakis et al., 2013 ), South America (da Silva Ribeiro et al., 2022 ), Africa, and Europe (Carapeta et al., 2015 ). In this study, we report the first isolation of DEZV and NEGLV from mosquitoes in Xinjiang, China. Phylogenetic analysis revealed that DEZV/XJ-ALT23-420-01 clusters with German strain 8345 (98.96% nucleotide identity), while NEGLV/XJ-JH20-91-01 forms a distinct branch with only 79.39% identity to known Negevirus strains, suggesting it represents a novel species. Phenotypically, plaque morphology differed significantly: DEZV produced fuzzy, irregular plaques whereas NEGLV formed distinct plaques with well-defined edges. This observation aligns with our prior report that Tanay virus (same subgroup as DEZV) similarly exhibits fuzzy plaques (Zhao et al., 2019 ). Genome of both DEZV/XJ-ALT23-420-01 and NEGLV/XJ-JH20-91-01 contain an SP24 element in the ORF3, which is a putative membrane protein of plant or insect viruses. There are studies revealed the potential evolutionary relationship of Negevirus with two plant virus families ( Kitaviridae and Virgaviridae ) based on SP24 and CP proteins, suggesting that the plant viruses may have originated from the horizontal transmission of ancient Negevirus viruses that occurred (Lu et al., 2024 ). Negevirus and the mentioned plant viruses may share a common ancestor capable of infecting both insects and plants. In future, identify the host range of Negevirus can help us to answer this evolutionary question. DEZV/XJ-ALT23-420-01 and NEGLV/XJ-JH20-91-01 demonstrate vertebrate cell-restricted tropism, showing no replication in tested mammalian cell lines. In contrast, both achieve a high viral loads (> 10⁹ copies/mL) in mosquito cells. Field studies confirm that DEZV naturally persists in multiple mosquito species and can coexist with medically important arbovirus such as Zika virus (ZIKV). Futermore, in vitro the co-infection experiment with Bunyamwera virus (BUNV), and Semliki Forest virus (SFV) showed that DEAV had minimal impact on BUNV or SFV replication (Shrivastava et al., 2017b ; Agboli et al., 2023 ). Next studies should investigate DEZV or NEGLV co-infection with additional arbovirus, both in cell lines and mosquitoes, to explore their potential impacts on arbovirus transmission. In summary, we isolated two new Negevirus strains, DEZV/XJ-ALT23-420-01 and NEGLV/XJ-JH20-91-01 and further investigated the global Negevirus distribution and host range. These findings expand our knowledge of this viral taxon. Declarations Ethics approval and consent to participate Not applicable. Consent for publication Not applicable. Availability of data and materials Sequence data that support the findings of this study have been deposited in the Genbase with the primary accession number PP908777 and PQ493505. The rest data and materials are provided within the manuscript or supplementary information files. Competing interests The authors declare no competing interests. Funding This work was supported by the National Key Research and Development Program of China (2022YFC2302700), the Youth Program of Wuhan Institute of Virology (2023QNTJ-03). Authors' contributions ZY and HX conceived the idea and coordinated the project. ZL, FT, FW, and XS performed the field work. 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Detection of a new insect flavivirus and isolation of Aedes flavivirus in Northern Italy. Parasit Vectors. 2012;5:223. https://doi.org/10.1186/1756-3305-5-223 . Shrivastava S, Puri V, Fedorova N, Amedeo P, Stockwell TB, Shabman RS, Rashid S, Pickett BE. Identification of Dezidougou Virus in a DAK AR 41524 Zika Virus Stock. Genome Announc. 2017a;5:e00605–17. https://doi.org/10.1128/genomeA.00605-17 . Shrivastava S, Puri V, Fedorova N, Amedeo P, Stockwell TB, Shabman RS, Rashid S, Pickett BE. Identification of Dezidougou Virus in a DAK AR 41524 Zika Virus Stock. Genome Announc. 2017b;5:e00605–17. https://doi.org/10.1128/genomeA.00605-17 . Stepanyuk MA, Legostaev SS, Karelina KV, Timofeeva NF, Emtsova KF, Ohlopkova OV, Taranov OS, Ternovoi VA, Protopopov AV, Loktev VB, Svyatchenko VA, Agafonov AP. Detection and characterization of the Dezidougou virus (genus Negevirus) in mosquitoes (Ochlerotatus caspius) collected in the Republic of Sakha (Yakutia). Vopr Virusol. 2025;70:47–56. https://doi.org/10.36233/0507-4088-280 . Stollar V, Thomas VL. An agent in the Aedes aegypti cell line (Peleg) which causes fusion of Aedes albopictus cells. Virology. 1975;64:367–77. https://doi.org/10.1016/0042-6822(75)90113-0 . Thuy NT, Huy TQ, Nga PT, Morita K, Dunia I, Benedetti L. A new nidovirus (NamDinh virus NDiV): Its ultrastructural characterization in the C6/36 mosquito cell line. Virology. 2013;444:337–42. https://doi.org/10.1016/j.virol.2013.06.030 . Vasilakis N, Castro-Llanos F, Widen SG, Aguilar PV, Guzman H, Guevara C, Fernandez R, Auguste AJ, Wood TG, Popov V, Mundal K, Ghedin E, Kochel TJ, Holmes EC, Walker PJ, Tesh RB. Arboretum and Puerto Almendras viruses: two novel rhabdoviruses isolated from mosquitoes in Peru. J Gen Virol. 2014;95:787–92. https://doi.org/10.1099/vir.0.058685-0 . Vasilakis N, Forrester NL, Palacios G, Nasar F, Savji N, Rossi SL, Guzman H, Wood TG, Popov V, Gorchakov R, González AV, Haddow AD, Watts DM, da Rosa APAT, Weaver SC, Lipkin WI, Tesh RB. Negevirus: a proposed new taxon of insect-specific viruses with wide geographic distribution. J Virol. 2013;87:2475–88. https://doi.org/10.1128/JVI.00776-12 . Xie J, Chen Y, Cai G, Cai R, Hu Z, Wang H. Tree Visualization By One Table (tvBOT): a web application for visualizing, modifying and annotating phylogenetic trees. Nucleic Acids Res. 2023;51:W587–92. https://doi.org/10.1093/nar/gkad359 . Zhang D, Gao F, Jakovlić I, Zou H, Zhang J, Li WX, Wang GT. PhyloSuite: An integrated and scalable desktop platform for streamlined molecular sequence data management and evolutionary phylogenetics studies. Mol Ecol Resour. 