Nucleotide sequence survey of perilla mosaic virus isolates in Japan unveils complex genetic structure and conserved RNA segments encoding homologous protein groups

Nucleotide sequence survey of perilla mosaic virus isolates in Japan unveils complex genetic structure and conserved RNA segments encoding homologous protein groups | 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 Nucleotide sequence survey of perilla mosaic virus isolates in Japan unveils complex genetic structure and conserved RNA segments encoding homologous protein groups Kenji Kubota, Yuya Chiaki This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4818253/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 08 Oct, 2024 Read the published version in Journal of General Plant Pathology → Version 1 posted 4 You are reading this latest preprint version Abstract Perilla mosaic virus (PerMV) is a fimovirus that harbors ten RNA segments (RNAs 1, 2, 3a, 3b, 4, 5, 6a, 6b, 6c, and 7). The presence of PerMV was first reported in Kochi, followed by Ibaraki, Aichi, and Oita prefectures in Japan, and most recently from South Korea. To understand the genetic diversity of these PerMV isolates, partial nucleotide sequences of RNAs 1 to 4 from 21 Japanese isolates were determined. Phylogenetic analysis revealed that the segments of the isolates are divided into two clades: the Eastern clade (isolates in Ibaraki) and the Western clade (isolates in Kochi, Oita, and South Korea). Isolates in Aichi appeared to be reassortants of both; RNAs 1 and 3b belonged to the Eastern clade, while the others were of the Western clade. Isolates lacking RNA2, found in four prefectures, suggest RNA2 is not essential for plant infection. Near-complete nucleotide sequences from four isolates in Ibaraki, Aichi, and Oita were determined. They maintained ten RNA segments, including RNAs encoding proteins of P3s, P6s, and P7. This indicates that these proteins, encoded by an apparently redundant segment, are crucial for PerMV’s infection and transmission cycles. fimovirus emaravirus genetic diversity glycoprotein homologous proteins Figures Figure 1 Figure 2 Introduction Fimoviruses, also referred to as emaraviruses, are plant-infecting, multipartite, negative-sense RNA viruses, and are members of the Fimoviridae family, consisting of only the Emaravirus genus. (Rehanek et al. 2022 ). The number of species in the Fimoviridae family has been increasing due to the continuous discovery of new species, reaching up to 32 species (Digiaro et al. 2024 ; Kuhn et al. 2023 ). While not all fimoviruses are transmitted this way, some have been shown to be transmitted by specific eriophyid mites (families Eriophydae and Diptilomiopidae) (Rehanek et al. 2022 ). Fimoviruses are related to plant-infecting viruses in the class Bunyaviricetes , which encompasses orthotospoviruses (family Tospoviridae , order Elliovirales ), fimoviruses ( Elliovirales ), and tenuiviruses (family Phenuiviridae , order Hareavirales ) (Kormelink et al. 2021 ). Unlike orthotospoviruses and tenuiviruses, fimoviruses’ genome varies in RNA segments (five to ten) depending on species, and RNA segments are primarily monocistronic, not ambisense. Among fimoviruses segments, RNA1 to RNA4 encode proteins P1; RNA-dependent RNA polymerase (RdRp), P2; glycoprotein precursor (GPP), P3; nucleocapsid protein (NP), and P4; movement protein (MP), respectively. While each fimovirus harbors additional protein-encoding RNA segments (e.g., RNA5 encoding protein P5), their amino acid sequences are less conserved, and their functions remain unclear (Rehanek et al. 2022 ). However, some fimovirus proteins share amino acid sequence homology among different species [e.g., P5s of rose rosette