Construction of an infectious full‑length and eGFP-tagged cDNA clone of a chilli ringspot virus isolate from Yunnan province, China

Construction of an infectious full‑length and eGFP-tagged cDNA clone of a chilli ringspot virus isolate from Yunnan province, China | 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 Research Article Construction of an infectious full‑length and eGFP-tagged cDNA clone of a chilli ringspot virus isolate from Yunnan province, China Mengying Hua, Shanshan Jiang, Enping Yuan, Qionglian Wan, Liyan Wang, and 7 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-1255906/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 5 You are reading this latest preprint version Abstract Chilli ringspot virus (ChiRSV; genus Potyvirus ) was one of several viruses previously detected in pepper samples with severe yellowing and curling symptoms growing in Wenshan, Yunan province, China. We now report the full-length sequence of ChiRSV-YN/Wenshan (MZ269480) which has 88.5-98.9%. nucleotide identity to other published ChiRSV isolates. A full-length cDNA infectious clone was constructed. This cDNA and an eGFP-tagged clone were infectious leading to systemic symptoms in both Nicotiana benthamiana and Capsicum spp. Single infection by ChiRSV caused mild mosaic or leaf crinkling in Capsicum frutescens L. and Capsicum annuum L. chilli ringspot virus potyvirus infectious clone Figures Figure 1 Figure 2 Figure 3 Main text Chilli ringspot virus (ChiRSV) was first reported in Vietnam in 2007 [6] and was classified as a distinct species in the genus Potyvirus in 2009 [3]. In 2011, the determination of the genomic sequence of ChiRSV-HN/14 isolate (from Hainan, China) provided further evidence that ChiRSV is a distinctive potyvirus [5]. The ChiRSV genome has 9,571 nucleotides (nt) of ssRNA and the virus has typical filamentous particles 780 nm in length [5, 8]. Recently, a further isolate of ChiRSV was reported from Yunnan province [8] which groups phylogenetically with the Hainan isolate. It has a broad host range among several economically important crops including chilli, tomato, tobacco, and cucurbitaceous vegetables [8, 11]. In a previous study using Next-generation RNA-Seq sequencing (NGS) and RT-PCR we showed that ChiRSV, pepper vein yellows virus, chilli veinal mottle virus, tomato zonate spot virus and cucumber mosaic virus were prevalent in samples of pepper ( Capsicum frutescens L.) plants from Wenshan city Yunnan province, China which had severe yellowing and curling symptoms [12]. ChiRSV was detected by RT-PCR in 31 of the 89 symptomatic samples. In this study we first obtained the complete genomic sequence of ChiRSV from Wenshan city. Total RNA was isolated using RTIzol TM Reagent (Invitrogen) and first strand cDNA was synthesized using ReverTra Ace-α-®(Toyobo) following the manufacturer’s protocol. Reverse transcription (RT) was performed at 42°C for 60 min with M4T primers (Supplementary Table S1) followed by 72°C for 10 min using KOD-plus-Neo (Toyobo) as specified by the manufacturer. The subsequent PCR used primers DP-ChiRSV f/DP-ChiRSV r (Supplementary Table S1) and incubation at 98°C for 3min, followed by 35 cycles of 98°C for 30s, 55°C for 30s, 68°C for 1kb/min and a final incubation at 68°C for 10min. 5’ and 3’ RACE reactions were performed to obtain the complete 5’and 3’ terminal sequences and three overlapping sections were amplified to verify the full-length sequence as described before [12]. The genome of ChiRSV-YN/Wenshan was 9653 nt long (Supplementary Figure S1A) and the sequence was deposited in GenBank with accession number: MZ269480. ChiRSV-YN/Wenshan has 88.5-98.9% nucleotide identity to the other reported full-length genome sequence of ChiRSV (Supplementary Table S2) and with 93.0-99.3% amino acid identity in comparisons of their polyproteins. In comparisons among the predicted mature proteins, only the P1 protein was less than 80% identical (Supplementary Table S2). In a Maximum Likelihood (ML) phylogenetic analysis, ChiRSV-YN/Wenshan clustered with other published ChiRSV isolates in a group distinct from other potyviruses (Supplementary Fig. S2). To examine the biological characteristics of