2020;20:348–55. https://doi.org/10.1111/1755-0998.13096 . Zhao L, Mwaliko C, Atoni E, Wang Y, Zhang Y, Zhan J, Hu X, Xia H, Yuan Z. 2019. Characterization of a Novel Tanay Virus Isolated From Anopheles sinensis Mosquitoes in Yunnan, China. Front Microbiol 10, 1963. https://doi.org/10.3389/fmicb.2019.01963 Additional Declarations No competing interests reported. Supplementary Files Supplementarymaterials.docx Cite Share Download PDF Status: Published Journal Publication published 03 Nov, 2025 Read the published version in Virology Journal → Version 1 posted Editorial decision: Revision requested 21 Jul, 2025 Reviews received at journal 20 Jul, 2025 Reviewers agreed at journal 03 Jul, 2025 Reviews received at journal 28 Jun, 2025 Reviewers agreed at journal 28 Jun, 2025 Reviewers invited by journal 26 Jun, 2025 Editor assigned by journal 26 Jun, 2025 Submission checks completed at journal 26 Jun, 2025 First submitted to journal 25 Jun, 2025 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. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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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-6970905","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":478432805,"identity":"e7b72ee2-24b0-47c6-81d2-bfb999119931","order_by":0,"name":"Zhaolin Li","email":"","orcid":"","institution":"Wuhan Institute of Virology","correspondingAuthor":false,"prefix":"","firstName":"Zhaolin","middleName":"","lastName":"Li","suffix":""},{"id":478432806,"identity":"4beeb6a1-f828-4920-ac0e-f2411d8479f9","order_by":1,"name":"Feng Tian","email":"","orcid":"","institution":"Xinjiang International Travel Health Care Center","correspondingAuthor":false,"prefix":"","firstName":"Feng","middleName":"","lastName":"Tian","suffix":""},{"id":478432808,"identity":"890a3aa0-d807-47e3-ad78-c9c908a8861e","order_by":2,"name":"Yi Huang","email":"","orcid":"","institution":"Westlake University","correspondingAuthor":false,"prefix":"","firstName":"Yi","middleName":"","lastName":"Huang","suffix":""},{"id":478432809,"identity":"f1244edb-817a-4061-a459-9b27bf1cba04","order_by":3,"name":"Doudou Huang","email":"","orcid":"","institution":"Wuhan Institute of Virology","correspondingAuthor":false,"prefix":"","firstName":"Doudou","middleName":"","lastName":"Huang","suffix":""},{"id":478432810,"identity":"6b1ac87c-fa3f-497b-a0e6-57c4243db73d","order_by":4,"name":"Fei Wang","email":"","orcid":"","institution":"Wuhan Institute of Virology","correspondingAuthor":false,"prefix":"","firstName":"Fei","middleName":"","lastName":"Wang","suffix":""},{"id":478432812,"identity":"43d17b1d-1812-4bb9-bd5e-0f270949e2f1","order_by":5,"name":"Zhiming Yuan","email":"","orcid":"","institution":"Wuhan Institute of Virology","correspondingAuthor":false,"prefix":"","firstName":"Zhiming","middleName":"","lastName":"Yuan","suffix":""},{"id":478432814,"identity":"538eb07b-4fbc-4331-a779-8d6fea9428d3","order_by":6,"name":"Xiang Sun","email":"","orcid":"","institution":"Xinjiang military command center for disease control and prevention","correspondingAuthor":false,"prefix":"","firstName":"Xiang","middleName":"","lastName":"Sun","suffix":""},{"id":478432816,"identity":"fcbaa861-c56e-486f-8e6d-5ecc42280d0b","order_by":7,"name":"Han Xia","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAsElEQVRIiWNgGAWjYBACAxCRwGADZDA2kKQljVQtDAyHYQwigDn72WMSD2rOJ25nYG58zMNgl0dQi2VPXrJBwrHbiTsbGJuNeRiSiwk77ECO4YMEttu5Gw4wtknOYDiQ2EBQy/k3BgcS/p0jRcsNoC2JbQfAWiQ+EKfljbFBYl9y/YbDjM0GHwySiXFYjpnkj292xgbH2x8+SKiwI6wFAZjBJhCvfhSMglEwCkYBHgAAiONAHJVFpA4AAAAASUVORK5CYII=","orcid":"","institution":"Wuhan Institute of Virology","correspondingAuthor":true,"prefix":"","firstName":"Han","middleName":"","lastName":"Xia","suffix":""}],"badges":[],"createdAt":"2025-06-25 05:53:17","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6970905/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6970905/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s12985-025-02961-x","type":"published","date":"2025-11-03T15:57:05+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":85776419,"identity":"7a96969c-ac24-4016-8b09-b46bd153bcac","added_by":"auto","created_at":"2025-07-01 14:30:13","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1328644,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCharacterized the CPE, plaque, and viral particle for DEZV/XJ23-420-01 and NEGLV/XJ-JH20-91-01 in C6/36 cells. (\u003c/strong\u003eA) CPE caused by DEZV and NEGLV in C6/36 cells at 2 days post infection (d.p.i). (B) Plaques of DEZV and NEGLV in C6/36 cells stained at 2 d.p.i. Cells were fixed with 10% formalin and stained with crystal violet. (C) Electron micrographs of DEZV and NEGLV viral particles visualized by PTA negative staining.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-6970905/v1/ee63309e6be96bcd3b1a9726.png"},{"id":85774583,"identity":"07136ae8-552e-41f5-a77c-8d30ff70215b","added_by":"auto","created_at":"2025-07-01 14:14:12","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":200053,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eReplication of DEZV/XJ23-420-01 and NEGLV/XJ-JH20-91-01 with different R/C in mosquito and vertebrate cell lines. \u003c/strong\u003e(A-C) The growth curves of DEZV in different cell lines; (D-F) The growth curves of NEGLV in different cell lines.