virus (RRV), fig mosaic virus (FMV), etc.] [summarized by Rehanek et al. ( 2022 )], suggesting they play crucial roles in virus infection and transmission. Another unique fimovirus genome feature is the presence of RNA segments encoding likely redundant proteins. High Plains wheat mosaic virus (HPWMoV) was the first reported to harbor two RNA3 variants, both encoding putative NP (P3) with ~ 80% amino acid sequence identities (Tatineni et al. 2014 ), followed by the discovery of three other fimoviruses encoding two P3s (Buzkan et al. 2019 ; Kubota et al. 2020 , 2021c ). As FMV encodes only one P3, enabling full ribonucleoprotein complex composition (Izhaki-Tavor et al. 2023 ), the reason for some fimoviruses encoding two NP variants remains unclear. Moreover, some fimoviruses possess two to four homologous proteins; e.g., P6, P7, P8a, and P8b of raspberry leaf blotch virus (Lu et al. 2015 ) or P5a, P5b, and P7 of Pistachia virus B (PiVB) (Buzkan et al. 2019 ). At least eight fimoviruses harbor RNAs encoding homologous protein groups (Rehanek et al. 2022 ). Although the functions and reasons for these homologous proteins’ presence are unknown, they likely hold biological significance and importance. Fimoviruses possess enveloped virions, also known as double-membrane-bound bodies (DMBs) (Rehanek et al. 2022 ). Similar to the role of glycoproteins (G N /G C ) of orthotospoviruses in enveloped virion formation (Bahat et al. 2020 ), fimovirus GPs are presumed to be responsible for DMB formation (Rehanek et al. 2022 ). Among fimoviruses, perilla mosaic virus (PerMV, the species Emaravirus perillae )has the most RNA segments, totaling ten; RNAs 1, 2, 3a, 3b, 4, 5, 6a, 6b, 6c, and 7 (Kubota et al. 2020 ). Proteins P1 to P4 are putative RdRp, GPP, NP, and MP, respectively. Proteins P3a and P3b are putative NPs, sharing 83.0% amino acid sequence identity. While redundancy in RNA3 has been observed in some other fimoviruses [HPWMoV, PiVB, and chrysanthemum mosaic-associated virus (ChMaV)] (Buzkan et al. 2019 ; Kubota et al. 2021c ; Tatineni et al. 2014 ), its biological significance remains unclear. PerMV P5 contains a domain homologous to Glu2-Pro, a glutamic protease encoded by a sadwavirus, and Glu-2 Pro is shared by some other fimovirus proteins (Rehanek et al. 2022 ). P6a–c of PerMV are proteins with molecular masses of 26.8–30.2 kDa, sharing amino acid sequence identities of 24.4–62.3% with each other (Kubota et al. 2020 ). PerMV P7, a 28.5 kDa protein, shares weak sequence homology (Rehanak et al. 2022). However, the molecular functions of P5, P6s, and P7 of PerMV remain elusive to date. PerMV was first identified in glasshouse grown shiso ( Perilla frutescence L.) plants exhibiting mosaic symptoms in Kochi Prefecture, Japan (Kubota et al. 2020 ). It has also been found in other shiso production areas in Aichi, Ibaraki, Oita, and Ehime prefectures (Ehime Plant Protection Office 2022 ). PerMV is effectively transmitted by the perilla rust mite (PRM), Aculops thymi (Nalepa 1889) (previously referred to as Shevtchenkella sp.) (Kadono et al. 2022 ; Kubota et al. 2020 ), and PRM has also been discovered in cultivated or wild-grown shiso plants in Kochi, Aichi, and Oita prefectures (Suzuki et al. 2018 ). In Japan, six fimoviruses have been reported: PerMV, FMV ( Emaravirus fici ), pear chlorotic leaf spot-associated virus (PCLSaV, Emaravirus pyri ), ChMaV ( Emaravirus chrysanthemi ), Vitis emaravirus (VEV, Emaravirus vitis ), and Japanese star anise ringspot-associated virus (JSARaV, Emaravirus illicii ) (Ishikawa et al. 2012 ; Kubota et al. 2021a , 2021b , 2021c ; Nabeshima and Abe 2021 ; Shimomoto et al. 2022 ). Takeyama et al. ( 2022 ) reported that the nucleotide sequences