ChiRSV-YN/Wenshan and to provide a tool for future investigations of mixed virus infection in pepper plant, a full-length infectious clone (pChiRSV) was prepared. The recombination cloning method was used to insert the complete ChiRSV cDNA sequence between the duplicated 35S promoter and hepatitis delta virus ribozyme (HDV-Rz) as described previously [12]. Then, pChiRSV was transformed into Agrobacterium tumefaciens which was then delivered to Nicotiana benthamiana plantlets by infiltration. Symptoms of witches’ broom and systemic mosaic were observed in systemic leaves at 6 dpi, and infected plants were significantly smaller at 10 dpi (Fig. 1A and Supplementary Table S4). RT-PCR and Western blot detected viral RNA and coat protein in the new non-inoculated leaves confirming that systemic infection had been established (Fig. 1A and 1B). Typical flexuous filamentous virions approximately 780 nm long were observed by transmission electron microscopy (TEM) in negatively-stained samples of the upper leaves (Fig. 1C). To investigate the symptoms of ChiRSV on peppers, seedlings of Capsicum frutescens L. and Capsicum annuum L. were mechanically inoculated using sap from infected N. benthamiana plants. At 15 dpi (day post inoculation), inoculated plants had mild mosaic ( C. annuum ) or leaf crinkling ( C. frutescens ) (Figure 2A and Supplementary Table S4), RT-PCR and Western blot confirmed that virus had spread systemically in the inoculated plants (Fig. 2B) and flexuous filamentous virions were observed in new non-inoculated leaves by TEM (Fig. 2C). These results show that the full-length cDNA clone of ChiRSV-YN/Wenshan could successfully infect both N. benthamiana and pepper plants. To trace and observe infection by ChiRSV-YN/Wenshan more conveniently, an eGFP-tagged cDNA infectious clone was then constructed. The sequence encoding a NIa protease cleavage site (TTVYHQ/A) was introduced between the eGFP and CP coding sequences (Supplementary Fig. S1C). The infectious clone pChiRSV-GFP was transformed into Agrobacterium tumefaciens which was then delivered to N. benthamiana plantlets by infiltration. At 12 dpi, examination under a UV lamp showed eGFP fluorescence in the upper non-inoculated leaves of most plants (Fig. 3A; Supplementary Table S4). The symptoms on these leaves were similar to those produced by the untagged cDNA clone. Western blots showed that the coat protein and GFP could be detected in the new non-inoculated leaves (Fig. 3B) and typical flexuous filamentous virions were also observed in them by TEM (Fig. 3C). In this study of ChiRSV-YN/Wenshan, only the P1 protein was significantly different to the mature proteins of other ChiRSV isolates. P1 is a multifunctional protein that participates in RNA binding, is associated with inclusion bodies, and plays roles in cell-to-cell and systemic movement [1, 2, 4, 9, 10]. The full-length cDNA clone of pChiRSV constructed in this study will provide a useful tool to investigate whether the diversity of P1 sequences among the isolates indicates that they have diverse roles. The ChiRSV-YN/Wenshan isolate was obtained in 2019 from plants with severe symptoms of PeVYD (pepper vein yellows disease). This disease is associated with co-infection by several viruses but especially the phloem-limited PoPeVYV (pod pepper vein yellows virus) together with PoPeVYVaRNA (pod pepper vein yellows virus-associated RNA) [7, 12]. Our infectious clone will also be a useful tool for examining whether ChiRSV can assist infection by PoPeVYV in the absence of PoPeVYVaRNA. Declarations Acknowledgments This work was supported by Chinese Agriculture Research System of MOF and MARA (CARS-24-C-04). This work also was financially supported by National Key R&D Program of China (2019YFD1001800) and K. C.Wong education foundation. We thank Professor Mike Adams for manuscript correction. Data availability statement The data that support the findings of this study are available from the corresponding author upon reasonable request. Ethics approval and consent to participate Not applicable. Consent for publication Not applicable. Competing interests The authors declare that they have no conflict of interest. Authors' contributions