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-6970905/v1/fac9cc5109910523fb8b4dfa.png"},{"id":85775493,"identity":"0454c170-50c4-46e4-ae97-8f02424146f0","added_by":"auto","created_at":"2025-07-01 14:22:13","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":61764,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGenome structure of DEZV/XJ-ALT23-420-01 and NEGLV/XJ-JH20-91-01.\u003c/strong\u003e (A) Genome structure of DEZV, with length of 9,066 nt ; (B) Genome structure of NEGLV (isolate XJ-JH20-91-01), with length of 9,531 nt. vMet: Vmethyltransf super family; AdoMet: AdoMet_MTases super family; FtsJ: FtsJ-like methyltransferase; RdRp: RNA-dependent RNA polymerase; DisA: DisA glycoprotein; DisB: DiSB-ORF2_chro, Putative virion glycoprotein of insect viruses; SP24: Putative virion membrane protein of plant and insect virus SP24, or structural protein of 24kD.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-6970905/v1/46134d6d5c67d75774f64ae9.png"},{"id":85774587,"identity":"19223eb6-62df-4afc-a665-5f032d010869","added_by":"auto","created_at":"2025-07-01 14:14:13","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":493518,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePhylogenetic tree for DEZV/XJ-ALT23-420-01) and NEGLV/XJ-JH20-91-01) based on RdRp coding region using the maximum likelihood method\u003c/strong\u003e. Red-highlighted entries in the figure represent the two virus isolates in this study.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-6970905/v1/46e1c5d4220c6079df96815b.png"},{"id":85774590,"identity":"8ebb8032-01e6-4cb2-9b93-1eb549edec85","added_by":"auto","created_at":"2025-07-01 14:14:13","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":628826,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGeographical distribution and host diversity of Negevirus group.\u003c/strong\u003e Based on publicly available sequence data from NCBI Virus (current as of 17 January 2025).\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-6970905/v1/0efeeafa31b21a1b712b663b.png"},{"id":95564158,"identity":"d27ab04c-b0c0-4e30-b148-d092fd717140","added_by":"auto","created_at":"2025-11-10 16:08:31","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3690350,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6970905/v1/915ee188-9495-4b6f-b10a-30ddfe6bcbc4.pdf"},{"id":85774582,"identity":"5cd9aa58-9215-42ee-a10f-04f44f803bab","added_by":"auto","created_at":"2025-07-01 14:14:12","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":23692,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarymaterials.docx","url":"https://assets-eu.researchsquare.com/files/rs-6970905/v1/3f7853af4920739cb94855e9.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Characterization of two novel Negevirus strains and analysis of global Negevirus distribution and host diversity","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eThere are two major groups of viruses that circulated in mosquito: mosquito-borne viruses (MBV) and mosquito-specific viruses (MSV) (Moonen et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). MBV, a subset of arbovirus, can infect both mosquito and vertebrate, whereas MSV belong to the insect-specific virus (ISV) group and replicate exclusively in mosquito (Stollar and Thomas, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e1975\u003c/span\u003e). The first discovered ISV was Cell-fusing agent virus (CFAV), which was identified by Stollar and colleagues in 1975 from \u003cem\u003eAedes aegypti\u003c/em\u003e cell lines. With advances in high-throughput sequencing and the implementation of mosquito virome surveys, an increasing number of ISVs have since been discovered. ISVs are widely distributed across diverse viral taxonomic groups, such as \u003cem\u003eFlaviviridae\u003c/em\u003e (Anakha et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Espinoza-G\u0026oacute;mez et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Roiz et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2012\u003c/span\u003e), \u003cem\u003eBunyaviridae\u003c/em\u003e (Auguste et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2014\u003c/span\u003e), \u003cem\u003eTogaviridae\u003c/em\u003e (Bennouna et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), \u003cem\u003eMesoniviridae\u003c/em\u003e (Lauber et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Thuy et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2013\u003c/span\u003e), \u003cem\u003eReoviridae\u003c/em\u003e (Hermanns et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Langat et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), \u003cem\u003eRhabdoviridae\u003c/em\u003e (Vasilakis et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2014\u003c/span\u003e), and new taxons such as \u003cem\u003eNegevirus\u003c/em\u003e (Bishop et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; da Silva Ribeiro et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Ribeiro et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Vasilakis et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Zhao et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eResearchs have demonstrated that the presence of MSVs can influence the replication and proliferation of MBVs. For example, Nhumirim virus (NHUV), isolated from mosquitoes in Brazil, significantly suppresses the amplification of West Nile virus (WNV), Japanese encephalitis virus (JEV), and St. Louis encephalitis virus (SLEV) when co-infecting C6/36 cells. The inhibitory effect was most pronounced against WNV and SLEV, with peak viral titers reduced by 10\u003csup\u003e6\u003c/sup\u003e PFU/mL and 10\u003csup\u003e4\u003c/sup\u003e PFU/mL, respectively (Kenney et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). These findings suggest that studying MSV-MBV interactions may offer novel strategies for controlling spreading of MBV. However, the mechanisms by which MSVs modulate MBV replication, dissemination, and transmission remain unclear.