of full-length ORFs encoded by RNAs 1 to 5 of 16 PCLSaV isolates collected from the Tohoku to Kyushu regions in Japan showed significantly lower genetic diversity compared to three Chinese isolates (Liu et al. 2020 ). This suggests that PCLSaV might have been introduced from China and spread only recently in Japan. Chlorotic leaf spot symptoms of pear trees, presumably caused by PCLSaV infection, have been noticed only after ~ 2010 in Japan. In contrast, mosaic diseases on shiso crops caused by PerMV occurred in the late 1980s and around 2000 in Aichi and Kochi prefectures, respectively (Kubota et al. 2020 ). Therefore, investigating the genetic diversity of PerMV isolates in Japan provides significant insight into the origin and distribution of PerMV. The first objective of this study is to overview the genetic diversity of PerMV populations in Japan by comparing nucleotide sequences of RNAs 1, 2, 3a, 3b, and 4 of 21 field isolates of PerMV collected from Ibaraki, Aichi, Kochi, and Oita prefectures from 2011 to 2015. During our above investigation, an occurrence of PerMV in South Korea was reported. The RNA segments of the South Korean isolate (PerMV-IS) shared nucleotide sequence identities of 92.09–97.37% with the Japanese isolate, Kochi_Nankoku_2011 (Oh et al. 2023 ). However, the nucleotide sequences of PerMV-IS were reported only for seven segments (each of RNAs 1 to 7), raising a question about whether PerMV indeed possesses 10 segments as we previously reported (Kubota et al. 2020 ). The complete nucleotide sequences of PerMV in Japan were determined only for one isolate, i.e., Kochi_Nankoku_2011, which harbors two RNA3s (RNA3a and 3b) and three RNA6s (RNA6a–c). These RNAs and proteins may share redundant functions and be dispensable for infection and transmission cycles; hence, isolates lacking these RNAs might exist. To clarify this possibility, we further attempted to amplify and determine the near-complete nucleotide sequences of all RNA segments of selected isolates in Ibaraki, Aichi, and Oita prefectures and also compared them to those of the previously identified Kochi and South Korean isolates. Materials and methods Collection of virus isolates and sequencing Leaves of green varieties of shiso plants exhibiting typical mosaic symptoms were collected from production greenhouses, an experimental greenhouse, or home gardens in Kochi, Oita, Aichi, and Ibaraki prefectures in Japan between 2011 and 2015 (Table 1 , Supplementary Fig. S1 ). Total RNA was extracted from the collected leaves using Trizol Reagent (ThermoFisher Scientific, Waltham, MA, USA), as previously described (Kubota et al. 2020 ). To obtain cDNAs of the PerMV segments, the total RNAs underwent reverse transcription using PrimeScript Reverse Transcriptase (TakaraBio, Shiga, Japan) with primers complementary to the 11-nt conserved 5′ and 3′ termini of RNA segments of PerMV, essentially as previously described (Kubota et al. 2020 ). Using these cDNAs as templates, attempts were made to amplify the cDNA of partial nucleotide sequences of RNAs 1, 2, 3a, 3b, and 4 of all the collected isolates by PCR, followed by direct sequencing with primers shown in Supplementary Table S1 . Additionally, for a total of four selected isolates (two from Ibaraki and one each from Aichi and Oita prefectures), near full-length sequences of all ten segments (hereafter, near-complete sequence) were determined by RT-PCR amplification followed by direct sequencing with previously reported primers (Kubota et al. 2020 ). PCR amplification was conducted using Tks Gflex DNA Polymerase (TakaraBio) or KOD One PCR Master Mix (Toyobo, Osaka, Japan). The amplified products underwent purification using ExoSAP-IT (ThermoFisher) and