MH, SJ, SW and JP conceived and designed the experiments. EY, HZ, QW, YL and HC collected the samples. MH and SJ performed the experiments. FY and JC analyzed the data. MH, SJ, SW and JP wrote the paper. All authors read and approved the final manuscript. References Arbatova J, Lehto K, Pehu E, Pehu T (1998) Localization of the P1 protein of potato Y potyvirus in association with cytoplasmic inclusion bodies and in the cytoplasm of infected cells. J Gen Virol 79(Pt 10):2319–2323 Brantley JD, Hunt AG (1993) The N-terminal protein of the polyprotein encoded by the potyvirus tobacco vein mottling virus is an RNA-binding protein. J Gen Virol 74(Pt 6):1157–1162 Carstens EB (2010) Ratification vote on taxonomic proposals to the International Committee on Taxonomy of Viruses (2009). Archives of Virology Germundsson A, Valkonen JP (2006) P1- and VPg-transgenic plants show similar resistance to Potato virus A and may compromise long distance movement of the virus in plant sections expressing RNA silencing-based resistance. Virus Res 116:208–213 Gong D, Wang JH, Lin ZS, Zhang SY, Zhang YL, Yu NT, Xiong Z, Liu ZX (2011) Genomic sequencing and analysis of Chilli ringspot virus, a novel potyvirus. Virus Genes 43:439–444 Ha C, Revill P, Harding RM, Vu M, Dale JL (2008) Identification and sequence analysis of potyviruses infecting crops in Vietnam. Arch Virol 153:45–60 Peng J, Bu S, Yin Y, Hua M, Zhao K, Lu Y, Zheng H, Wan Q, Zhang S, Chen H, Liu Y, Chen J, Mo X, Yan F (2021) Biological and Genetic Characterization of Pod Pepper Vein Yellows Virus-Associated RNA From Capsicum frutescens in Wenshan, China. Front Microbiol 12:662352 Rahman MS, Su X, Zheng K, Cheng X, Li T, Zhao L, Dong J, Zhang Z (2020) Characterization of a New Isolate of Chilli ringspot virus in Yunnan, China. J Crop Sci Biotechnol 23:57–63 Verchot J, Carrington JC (1995) Debilitation of plant potyvirus infectivity by P1 proteinase-inactivating mutations and restoration by second-site modifications. J Virol 69:1582–1590 Verchot J, Carrington JC (1995) Evidence that the potyvirus P1 proteinase functions in trans as an accessory factor for genome amplification. J Virol 69:3668–3674 Wang J-H, Zhang S-Y, Gong D, Wu Y-P, Zhang Y-L, Yu N-T, Liu Z-X, Xiong Z (2012) First Report of Chilli ringspot virus on Chili Pepper in China. Plant Dis 96:462–462 Zhao K, Yin Y, Hua M, Wang S, Mo X, Yuan E, Zheng H, Lin L, Chen H, Lu Y, Chen J, Peng J, Yan F (2021) Pod pepper vein yellows virus, a new recombinant polerovirus infecting Capsicum frutescens in Yunnan province, China. Virol J 18:42 Supplementary Files FIGS1.tif Fig. S1. (A) Genome organization ChiRSV. Diagram showing how the full-length sequence was constructed by RACE and overlap extension RT-PCR. (B) Diagrammatic representation of the full-length cDNA infectious clone pChiRSV. (C) Diagrammatic representation showing the insertion of the eGFP gene into the infectious clone pChiRSV. FIGS2.tif Fig. S2. Maximum Likelihood phylogenetic tree constructed using MEGA X, showing the relationship of ChiRSV-YN/Wenshan to other potyviruses using their nt sequences. Numbers on branches are bootstrap support values (1,000 replicates). Multiple nucleotide sequences were aligned using MUSCLE, the best model (GTR+G+I) and subsequent analysis was determined by MEGAX. Virus acronyms and GenBank Accessions Nos are listed in Table S3. TableS1.docx TableS2.docx TableS3.docx TableS4.docx Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Minor Revision 30 Jan, 2022 Reviews received at journal 16 Jan, 2022 Reviewers invited by journal 16 Jan, 2022 Editor assigned by journal 13 Jan, 2022 First submitted to journal 12 Jan, 2022 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. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-1255906","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":76872895,"identity":"c89d0d54-c460-4af9-98ce-a8ca8f8f533e","order_by":0,"name":"Mengying Hua","email":"","orcid":"","institution":"Ningbo University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Mengying","middleName":"","lastName":"Hua","suffix":""},{"id":76872896,"identity":"4d3a559e-ecb9-4b27-8784-54999516a2d2","order_by":1,"name":"Shanshan Jiang","email":"","orcid":"","institution":"Shandong Academy of Agricultural 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05:37:31","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-1255906/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-1255906/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":17437320,"identity":"57aec4af-007b-4bc0-8008-6e9fc0710d43","added_by":"auto","created_at":"2022-01-18 18:15:38","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1086782,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eInfectivity and symptoms of ChiRSV-YN/Wenshan following inoculation of \u003cem\u003eNicotiana benthamiana\u003c/em\u003e by pChiRSV. \u003c/strong\u003e(A) Phenotype of \u003cem\u003eN. benthamiana\u003c/em\u003e plants agroinfiltrated with viral infectious clone combinations or empty agrobacterium (CK) at 10 days post infiltration. (B) RT-PCR and Western blot confirming the presence of viral RNAs in systemic leaves of inoculated plants. (C) Typical potyvirus virions in negatively-stained samples of systemic leaves of plants inoculated with the ChiRSV infectious clone. Bars represent 100nm.\u0026nbsp;\u003c/p\u003e","description":"","filename":"FIG1.png","url":"https://assets-eu.researchsquare.com/files/rs-1255906/v1/0886d0d95c129532efe91706.png"},{"id":17437322,"identity":"0a1fea44-aae2-4e7e-99dc-aa04bc7050ce","added_by":"auto","created_at":"2022-01-18 18:15:38","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1362961,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSymptoms caused by ChiRSV in peppers\u003cem\u003e.\u003c/em\u003e \u003c/strong\u003e(A) Symptoms on plants of \u003cem\u003eCapsicum annuum L \u003c/em\u003eand \u003cem\u003eC. frutescens L\u003c/em\u003e. inoculated with ChiRSV and photographed at 15 dpi (B) RT-PCR and Western blot confirming the presence of viral RNAs in systemic leaves of inoculated plants. (C) Virions observed by TEM in negatively-stained samples of systemic leaves. Bars represent 200nm.\u003c/p\u003e","description":"","filename":"FIG2.png","url":"https://assets-eu.researchsquare.com/files/rs-1255906/v1/cf4317896b846e16b86f6913.png"},{"id":17437654,"identity":"f535f225-1ea4-4eea-b730-49abe3438b3c","added_by":"auto","created_at":"2022-01-18 18:18:38","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1371256,"visible":true,"origin":"","legend":"\u003cp\u003eExpression of eGFP by the infectious clone pChiRSV-GFP in \u003cem\u003eN. benthamiana\u003c/em\u003e. (A) Symptoms in \u003cem\u003eN. benthamiana\u003c/em\u003e plants inoculated with pCB301-CH (Mock) or pChiRSV-GFP under UV and natural light at 8 dpi. (B) RT-PCR and Western blot analysis confirming the presence of ChiRSV-GFP in the infected leaves of \u003cem\u003eN. benthamiana\u003c/em\u003e plants. (C) Virions observed by TEM in negatively-stained samples of systemic leaves of plants inoculated with the pChiRSV-GFP infectious clone. Bars represent 200nm.\u003c/p\u003e","description":"","filename":"FIG3.png","url":"https://assets-eu.researchsquare.com/files/rs-1255906/v1/63e062b377eb121fd788c45e.png"},{"id":17437657,"identity":"5ff092d5-989f-4641-9687-0c77e606eda7","added_by":"auto","created_at":"2022-01-18 18:18:41","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":245124,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-1255906/v1/8257920c-a235-4985-be46-a6f1d66e0607.pdf"},{"id":17437323,"identity":"5a385814-01d9-4220-8059-32b47e9a55bb","added_by":"auto","created_at":"2022-01-18 18:15:38","extension":"tif","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":209258,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFig. S1.\u003c/strong\u003e (A) Genome organization ChiRSV. Diagram showing how the full-length sequence was constructed by RACE and overlap extension RT-PCR. (B) Diagrammatic representation of the full-length cDNA infectious clone pChiRSV. (C) Diagrammatic representation showing the insertion of the eGFP gene into the infectious clone pChiRSV.\u003c/p\u003e","description":"","filename":"FIGS1.tif","url":"https://assets-eu.researchsquare.com/files/rs-1255906/v1/5b90ef1b0b2ca958e705c78e.tif"},{"id":17437655,"identity":"e9332bf1-14f4-4154-ab18-45fcbe900d19","added_by":"auto","created_at":"2022-01-18 18:18:38","extension":"tif","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":1701441,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFig. S2.