\u003c/p\u003e \u003cp\u003eNegevirus is a new taxon group of non-segmented, single-stranded positive-sense RNA viruses with wide geographic distribution. The genome size for Negevirus is around 9\u0026ndash;10 kb, containing three open reading frames (ORFs), flanked by 5\u0026prime; and 3\u0026prime; untranslated regions (UTRs), and terminates in a 3\u0026prime; poly (A) tail. The virus particles are ellipsoidal or spherical in shape, with a diameter of 45\u0026ndash;55 nm (Vasilakis et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). The genus \u003cem\u003eNegevirus\u003c/em\u003e has been classified into two distinct clades: \u003cem\u003eNelorpivirus\u003c/em\u003e and \u003cem\u003eSandewavirus\u003c/em\u003e (Kallies et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2014\u003c/span\u003e) Based on genomic organization and phylogenetic analysis, which are distantly related to some plant viruses, such as \u003cem\u003eCileviruses\u003c/em\u003e, \u003cem\u003eHigrevirus\u003c/em\u003e and \u003cem\u003eBlunervirus\u003c/em\u003e. While primarily detected in mosquitoes, they have also been identified in sandflies, bees, and other arthropods.\u003c/p\u003e \u003cp\u003eIn this study, during mosquito and virus surveillance conducted in Xinjiang, China, we isolated one strain of Dezidougou virus (DEZV) from \u003cem\u003eAedes vexans\u003c/em\u003e and one strain of Negev-like virus (NEGLV) from \u003cem\u003eCulex pipiens\u003c/em\u003e. The genome sequencing, viral purification, growth characteristics, and phylogenetic relationships of these two Negevirus strains were analyzed. Additionally, the global distribution patterns and host diversity of all known Negevirus members were investigated.\u003c/p\u003e"},{"header":"2. Method","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Cells\u003c/h2\u003e \u003cp\u003eThe C6/36 and Aag2 (mosquito cell lines), VeroE6, BHK-21, SW13, and PK15 (vertebrate cell lines) were used in this study.\u003c/p\u003e \u003cp\u003eC6/36 and Aag2 cells were maintained in CO₂ incubator at 28\u0026deg;C in Roswell Park Memorial Institute (RPMI) 1640 medium containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (PS). BHK-21, VeroE6, SW13, and PK15 cells were cultured in CO₂ incubator at 37\u0026deg;C in Dulbecco\u0026rsquo;s minimal essential medium (DMEM) containing 10% FBS and 1% penicillin-streptomycin.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Mosquito collection and preparation\u003c/h2\u003e \u003cp\u003eFrom June 2020 to July 2023, mosquito collections were conducted in Xinjiang Uygur Autonomous Region, China, focusing on the Ebinur Lake and Takeshiken Port area. Mosquitoes were captured using CO₂-baited traps and subsequently morphology identification was conducted.\u003c/p\u003e \u003cp\u003eApproximately 50\u0026ndash;60 mosquitoes were pooled per sample and homogenized in RPMI 1640 medium supplemented with 2% penicillin-streptomycin. The homogenate was centrifuged at 4\u0026deg;C and 15,000 g for 30 min, then the debris in the bottom was discarded and centrifuged again at 15,000 g for 10 min to get the supernatant.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Virus isolation and purification\u003c/h2\u003e \u003cp\u003eCells were seeded into 24-well plates (C6/36: 2\u0026times;10\u003csup\u003e5\u003c/sup\u003e cells/well; VeroE6 and BHK-21: 1.6\u0026times;10\u003csup\u003e5\u003c/sup\u003e cells/well). The supernatant acquired in section \u003cspan refid=\"Sec4\" class=\"InternalRef\"\u003e2.2\u003c/span\u003e was used to inoculate cell monolayers in 24-well plates. After a 1-hour incubation at 28\u0026deg;C (C6/36) or 37\u0026deg;C (VeroE6 and BHK-21), the inoculum was removed and replaced with maintenance medium (RPMI 1640 for C6/36 and DMEM for mammalian cells) containing 2% FBS. Cells were observed daily for 7 days to examine cytopathic effect (CPE), with all procedures replicated three times.\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eTo acquire pure virus stocks, the supernatant in the well showing CPE was harvested, diluted, and subjected to plaque purification on C6/36 cells. Virus-specific PCR primers (STable 1) were designed for viral identification.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Virus sequencing\u003c/h2\u003e \u003cp\u003eSupernatant from infected cells were harvested for viral RNA extraction using an automated nucleic acid extraction system (NanoMagBio, S-48 and NMG0966-16). RNA sequencing libraries were developed by Institutional Center for Shared Technologies and Facilities of Wuhan Institute of Virology, CAS. Sequencing was performed at Center for Instrumental Analysis and Metrology of Wuhan Institute of Virology, CAS, using the Illumina (Illumina Novaseq) platform. The resulting sequences were assembled \u003cem\u003ede novo\u003c/em\u003e using Trinity v2.15.2 (Grabherr et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2011\u003c/span\u003e) and subjected to sequence comparison by Blast-n.\u003c/p\u003e \u003cp\u003eOverlapping primers (STable 2 and STable 3) were designed according to the assembled contigs using Primalscheme (Quick et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2017\u003c/span\u003e) (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://primalscheme.com/\u003c/span\u003e\u003cspan address=\"https://primalscheme.com/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Purified viral RNA was reverse transcribed and amplified by PCR, followed by purification of PCR products using the E.Z.N.A.