were then subjected to a direct sequencing reaction utilizing a BigDye Terminator v3.1 Cycle Sequencing Kit (ThermoFisher). The primers indicated in Table S1 , along with those previously outlined by Kubota et al. ( 2020 ), were employed to determine partial or near-complete sequences. The sequencing process was executed using an ABI 3130xl Genetic Analyzer (Applied Biosystems, Waltham, MA, U.S.A.). Table 1 Collection of field isolates of perilla mosaic virus utilized in the study Place (Condition) Year Isolate a RNA amplified Sequence determined Accession Reference Nankoku, Kochi, Japan (Production greenhouse) 2011 Kochi_Nankoku_2011 1–7 b Complete NC_078367–NC_078376 Kubota et al. ( 2020 ) Kochi_Nankoku_2011_2 1, 3a, 3b, 4 c Partial LC830810–LC830813 This study 2015 Kochi_Nankoku_2015_1 1–4 d Partial LC776290–LC776294 This study Kochi_Nankoku_2015_2 1, 3a, 3b, 4 Partial LC776295–LC776298 This study Oita, Oita, Japan (Production greenhouse) 2013 Oita_Oita_2013_1 1–7 Near-complete LC830770–LC830779 This study 2015 Oita_Oita_2015_1 1–4 Partial LC776304–LC776308 This study Oita_Oita_2015_2 1–4 Partial LC776309–LC776313 This study Oita_Oita_2015_3 1, 3a, 3b, 4 Partial LC776314–LC776317 This study Oita_Oita_2015_4 1–4 Partial LC776318–LC776322 This study Toyohashi, Aichi, Japan (Production greenhouse) 2013 Aichi_Toyohashi_2013_1 1–4 Partial LC776323–LC776327 This study Aichi_Toyohashi_2013_2 1–7 Near-complete LC830780–LC830789 This study 2015 Aichi_Toyohashi_2015_1 1–4 Partial LC776333–LC776337 This study Aichi_Toyohashi_2015_2 1, 3a, 3b, 4 Partial LC776338–LC776341 This study Ushiku, Ibaraki, Japan (Experimental greenhouse) 2013 Ibaraki_Ushiku_2013_1 1–4 Partial LC776347–LC776351 This study Ibaraki_Ushiku_2013_2 1–4 Partial LC776352–LC776356 This study Namegata, Ibaraki, Japan (Production greenhouse) 2014 Ibaraki_Namegata_2014_1 1–7 Near complete LC830800–LC830809 This study 2015 Ibaraki_Namegata_2015_1 1–4 Partial LC776367–LC776371 This study Ibaraki_Namegata_2015_2 1, 3a, 3b, 4 Partial LC776372–LC776375 This study Miho, Ibaraki, Japan (Home garden, wild grown) 2013 Ibaraki_Miho_2013_1 1–7 Near-complete LC830790–LC830799 This study 2015 Ibaraki_Miho_2015_1 1–4 Partial LC776357–LC776361 This study Tsukuba, Ibaraki, Japan (Flower bed) (Home garden, wild grown) 2015 Ibaraki_Tsukuba_2015_1 1–4 Partial LC776376–LC776380 This study Ibaraki_Tsukuba_2015_2 1–4 Partial LC833855, LC776381–2, LC833856, LC776383 This study Iseo-myeon, South Korea 2021 IS 1–7e Near-complete LC721296–LC721302 Oh et al. ( 2023 ) a Isolates sharing with same place names (e.g., Kochi_Nankoku) were collected in the same greenhouse or a home garden, except that Ibaraki_Tukuba_2015_1 and Ibaraki_Tsukuba_2015_2. b RNAs 1, 2, 3a, 3b, 4, 5, 6a, 6b, 6c, and 7. c Among RNAs 1, 2, 3a, 3b, and 4, RNA2 was not amplified by RT-PCR. d RNAs 1, 2, 3a, 3b, and 4. e Only RNAs 1, 2, 3, 4, 5, 6, and 7 were reported for the isolate IS. Sequence analyses Nucleotide sequence reads were assembled using CodonCode Aligner Software (CodonCode Corporation, Centerville, MA, USA). The analysis utilized a total of 23 isolates, including the previously reported Kochi_Nankoku_2011 (Kubota et al. 2020 ; NC_078364–NC_078373) and IS (Oh et al. 2023 ; LC721296–LC721303). Nucleotide and amino acid sequence identities were calculated using SDTv1.2 (Muhire et al. 2014 ), with the alignment option set to ClustalW (Thompson et al. 1994 ). Nucleotide sequences were aligned using ClustalW, as implemented in MEGA11 (Tamura et al. 2021 ), and phylogenetic trees were reconstructed using the neighbor-joining method (Saitou and Nei 1987 ) with Kimura’s two parameter model (Kimura 1980 ). The phylogeny was tested by performing 1,000 bootstrap