\u003c/strong\u003e Maximum Likelihood phylogenetic tree constructed using MEGA X, showing the relationship of ChiRSV-YN/Wenshan to other potyviruses using their nt sequences. Numbers on branches are bootstrap support values (1,000 replicates). Multiple nucleotide sequences were aligned using MUSCLE, the best model (GTR+G+I) and subsequent analysis was determined by MEGAX. Virus acronyms and GenBank Accessions Nos are listed in Table S3.\u003c/p\u003e","description":"","filename":"FIGS2.tif","url":"https://assets-eu.researchsquare.com/files/rs-1255906/v1/ee055586af232b2d1e9ec55b.tif"},{"id":17437327,"identity":"cc116fa6-2196-43b1-961c-53e197f98af0","added_by":"auto","created_at":"2022-01-18 18:15:38","extension":"docx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":20862,"visible":true,"origin":"","legend":"","description":"","filename":"TableS1.docx","url":"https://assets-eu.researchsquare.com/files/rs-1255906/v1/3cbb71edbf3686a18150ed9c.docx"},{"id":17437321,"identity":"c893fa09-16b2-4359-97ef-a24566334b75","added_by":"auto","created_at":"2022-01-18 18:15:38","extension":"docx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":19357,"visible":true,"origin":"","legend":"","description":"","filename":"TableS2.docx","url":"https://assets-eu.researchsquare.com/files/rs-1255906/v1/5d7d46e064330533004a60cb.docx"},{"id":17437653,"identity":"bb4e6f28-83d3-47e1-ba40-26f5a8b7e918","added_by":"auto","created_at":"2022-01-18 18:18:38","extension":"docx","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":19131,"visible":true,"origin":"","legend":"","description":"","filename":"TableS3.docx","url":"https://assets-eu.researchsquare.com/files/rs-1255906/v1/c8dc0549a9e9ed3cbe95a44d.docx"},{"id":17437324,"identity":"b971783e-015a-412e-86d2-1ee5dfea4f57","added_by":"auto","created_at":"2022-01-18 18:15:38","extension":"docx","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":19351,"visible":true,"origin":"","legend":"","description":"","filename":"TableS4.docx","url":"https://assets-eu.researchsquare.com/files/rs-1255906/v1/ea40b754cc98c7c35cf8def1.docx"}],"financialInterests":"","formattedTitle":"Construction of an infectious full‑length and eGFP-tagged cDNA clone of a chilli ringspot virus isolate from Yunnan province, China","fulltext":[{"header":"Main text","content":"\u003cp\u003eChilli ringspot virus (ChiRSV) was first reported in Vietnam in 2007\u0026nbsp;[6]\u0026nbsp;and was classified as a distinct species in the genus \u003cem\u003ePotyvirus\u003c/em\u003e in 2009\u0026nbsp;[3]. In 2011, the determination of the genomic sequence of ChiRSV-HN/14 isolate (from Hainan, China) provided further evidence that ChiRSV is a distinctive potyvirus\u0026nbsp;[5]. The ChiRSV genome has 9,571 nucleotides (nt) of ssRNA and the virus has typical filamentous particles 780 nm in length\u0026nbsp;[5, 8]. Recently, a further isolate of ChiRSV was reported from Yunnan province\u0026nbsp;[8]\u0026nbsp;which groups phylogenetically with the Hainan isolate. It has a broad host range among several economically important crops including chilli, tomato, tobacco, and cucurbitaceous vegetables\u0026nbsp;[8, 11].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn a previous study using Next-generation RNA-Seq sequencing (NGS) and RT-PCR we showed that ChiRSV, pepper vein yellows virus, chilli veinal mottle virus, tomato zonate spot virus and cucumber mosaic virus were prevalent in samples of pepper\u003cem\u003e\u0026nbsp;\u003c/em\u003e(\u003cem\u003eCapsicum frutescens\u0026nbsp;\u003c/em\u003eL.) plants from Wenshan city Yunnan province, China which had severe yellowing and curling symptoms\u0026nbsp;[12]. ChiRSV was detected by RT-PCR in 31 of the 89 symptomatic samples. In this study we first obtained the complete genomic sequence of ChiRSV from Wenshan city. Total RNA was isolated using RTIzol\u003csup\u003eTM\u003c/sup\u003e Reagent (Invitrogen) and first strand cDNA was synthesized using ReverTra Ace-\u0026alpha;-\u0026reg;(Toyobo) following the manufacturer\u0026rsquo;s protocol. Reverse transcription (RT) was performed at\u0026nbsp;42\u0026deg;C for 60 min with M4T primers (Supplementary Table S1) followed by 72\u0026deg;C for 10 min using KOD-plus-Neo (Toyobo) as specified by the manufacturer. The subsequent PCR used primers DP-ChiRSV f/DP-ChiRSV r (Supplementary Table S1) and incubation at 98\u0026deg;C for 3min, followed by 35 cycles of 98\u0026deg;C for 30s, 55\u0026deg;C for 30s, 68\u0026deg;C for 1kb/min and a final incubation at 68\u0026deg;C for 10min.