\u0026reg; Gel Extraction Kit (Omega Bio-Tek, D2500-02). Missing terminal sequences were obtained using the HiScript-TS 5'/3' RACE Kit (Vazyme, RA101) according to the manufacturer's protocol. The complete viral genome was obtained through sequence splicing and assembly using DNASTAR Lasergene SeqMan Pro v7.1.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Viral morphological characteristics\u003c/h2\u003e \u003cp\u003eThe virus was amplified using C6/36 cell lines. After 2 days of cultivation, the supernatant from samples exhibiting CPE was collected and purified by sucrose density gradient centrifugation (Beckman Coulter SW32, 4\u0026deg;C, 32,000 rpm for 3 hours). The supernatant was then discarded, and the precipitates were resuspended in 400 \u0026micro;L phosphate-buffered saline (PBS). Prior to ultracentrifugation, a sucrose gradient solution was prepared, and the viral supernatant was carefully layered onto it.\u003c/p\u003e \u003cp\u003eUltracentrifugation was performed at 4\u0026deg;C for 4 hours using a Beckman MLS50 rotor at 45,000 rpm. After centrifugation, the position of the viral band in the centrifuge tube was observed and recorded. A 1 mL syringe was then inserted into the band to aspirate the target viral band. Excess sucrose was removed using an Amicon Ultra-0.5 Centrifugal Filter Unit (50 kDa), following the manufacturer\u0026rsquo;s protocol, and this step was repeated five times.\u003c/p\u003e \u003cp\u003eThe purified viruses were loaded onto Formvar carbon-coated copper grid and negatively stained with 1% uranyl acetate. Morphological analysis was conducted using a Thermo Scientific Talos L120C transmission electron microscope operated at 120 kV.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Plaque and TCID50 assay\u003c/h2\u003e \u003cp\u003eVirus titration was performed as described by Vasilakis\u0026rsquo; team (Vasilakis et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Viral progeny formed plaques on C6/36 cell monolayers in 24-well plates. The viral supernatant was serially diluted 10-fold in culture medium. For each well, 100 \u0026micro;L of virus dilution in different fold was added, and the plates were incubated at 28\u0026deg;C for 1 hour, gently rocked every 15 minutes. Afterward, the supernatant was discarded and replaced with 100 \u0026micro;L of a medium consisting of a 1:1 mixture of 2% tragacanth suspension and 2 \u0026times; RPMI with 5% FBS, 2% tryptose phosphate broth (TPB), and 2% penicillin-streptomycin. The cells were incubated at 28\u0026deg;C for 2 days to allow plaque formation. After incubation, the overlay was discarded, and the cells were fixed with 10% formaldehyde for 1 hour. Staining was done overnight at room temperature with 2% crystal violet and plaques were counted and recorded after removing excess stain under running water.\u003c/p\u003e \u003cp\u003eViral titers were additionally quantified using the 50% tissue culture infectious dose (TCID50) endpoint dilution method. A monolayer of C6/36 cells was spread in a 96-well plate. The viral supernatant was serially diluted 10-fold from 1\u0026times;10⁻\u0026sup1; to 1\u0026times;10⁻\u0026sup1;\u0026sup1;, repeated eight wells per gradient. For each column, 100 \u0026micro;l of the viral supernatant was added and incubated for 1 hour. The plate was gently shaken every 15 min, then the supernatant was discarded, and the plate was replaced with 200 \u0026micro;L RPMI (2% FBS and 2% PS) medium for incubation at 28\u0026deg;C. The cell growth status was monitored daily and quantified for each well.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7 Virus grow kinetics\u003c/h2\u003e \u003cp\u003eThe growth kinetics of DEZV/XJ-ALT23-420-01 and NEGLV/XJ-JH20-91-01 were determined in two mosquito cell lines: C6/36 (\u003cem\u003eAedes albopictus\u003c/em\u003e) and Aag2 (\u003cem\u003eAedes aegypti\u003c/em\u003e), and four vertebrate cell lines: VeroE6 (monkey), BHK-21 (baby hamster), SW13 (human), and PK-15 (pig).\u003c/p\u003e \u003cp\u003eBriefly, cells were seeded into T-25 flasks (10\u003csup\u003e5\u0026ndash;6\u003c/sup\u003e cells/per flask). After cultivation for 24h, the cells were incubated with virus at a ratio of viral RNA copies to cell number (R/C) from 0.001 to 1000. After incubation for 1 h, the inoculation was removed, washed three times with PBS, and replaced by fresh RPMI-1640 medium containing 2% FBS. Supernatant from the infected cell cultures were collected daily and stored at 80 \u0026ordm;C for viral RNA detection. The RNA template for qRT-PCR standard curve of NEGV and DEZV were produced by Takara In Vitro Transcription T7 kit and the designed primers (STable 4).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8 Phylogenetic analysis, geographical distribution, and host diversity of Negevirus\u003c/h2\u003e \u003cp\u003eFirst, RNA-dependent RNA polymerase (RdRp) sequences from both isolates were predicted using NCBI's Conserved Domain Search (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi\u003c/span\u003e\u003cspan address=\"https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Subsequently, a maximum likelihood phylogenetic tree was constructed using the complete RdRp coding regions of: (1) selected Negevirus isolates, and (2) representative members of two phylogenetically related plant virus families (\u003cem\u003eKitaviridae\u003c/em\u003e and \u003cem\u003eVirgaviridae\u003c/em\u003e).\u003c/p\u003e \u003cp\u003ePhylogenetic analysis was performed in PhyloSuite v1.2.3 (Zhang et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) with 1,000 standard bootstrap replicates. Trees were visualized and edited using Normal tree visualization software (Xie et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAll publicly available Negevirus related sequences (as of January 17, 2025) were retrieved from NCBI Virus, with associated host and geographical metadata. Sankey diagrams for distribution and host diversity analyses were generated using Origin 2024b.