pseudoreplicates. Recombination analysis of isolates with nearly complete sequences (five Japanese and one South Korean isolates) was conducted using RDP5 (Martin et al. 2021 ). Results Genetic diversities of the Japanese isolates based on partial RNA1–RNA4 sequences Out of the 21 newly collected isolates, 17 yielded partial nucleotide sequences of RNAs 1, 2, 3a, 3b, and 4. Full-length RT-PCR products for RNA2 were not detected from five isolates (Oita_Oita_2015_3, Kochi_Nankoku_2011_2, Kochi_Nankoku_2015_2, Aichi_Toyohashi_2015_2, and Ibaraki_Namegata_2015_2). Further attempts to amplify the regions nts 948–1369 or nts 1258–1714 using two other primer sets shown in Table S1 yielded consistent results across the 21 tested isolates (positive, 16; negative, 5). The lengths of the obtained partial nucleotide sequences used for the subsequent analysis are as follows: RNA1, 786 nt (corresponding to nts 3375–4112 of Kochi_Nankoku_2011); RNA2, 661 or 658 nt (nts1064–1724); RNA3a, 520 nt (nts 430–949); RNA3b, 520 nt (nts 429–948); RNA4, 878 nt (nts 391–1268) (Table 1 ). RNA2 of 14 isolates was 661 nt, while the isolates Aichi_Toyobashi_2013_1 and Aichi_Toyohashi_2013_2 harbored a common 3-nt deletion, leading to a deletion of the aspartic acid residue at aa 204 of P2. Nucleotide sequence identities among the Japanese isolates were 93.7–100% for RNA1, 91.1–100% for RNA2, 92.5–100% for RNA3a, 91.7–100% for RNA3b, and 93.7–100% for RNA4. Phylogenetic analysis of the partial nucleotide sequences of RNAs 1–4 from the PerMV isolates revealed that the segments of the PerMV isolates were divided into two clades (Fig. 1 ). The sequences of RNA1 fell into one clade consisting of isolates collected from Ibaraki and Aichi prefectures, and another clade composed of isolates from Kochi and Oita prefectures (Fig. 1 a). The isolate IS from South Korea grouped with the latter, although it appeared distantly related to the Japanese isolates. However, RNA2 displayed a different phylogenetic pattern; one clade consisted of isolates only from Ibaraki, and another was composed of isolates from Aichi, Kochi, and Oita prefectures (Fig. 1 b). The phylogeny of RNA3a and RNA3b was similar to that of RNA2 and RNA1, respectively (Fig. 1 c), except that RNA3a of the isolate Aichi_Toyohashi_2015_1 fell into the clade with the isolates from Ibaraki. The phylogeny of RNA4 was similar to that of RNA2 (Fig. 1 d). Near-complete nucleotide sequences of the four isolates We determined the near-complete nucleotide sequences of all segments from four selected isolates, namely Oita_Oita_2013_1, Aichi_Toyohashi_2013_2, Ibaraki_Miho_2013_1, and Ibaraki_Namegata_2014_1, collected from distant areas in Japan. PCR amplification from the cDNAs of these isolates generated products of corresponding sizes for each segment (data not shown), and their nucleotide sequences were determined through direct sequencing. The lengths of the nucleotide sequences used for alignment were as follows: RNA1, 7131 nt (corresponding to nts 115–7245 of Kochi_Nankoku_2011); RNA2, 2048 nt (nts 23–2070); RNA3a, 1036 nt (nts 23–1058); RNA3b, 1034 nt (nts 23–1056); RNA4, 1250 nt (nts 23–1272), RNA5, 1022 nt (nts 23–1044); RNA6a, 981 nt (nts 126–1106); RNA6b, 931 nt (nts 145–1075); RNA6c, 971 nt (nts 58–1028); RNA7, 954 nt (nts 23–976) (Table 1 ). All sequenced segments encoded a single protein, sharing amino acid sequence identities with proteins P1 to P7 of Kochi_Nankoku_2011 (Table 2 ). Notably, the segments encoding homologous proteins (i.e., RNAs 3a/b and RNAs 6a–c/7) were present in all isolates, regardless of collection area, year of collection, or position in phylogenetic clades. Among the proteins, P4 was the most highly conserved (> 99.4%), while