\u0026nbsp;5\u0026rsquo; and 3\u0026rsquo; RACE reactions were performed to obtain the complete 5\u0026rsquo;and 3\u0026rsquo; terminal sequences and three overlapping sections were amplified to verify the full-length sequence as described before\u0026nbsp;[12]. The genome of ChiRSV-YN/Wenshan was 9653 nt long (Supplementary\u0026nbsp;Figure S1A) and the sequence was deposited in GenBank with accession number: MZ269480.\u003c/p\u003e\n\u003cp\u003eChiRSV-YN/Wenshan has 88.5-98.9% nucleotide identity to the other reported full-length genome sequence of ChiRSV (Supplementary\u0026nbsp;Table S2) and with 93.0-99.3% amino acid identity in comparisons of their polyproteins. In comparisons among the predicted mature proteins, only the P1 protein was less than 80% identical (Supplementary\u0026nbsp;Table S2). In a Maximum Likelihood (ML) phylogenetic analysis, ChiRSV-YN/Wenshan clustered with other published ChiRSV isolates in a group distinct from other potyviruses (Supplementary\u0026nbsp;Fig. S2).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTo examine the biological characteristics of ChiRSV-YN/Wenshan and to provide a tool for future investigations of mixed virus infection in pepper plant, a full-length infectious clone (pChiRSV) was prepared. The recombination cloning method was used to insert the complete ChiRSV cDNA sequence between the duplicated 35S promoter and hepatitis delta virus ribozyme (HDV-Rz) as described previously\u0026nbsp;[12]. Then, pChiRSV was transformed into \u003cem\u003eAgrobacterium tumefaciens\u003c/em\u003e which was then delivered to \u003cem\u003eNicotiana benthamiana\u003c/em\u003e plantlets by infiltration. Symptoms of witches\u0026rsquo; broom and systemic mosaic were observed in systemic leaves at 6 dpi, and infected plants were significantly smaller at 10 dpi (Fig. 1A and Supplementary\u0026nbsp;Table S4). RT-PCR and Western blot detected viral RNA and coat protein in the new non-inoculated leaves confirming that systemic infection had been established (Fig. 1A and 1B). Typical flexuous filamentous virions approximately 780 nm long were observed\u0026nbsp;by\u0026nbsp;transmission electron microscopy (TEM) in negatively-stained samples of the upper leaves (Fig. 1C).\u0026nbsp;To investigate the symptoms of ChiRSV on peppers, seedlings of \u003cem\u003eCapsicum frutescens L.\u0026nbsp;\u003c/em\u003eand\u003cem\u003e\u0026nbsp;Capsicum annuum L.\u0026nbsp;\u003c/em\u003ewere mechanically inoculated using sap from infected \u003cem\u003eN. benthamiana\u003c/em\u003e plants. At 15 dpi (day post inoculation), inoculated\u003cem\u003e\u0026nbsp;\u003c/em\u003eplants had mild mosaic (\u003cem\u003eC. annuum\u003c/em\u003e) or leaf crinkling (\u003cem\u003eC. frutescens\u003c/em\u003e) (Figure 2A and\u0026nbsp;Supplementary\u0026nbsp;Table S4), RT-PCR and Western blot confirmed that virus had spread systemically in the inoculated plants (Fig. 2B) and flexuous filamentous virions\u0026nbsp;were observed\u0026nbsp;in new non-inoculated leaves\u0026nbsp;by\u0026nbsp;TEM (Fig. 2C). These results show that the full-length cDNA clone of ChiRSV-YN/Wenshan could successfully infect both\u0026nbsp;\u003cem\u003eN. benthamiana\u003c/em\u003e and pepper plants.\u003c/p\u003e\n\u003cp\u003eTo trace and observe infection by\u0026nbsp;ChiRSV-YN/Wenshan more conveniently, an eGFP-tagged cDNA infectious clone was then constructed. The sequence encoding a NIa protease cleavage site (TTVYHQ/A) was introduced between the eGFP and CP coding sequences (Supplementary\u0026nbsp;Fig. S1C). The infectious clone pChiRSV-GFP\u0026nbsp;was transformed into \u003cem\u003eAgrobacterium tumefaciens\u003c/em\u003e which was then delivered to \u003cem\u003eN. benthamiana\u003c/em\u003e plantlets by infiltration. At 12 dpi, examination under a UV lamp showed eGFP fluorescence in the upper non-inoculated leaves of most plants (Fig. 3A;\u0026nbsp;Supplementary\u0026nbsp;Table S4). The symptoms on these leaves were similar to those produced by the\u0026nbsp;untagged cDNA clone.