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\n \u003ch2\u003e3.1 Virus isolation and morphology\u003c/h2\u003e\n \u003cp\u003eA total of three C6/36 wells inoculated with \u003cem\u003eAedes vexans\u003c/em\u003e homogenate tested positive for DEZV, while thirteen wells inoculated with \u003cem\u003eCulex pipiens\u003c/em\u003e sample tested positive for NEGLV. Subsequently, one DEZV strain (XJ23-420-01) and one NEGLV strain (XJ-JH20-91-01) were successfully isolated. Both DEZV/XJ23-420-01) and NEGLV/XJ-JH20-91-01) induced prominent CPE in C6/36 cells at 2 days post-inoculation (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eA).\u003c/p\u003e\n \u003cp\u003eIn NEGLV infected C6/36 monolayers, plaques displayed distinct edges and were readily quantifiable. In contrast, DEZV infected cells produced heterogeneous plaques with fuzzy, indistinct boundaries (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eB), making plaque-based titer determination unreliable. Therefore, titer for DEZV was quantified using the TCID assay. In C6/36 cells, the titer of DEZV can reach 10\u003csup\u003e7.9\u003c/sup\u003e TCID\u003csub\u003e50\u003c/sub\u003e/mL (equal to 1.4\u0026times; 10\u003csup\u003e7\u003c/sup\u003e PFU/mL) (STable 5) (Spearman, 1908; K\u0026auml;rber, 1931) (Lei et al., \u003cspan class=\"CitationRef\"\u003e2021\u003c/span\u003e), and NEGLV was 10\u003csup\u003e6\u003c/sup\u003e PFU/mL.\u003c/p\u003e\n \u003cp\u003eIn addition, electron microscopy revealed that both purified DEZV and NEGLV particles were exhibited elliptical shape (40\u0026ndash;60 nm in diameter), with envelope (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eC).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\n \u003ch2\u003e3.2 Viral replication in mosquito and vertebrate cell lines\u003c/h2\u003e\n \u003cp\u003eIn both C6/36 and Aag2 cells, DEZV/XJ23-420-01 and NEGLV/XJ-JH20-91-01 infection induced extensive CPE within 2 d.p.i whether low or high doses (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eA, \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eB, \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eD, and \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eE). Viral RNA copies in infected C6/36 cells increased rapidly by 1 d.p.i, reaching concentrations\u0026thinsp;\u0026ge;\u0026thinsp;10\u003csup\u003e8\u003c/sup\u003e copies/mL. In contrast, no significant CPE was observed in any vertebrate cell lines tested (Vero E6, SW13, BHK-21, PK-15) derived from human, monkey, hamster or pig, even at high infection doses (MOI\u0026thinsp;=\u0026thinsp;10) (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eC and \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eF).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\n \u003ch2\u003e3.3 Viral sequencing and genome structure\u003c/h2\u003e\n \u003cp\u003eThe complete genomes of both viruses were obtained through next-generation sequencing (NGS) and rapid amplification of cDNA ends (RACE), and the sequences were deposited in GenBank under accession numbers PP908777 (DEZV/XJ-ALT23-420-01) and PQ493505 (NEGLV/XJ-JH20-91-01). The genome structure was predicted through NCBI Conserved Region Prediction Tool (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e\n \u003cp\u003eDEZV/XJ-ALT23-420-01 possesses a 9,066 nt single-stranded, positive-sense RNA genome with a poly(A) tail. BLASTn analysis revealed 98.96% nucleotide identity to DEZV strain 8345 (from Germany), with high similarity. Its genome contains three open reading frames (ORFs): (1) ORF1 (nt 70-6810) encodes: FtsJ-like methyltransferase domain (nt positions 787\u0026ndash;994), viral RNA helicase domain (nt 1316\u0026ndash;1612), and RdRp domain (nt 1921\u0026ndash;2202); (2) ORF2 (nt 6842\u0026ndash;8125) contains DisA glycoprotein domain (nt 6989\u0026ndash;8116); (3) ORF3 (nt 8233\u0026ndash;8829) contains SP24 domain (nt 8353\u0026ndash;8772) (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eA). NEGLV/XJ-JH20-91-01 possesses a 9,531 nt single-stranded, positive-sense RNA genome with a poly(A) tail. BLASTn analysis revealed only 79.39% nucleotide identity to Negev virus strain BeAr805514 (from Brazilian Amazon), establishing it as a novel strain. Genomic annotation identified three open reading frames (ORFs): (1) ORF1 (nt 233\u0026ndash;7339) encodes: viral methyltransferase (vMet; nt 187\u0026ndash;539), AdoMet-dependent MTase superfamily domain (AdoMet; aa 914\u0026ndash;1101), viral RNA helicase (VRhel; nt 1471\u0026ndash;1733), and RdRp (nt 2113\u0026ndash;2379); (2) ORF2 (nt 7373\u0026ndash;8575) contains DisB glycoprotein domain (nt 7553\u0026ndash;7744); (3) ORF3 (nt 8624\u0026ndash;9250) contains SP24 domain (nt 8774\u0026ndash;9190) (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eB).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\n \u003ch2\u003e3.4 Phylogenetic analysis\u003c/h2\u003e\n \u003cp\u003eA maximum likelihood phylogenetic tree was constructed using the RdRp coding regions of representative members from \u003cem\u003eNegevirus\u003c/em\u003e, \u003cem\u003eVirgaviridae\u003c/em\u003e, and \u003cem\u003eKitaviridae\u003c/em\u003e, with branch support assessed by 1,000 bootstrap replicates (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e). The analysis revealed two distinct subgroup of \u003cem\u003eNegevirus\u003c/em\u003e, \u003cem\u003eSandewavirus\u003c/em\u003e and \u003cem\u003eNelorpivirus\u003c/em\u003e.