P6c was the least conserved (85.0–98.3%). Generally, two isolates from Ibaraki showed lower identities to Kochi_Nankoku_2011 compared to those from Oita and Aichi, but P1 and P3b of Aichi_Toyohashi_2013_2 showed lower identities. Consistent with these results, phylogenetic analysis using near-complete nucleotide sequences indicated that each segment divided into two groups (Fig. 2 ), as observed in the partial RNA1 to RNA4. RNA3 and RNA6 of the isolate IS fell into each clade together with RNA3a and RNA6b of the Japanese isolates, respectively. No recombination event was detected in all segments of the five Japanese isolates. Table 2 Comparison of amino acid sequence identities between Kochi_Nankoku_2011 and five other perilla mosaic virus isolates Kochi_Nankoku_2011 Isolate P1 P2 P3a P3b P4 P5 P6a P6b P6c P7 Oita_Oita_2013_1 99.6 98.3 99.6 99.6 100 98.2 98.4 98.4 98.3 99.6 Aichi_Toyohashi_2013_2 97.4 99.5 99.6 96.2 100 96.8 99.2 100 97.4 99.2 Ibaraki_Miho_2013_1 97.5 98.6 95.1 94.4 99.4 90.3 93.3 89.1 85.8 98.4 Ibaraki_Namegata_2014_1 97.6 96.2 94.7 96.6 99.4 90.3 93.3 91.1 85.0 98.4 IS 99.1 98.3 92.9 a 88.0 a 100 93.5 62.1 b 96.1 b 25.3 b 97.2 a Amino acid sequence identities between P3 of the IS isolate of PerMV (LC721296) and P3a and P3b of Kochi_Nankoku_2011. b Amino acid sequence identities between P6 of the IS isolate of PerMV (LC721301) and P6a, P6b, and P6c of Kochi_Nankoku_2011. Discussion In this study, we collected a total of 21 PerMV isolates from four prefectures in Japan between 2011 and 2015. We determined the partial nucleotide sequences of RNA1-RNA4 and the near-complete genome sequences of four selected isolates. The partial nucleotide sequences of RNA1–RNA4 from the Japanese isolates shared more than 90% identity. However, phylogenetic analysis revealed a clear division into two clades: one containing segments of isolates from Western Japan (Kochi and Oita prefectures), and another containing isolates from Eastern Japan (Ibaraki prefecture) (Fig. 1 ). Isolates from Aichi consisted of RNA1 and RNA3b from Eastern Japan and the others from Western Japan. This suggests that the PerMV population in Japan is genetically diverse, with at least two groups. Regarding the isolates in Aichi, they could be reassortants of isolates from Eastern and Western Japan. Alternatively, Aichi, located near the center of origin of PerMV, could be the point from which they diversified and spread to Eastern and Western regions in Japan. The latter scenario may be more likely, as occurrences of the mosaic disease, presumably caused by PerMV, were first noticed in Aichi prefecture as early as 1980s, followed by Kochi around 2000 (Kubota et al. 2020 ). However, further investigation into the phylogenetic diversities of the other segments and isolates in other regions is needed to elucidate the PerMV genetic structure in Japan. In contrast to the very low genetic diversity observed in 16 PCLSaV isolates collected from 13 prefectures in Japan (Takeyama et al. 2022 ), the PerMV isolates in four prefectures exhibited much higher genetic diversities. This could result from significant differences in the origin, spread, and distribution of these viruses. An interesting discovery in our survey was that PerMV isolates, likely lacking RNA2, were found in all four prefectures (Table 1 ). Although the three RNA2-specific primer sets used for RT-PCR were designed based on the Kochi_Nankoku_2011 isolate, the failure to detect RNA2 could not be attributed to sequence mismatches, as such RNA2-negative isolates were also found in Kochi and Oita prefectures. If these isolates indeed lack RNA2, they would not express GPs, suggesting that GPs are dispensable for systemic infection in shiso