\u0026nbsp;Western blots showed that the coat protein and GFP could be detected in the new non-inoculated leaves (Fig. 3B) and\u0026nbsp;typical\u0026nbsp;flexuous filamentous virions were also\u0026nbsp;observed in them by\u0026nbsp;TEM (Fig. 3C).\u003c/p\u003e\n\u003cp\u003eIn this study of ChiRSV-YN/Wenshan, only the P1 protein was significantly different to the mature proteins of other ChiRSV isolates. P1 is a multifunctional protein that participates in RNA binding, is associated with inclusion bodies, and plays roles in cell-to-cell and systemic movement [1, 2, 4, 9, 10]. The full-length cDNA clone of pChiRSV constructed in this study will provide a useful tool to investigate whether the diversity of P1 sequences among the isolates indicates that they have diverse roles. The ChiRSV-YN/Wenshan isolate was obtained in 2019 from plants with severe symptoms of PeVYD (pepper vein yellows disease). This disease is associated with co-infection by several viruses but especially the phloem-limited PoPeVYV (pod pepper vein yellows virus) together with PoPeVYVaRNA (pod pepper vein yellows virus-associated RNA) [7, 12]. Our infectious clone will also be a useful tool for examining whether ChiRSV can assist infection by PoPeVYV in the absence of PoPeVYVaRNA.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by Chinese Agriculture Research System of MOF and MARA (CARS-24-C-04). This work also was financially supported by National Key R\u0026amp;D Program of China (2019YFD1001800) and K. C.Wong education foundation. We thank Professor Mike Adams for manuscript correction.\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 upon reasonable request.\u003c/p\u003e\n\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\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMH, SJ, SW and JP conceived and designed the experiments. EY, HZ, QW, YL and HC collected the samples. MH and SJ performed the experiments. FY and JC analyzed the data. MH, SJ, SW and JP wrote the paper. All authors read and approved the final manuscript.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eArbatova J, Lehto K, Pehu E, Pehu T (1998) Localization of the P1 protein of potato Y potyvirus in association with cytoplasmic inclusion bodies and in the cytoplasm of infected cells. J Gen Virol 79(Pt 10):2319\u0026ndash;2323\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBrantley JD, Hunt AG (1993) The N-terminal protein of the polyprotein encoded by the potyvirus tobacco vein mottling virus is an RNA-binding protein. J Gen Virol 74(Pt 6):1157\u0026ndash;1162\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCarstens EB (2010) Ratification vote on taxonomic proposals to the International Committee on Taxonomy of Viruses (2009). Archives of Virology\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGermundsson A, Valkonen JP (2006) P1- and VPg-transgenic plants show similar resistance to Potato virus A and may compromise long distance movement of the virus in plant sections expressing RNA silencing-based resistance. Virus Res 116:208\u0026ndash;213\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGong D, Wang JH, Lin ZS, Zhang SY, Zhang YL, Yu NT, Xiong Z, Liu ZX (2011) Genomic sequencing and analysis of Chilli ringspot virus, a novel potyvirus. Virus Genes 43:439\u0026ndash;444\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHa C, Revill P, Harding RM, Vu M, Dale JL (2008) Identification and sequence analysis of potyviruses infecting crops in Vietnam. Arch Virol 153:45\u0026ndash;60\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePeng J, Bu S, Yin Y, Hua M, Zhao K, Lu Y, Zheng H, Wan Q, Zhang S, Chen H, Liu Y, Chen J, Mo X, Yan F (2021) Biological and Genetic Characterization of Pod Pepper Vein Yellows Virus-Associated RNA From Capsicum frutescens in Wenshan, China. Front Microbiol 12:662352\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRahman MS, Su X, Zheng K, Cheng X, Li T, Zhao L, Dong J, Zhang Z (2020) Characterization of a New Isolate of Chilli ringspot virus in Yunnan, China. J Crop Sci Biotechnol 23:57\u0026ndash;63\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVerchot J, Carrington JC (1995) Debilitation of plant potyvirus infectivity by P1 proteinase-inactivating mutations and restoration by second-site modifications. J Virol 69:1582\u0026ndash;1590\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVerchot J, Carrington JC (1995) Evidence that the potyvirus P1 proteinase functions in trans as an accessory factor for genome amplification. J Virol 69:3668\u0026ndash;3674\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang J-H, Zhang S-Y, Gong D, Wu Y-P, Zhang Y-L, Yu N-T, Liu Z-X, Xiong Z (2012) First Report of Chilli ringspot virus on Chili Pepper in China. Plant Dis 96:462\u0026ndash;462\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhao K, Yin Y, Hua M, Wang S, Mo X, Yuan E, Zheng H, Lin L, Chen H, Lu Y, Chen J, Peng J, Yan F (2021) Pod pepper vein yellows virus, a new recombinant polerovirus infecting Capsicum frutescens in Yunnan province, China. Virol J 18:42\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":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"archives-of-virology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"arvi","sideBox":"Learn more about [Archives of Virology](https://www.springer.com/journal/705)","snPcode":"705","submissionUrl":"https://submission.nature.com/new-submission/705/3","title":"Archives of Virology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"chilli ringspot virus, potyvirus, infectious clone","lastPublishedDoi":"10.21203/rs.3.rs-1255906/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-1255906/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eChilli ringspot virus (ChiRSV; genus \u003cem\u003ePotyvirus\u003c/em\u003e) was one of several viruses previously detected in pepper samples with severe yellowing and curling symptoms growing in Wenshan, Yunan province, China. We now report the full-length sequence of ChiRSV-YN/Wenshan (MZ269480) which has 88.5-98.9%. nucleotide identity to other published ChiRSV isolates. A full-length cDNA infectious clone was constructed. This cDNA and an eGFP-tagged clone were infectious leading to systemic symptoms in both \u003cem\u003eNicotiana benthamiana\u003c/em\u003e and \u003cem\u003eCapsicum\u003c/em\u003e spp. Single infection by ChiRSV caused mild mosaic or leaf crinkling in \u003cem\u003eCapsicum frutescens L.\u003c/em\u003e and \u003cem\u003eCapsicum annuum L.\u003c/em\u003e\u003c/p\u003e","manuscriptTitle":"Construction of an infectious full‑length and eGFP-tagged cDNA clone of a chilli ringspot virus isolate from Yunnan province, China","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2022-01-18 18:15:36","doi":"10.21203/rs.3.rs-1255906/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Minor Revision","date":"2022-01-31T00:25:39+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2022-01-16T09:18:04+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2022-01-16T08:48:17+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2022-01-13T18:04:10+00:00","index":"","fulltext":""},{"type":"submitted","content":"Archives of Virology","date":"2022-01-13T00:36:58+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"archives-of-virology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"arvi","sideBox":"Learn more about [Archives of Virology](https://www.springer.com/journal/705)","snPcode":"705","submissionUrl":"https://submission.nature.com/new-submission/705/3","title":"Archives of Virology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"ab119e9c-a624-471f-afc9-8d6de10d5e88","owner":[],"postedDate":"January 18th, 2022","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2022-03-21T07:42:19+00:00","versionOfRecord":[],"versionCreatedAt":"2022-01-18 18:15:36","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-1255906","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-1255906","identity":"rs-1255906","version":["v1"]},"buildId":"ApUGefWb6u5IBVtyqm6d5","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}