\u003c/p\u003e\n \u003cp\u003eDEZVs belong to \u003cem\u003eSandewavirus\u003c/em\u003e subgroup and formed into two clusters: our DEZV/XJ-ALT23-420-01 and German strain 8345 (WGH58589.1) in one cluster, and FIN/PP-2018/82 (UUV42173.1), Yakutsk_2023 (XCM48930.1), FTA2-3 (QIN93579.1), and the prototype strain (ARQ15945.1) in the second cluster. NEGVs and NEGLVs belong to \u003cem\u003eNelorpivirus\u003c/em\u003e subgroup, and NEGLV/XJ-JH20-91-01 formed a separate branch, distinguishing from other NEGV or NEGLV strains.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\n \u003ch2\u003e3.5 Geographical distribution and host diversity of Negevirus\u003c/h2\u003e\n \u003cp\u003eAnalysis of host diversity and geographic distribution of \u003cem\u003eNegavirus\u003c/em\u003e group revealed global occurrence across all continents except Antarctica. Most sequences (136/168; 81%) originated from mosquitoes, with \u003cem\u003eCulex\u003c/em\u003e species representing the highest proportion (68/168; 40.5%). Negevirus members were also detected in other invertebrates including bees, aphids, and flies, and notably in flatworms (Platyhelminthes). Piura virus was the most prevalent strain (45/168; 26.8%), detected in multiple mosquito vectors including \u003cem\u003eCulex\u003c/em\u003e and \u003cem\u003eAnopheles\u003c/em\u003e species (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e\n\u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003e \u003cem\u003eNegevirus\u003c/em\u003e comprises two subgroups: \u003cem\u003eSandewavirus\u003c/em\u003e and \u003cem\u003eNelorpivirus\u003c/em\u003e. DEZV, classified within \u003cem\u003eSandewavirus\u003c/em\u003e, has been reported in Africa (Shrivastava et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2017a\u003c/span\u003e), Asia (Stepanyuk et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2025\u003c/span\u003e), and Europe (Agboli et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). In contrast, NEGV/NEGLV belongs to \u003cem\u003eNelorpivirus\u003c/em\u003e and has been detected in North America (Vasilakis et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2013\u003c/span\u003e), South America (da Silva Ribeiro et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), Africa, and Europe (Carapeta et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2015\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eIn this study, we report the first isolation of DEZV and NEGLV from mosquitoes in Xinjiang, China. Phylogenetic analysis revealed that DEZV/XJ-ALT23-420-01 clusters with German strain 8345 (98.96% nucleotide identity), while NEGLV/XJ-JH20-91-01 forms a distinct branch with only 79.39% identity to known Negevirus strains, suggesting it represents a novel species. Phenotypically, plaque morphology differed significantly: DEZV produced fuzzy, irregular plaques whereas NEGLV formed distinct plaques with well-defined edges. This observation aligns with our prior report that Tanay virus (same subgroup as DEZV) similarly exhibits fuzzy plaques (Zhao et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eGenome of both DEZV/XJ-ALT23-420-01 and NEGLV/XJ-JH20-91-01 contain an SP24 element in the ORF3, which is a putative membrane protein of plant or insect viruses. There are studies revealed the potential evolutionary relationship of \u003cem\u003eNegevirus\u003c/em\u003e with two plant virus families (\u003cem\u003eKitaviridae\u003c/em\u003e and \u003cem\u003eVirgaviridae\u003c/em\u003e) based on SP24 and CP proteins, suggesting that the plant viruses may have originated from the horizontal transmission of ancient Negevirus viruses that occurred (Lu et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Negevirus and the mentioned plant viruses may share a common ancestor capable of infecting both insects and plants. In future, identify the host range of Negevirus can help us to answer this evolutionary question.\u003c/p\u003e\u003cp\u003eDEZV/XJ-ALT23-420-01 and NEGLV/XJ-JH20-91-01 demonstrate vertebrate cell-restricted tropism, showing no replication in tested mammalian cell lines. In contrast, both achieve a high viral loads (\u0026gt; 10⁹ copies/mL) in mosquito cells. Field studies confirm that DEZV naturally persists in multiple mosquito species and can coexist with medically important arbovirus such as Zika virus (ZIKV). Futermore, in vitro the co-infection experiment with Bunyamwera virus (BUNV), and Semliki Forest virus (SFV) showed that DEAV had minimal impact on BUNV or SFV replication (Shrivastava et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2017b\u003c/span\u003e; Agboli et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Next studies should investigate DEZV or NEGLV co-infection with additional arbovirus, both in cell lines and mosquitoes, to explore their potential impacts on arbovirus transmission.\u003c/p\u003e\u003cp\u003eIn summary, we isolated two new Negevirus strains, DEZV/XJ-ALT23-420-01 and NEGLV/XJ-JH20-91-01 and further investigated the global Negevirus distribution and host range. These findings expand our knowledge of this viral taxon.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSequence data that support the findings of this study have been deposited in the Genbase with the primary accession number PP908777 and PQ493505. The rest data and materials are provided within the manuscript or supplementary information files.\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\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the National Key Research and Development Program of China (2022YFC2302700), the Youth Program of Wuhan Institute of Virology (2023QNTJ-03).