plants. Elliovirus GPs on the surface of enveloped virions play pivotal roles in virus entry, virion assembly, and vector transmission (Hulswit et al. 2021 ). Mutants of tomato spotted wilt virus (TSWV), an orthotospovirus lacking enveloped virions and thrips-borne transmissibility, can be obtained through repeated mechanical passages (de Oliveira Resende et al. 1991 ; Ie 1982 ; Nagata et al. 2000 ; Verkleij and Peters 1983 ). These mutants harbor nucleotide substitutions or deletions in the GP ORF of the M RNA (Sin et al. 2005 ), demonstrating that orthotospovirus GPs are dispensable for systemic infection in plants after they invade plant cells. Since the M RNA encodes not only GPs but also NSm,which is required for cell-to-cell movement (Feng et al. 2023 ), TSWV mutants lacking the M RNA should no longer infect systemically. Unlike orthotospoviruses, fimovirus RNA2 encodes only GP, and the MP is encoded by RNA4. Thus, mutations, deletion of partial sequence, or complete loss of RNA2 might not affect systemic infection ability, suggesting the potential existence of fimovirus isolates lacking RNA2, given that GPs are unnecessary for replication and cell-to-cell and long-distance movement in an infected plant. Furthermore, once such RNA2-lacking isolates emerge from a wild-type, i.e., RNA2-retaining isolate in an infected plant, they may replicate and move faster than the wild-type, although they would not be transmissible to another host by mites. To our knowledge, the presence of fimovirus isolates lacking RNA2 under natural conditions (i.e., not under experimental condition) has not been reported yet. Analogous to orthotospoviruses, it is also expected that fimovirus isolates lacking RNA2 would lose the enveloped virion and mite-borne transmissibility, making the occurrence of RNA2-lacking isolates unusual except under specific conditions. However, we have observed that RNA2-lacking PerMV isolates can be reproducibly obtained by maintaining PerMV in infected shiso plants without infestation of PRMs (Kubota et al. 2024 ). Notably, the RNA2-lacking PerMV isolates were found only in production greenhouses, where insecticides were periodically applied (Table 1 ). Furthermore, Verchot et al. ( 2020 ) have reported that an agrobacteium-mediated infectious clone of RRV, that was composed only of the constructs of RNA1 (RdRp), RNA3 (NP), and RNA5 (whose ORF was replaced with that of a fluorescent protein), can infect in an agro-inoculated Nicotiana benthamiana leaf, indicating that RNA2 (GP) is dispensable at least for replication of fimovirus. Near-complete nucleotide sequence analysis of the four isolates from Ibaraki, Aichi, and Oita prefectures, compared with previously reported isolates Kochi_Nankoku_2011 and IS, clearly demonstrated that RNAs encoding homologous proteins (i.e., RNA3ab and RNA6a–c/7) were conserved in all five investigated Japanese isolates. Although Oh et al. ( 2023 ) reported only seven RNA segments for the IS isolate, we cannot specify whether it indeed harbors only seven RNAs, or possesses a total of 10 segments, including unreported homologs of RNA3 and RNA6. Regardless, all five Japanese isolates, collected from different areas and belonging to genetically distinct clades (Fig. 2 ), commonly possessed ten RNAs, including ones encoding homologous proteins. This strongly supports that all ten PerMV proteins are indispensable for the virus’s infection cycle. Thus, understanding the molecular functions of these proteins is of great importance. Investigating the differences in biological properties, such as host range, symptomatology, and transmissibility, between isolates belonging to different clades could be of interest. Another intriguing question