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eZY and HX conceived the idea and coordinated the project. ZL, FT, FW, and XS performed the field work. ZL, YH, and DH performed experiments. ZL, and FT analyzed data. FT, XS, ZY, and HX provided resources. ZL, FW, and HX drew the figures and drafted the manuscript. ZL, FT, XS, ZY and HX revised the manuscript. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors would like to thank the technical support from Xinjiang International Travel Health Care Center for the assistance in the field work, and the staff of the Institutional Center for Shared Technologies and Facilities of Wuhan Institute of Virology, CAS.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAgboli E, Schulze J, Jansen S, Cadar D, Sreenu VB, Leggewie M, Altinli M, Badusche M, J\u0026ouml;st H, B\u0026ouml;rstler J, Schmidt-Chanasit J, Schnettler E. Interaction of Mesonivirus and Negevirus with arboviruses and the RNAi response in Culex tarsalis-derived cells. 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Front Microbiol 10, 1963. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3389/fmicb.2019.01963\u003c/span\u003e\u003cspan address=\"10.3389/fmicb.2019.01963\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\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":"[email protected]","identity":"virology-journal","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"virj","sideBox":"Learn more about [Virology Journal](http://virologyj.biomedcentral.com/)","snPcode":"12985","submissionUrl":"https://submission.nature.com/new-submission/12985/3","title":"Virology Journal","twitterHandle":"@VirologyJ","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Dezidougou virus, Negev-like virus, Negevirus, insect specific virus, Aedes vexans, Culex pipiens, Xinjiang","lastPublishedDoi":"10.21203/rs.3.rs-6970905/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6970905/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eMosquitoes and mosquito-borne viruses pose a significant global public health concern. Furthermore, mosquitoes carry a wide range of insect-specific viruses (ISVs), and studying these ISVs is important due to their potential influence on mosquito behavior and the transmission of mosquito-borne viruses. In this study, we report the first isolation of two Negevirus strains from \u003cem\u003eAedes\u003c/em\u003e and \u003cem\u003eCulex\u003c/em\u003e mosquitoes in Xinjiang, China: Dezidougou virus (DEZV, isolate XJ-ALT23-420-01) and Negev-like virus (NEGLV, isolate XJ-JH20-91-01). Phylogenetically, the nucleotide sequence of DEZV exhibits high similarity (98.96%) with the DEZV isolate 8345 from Germany but shows lower similarity (\u0026lt;\u0026thinsp;91.91%) with other DEZV strains. In contrast, the NEGLV isolate XJ-JH20-91-01 displays significant divergence from other Nege virus (NEGV) or NEGLV, sharing only 79.39% nucleotide similarity with the most closely related strain, NEGV BeAr805514. Both DEZV/XJ-ALT23-420-01 and NEGLV/XJ-JH20-91-01 replicated rapidly in mosquito cell lines (C6/36 and Aag2), reaching viral loads of up to 10\u003csup\u003e9\u0026ndash;10\u003c/sup\u003e copies/mL, causing significant cytopathic effect (CPE) in these cell lines but failing to replicate in vertebrate cells. In addition, analysis of the location and potential hosts of all published Negevirus members indicated their wide distribution and diverse host range. These findings extend the current spectrum of Negevirus group and provide deeper insights into its geographical distribution and host diversity.\u003c/p\u003e","manuscriptTitle":"Characterization of two novel Negevirus strains and analysis of global Negevirus distribution and host diversity","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-01 14:14:08","doi":"10.21203/rs.3.rs-6970905/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-07-22T02:01:04+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-07-20T19:36:16+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"171585898577570245512422768477243652395","date":"2025-07-03T07:56:46+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-06-29T03:41:22+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"29844609647583667745269857041481402370","date":"2025-06-29T02:31:19+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-06-27T02:09:22+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-06-26T08:21:30+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-06-26T07:28:38+00:00","index":"","fulltext":""},{"type":"submitted","content":"Virology Journal","date":"2025-06-25T05:46:11+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"virology-journal","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"virj","sideBox":"Learn more about [Virology Journal](http://virologyj.biomedcentral.com/)","snPcode":"12985","submissionUrl":"https://submission.nature.com/new-submission/12985/3","title":"Virology Journal","twitterHandle":"@VirologyJ","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"a3df48a0-4ab1-46d7-bea8-e4430fed4b81","owner":[],"postedDate":"July 1st, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-11-10T16:04:24+00:00","versionOfRecord":{"articleIdentity":"rs-6970905","link":"https://doi.org/10.1186/s12985-025-02961-x","journal":{"identity":"virology-journal","isVorOnly":false,"title":"Virology Journal"},"publishedOn":"2025-11-03 15:57:05","publishedOnDateReadable":"November 3rd, 2025"},"versionCreatedAt":"2025-07-01 14:14:08","video":"","vorDoi":"10.1186/s12985-025-02961-x","vorDoiUrl":"https://doi.org/10.1186/s12985-025-02961-x","workflowStages":[]},"version":"v1","identity":"rs-6970905","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6970905","identity":"rs-6970905","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}