is whether reassortants can be obtained or if some genetic exclusion occurs when two isolates from different clades infect a plant. Since the first identification of a fimovirus two decades ago (Benthack et al. 2005 ), the number of species in the Emaravirus genus of the Fimoviridae family has rapidly expanded to 32, surpassing those of the Orthotospovirus (26) and Tenuivirus (8) genera (Kuhn et al. 2023 ). Fimoviruses appear to be more diverse than the other two; for instance, while the hosts of orthotospoviruses and tenuiviruses are primarily confined to herbaceous dicots and monocots, respectively, fimoviruses have a broader host range that includes herbaceous and woody dicots as well as monocots. Consequently, based on the high diversity in encoded proteins and genome sequences, the separation of the Emaravirus genus into two or more genera has been proposed (Rehanek et al. 2022 ). The genome’s plasticity, largely due to its monocistronic nature, may be a key driver for the rapid diversification and evolutionary adaptation of fimoviruses. Declarations List of Supplementary files Supplementary Table S1 : Primers list Supplementary Figure S1 : Geographical map Compliance with Ethical Standards This article does not contain any studies with human participants or animals performed by any of the authors. Conflicts of interest The authors declare that they have no conflict of interest. Acknowledgments The authors are grateful to M. Yasunaga, T. Usugi, M. Kubota, T. Kawano, H. Tanaka, Y. Shimomoto, and K. Tanaka for the collection of shiso samples, and would like to thank reviewers for valuable comments that improved our manuscript. This work was partly supported by JSPS KAKENHI Grant Number JP21K05605. References Bahat Y, Alter J, Dessau M (2020) Crystal structure of tomato spotted wilt virus G N reveals a dimer complex formation and evolutionary link to animal-infecting viruses. 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Plant Dis 105:1234. https://doi.org/10.1094/PDIS-09-20-2035-PDN Kubota K, Chiaki Y, Yanagisawa H, Yamasaki J, Horikawa H, Tsunekawa K, Morita Y (2021b) Novel degenerate primer sets for the detection and identification of emaraviruses reveal new chrysanthemum species. J Virol Methods 288:113992. https://doi.org/10.1016/j.jviromet.2020.113992 Kubota K, Usugi T, Tomitaka Y, Shimomoto Y, Takeuchi S, Kadono F, Yanagisawa H, Chiaki Y, Tsuda S (2020) Perilla mosaic virus is a highly divergent emaravirus transmitted by Shevtchenkella sp. (Acari: Eriophyidae). Phytopathology 110:1352–1361. https://doi.org/10.1094/PHYTO-01-20-0013-R Kubota K, Yanagisawa H, Chiaki Y, Yamasaki J, Horikawa H, Tsunekawa K, Morita Y, Kadono F (2021c) Complete nucleotide sequence of chrysanthemum mosaic-associated virus, a novel emaravirus infecting chrysanthemum. Arch Virol 166:1241–1245. https://doi.org/10.1007/s00705-021-04979-2 Kuhn JH, Abe J, Adkins S, et al. (2023) Annual (2023) taxonomic update of RNA-directed RNA polymerase-encoding negative-sense RNA viruses (realm Riboviria : kingdom Orthornavirae : phylum Negarnaviricota ). J Gen Virol 104. https://doi.org/10.1099/jgv.0.001864 Liu H, Wang G, Yang Z, Wang Y, Zhang Z, Li L, Waqas M, Hong N, Liu H, Wang G, Hong N, Hong J, Zhang J, Xu L, Qi L (2020) Identification and characterization of a pear chlorotic leaf spot-associated virus, a novel emaravirus associated with a severe disease of pear trees in China. Plant Dis 104:2786–2798. https://doi.org/10.1094/PDIS-01-20-0040-RE Lu Y, McGavin W, Cock PJA, Schnettler E, Yan F, Chen J, MacFarlane S (2015) Newly identified RNAs of raspberry leaf blotch virus encoding a related group of proteins. J Gen Virol 96:3432–3439. https://doi.org/10.1099/jgv.0.000277 Martin DP, Varsani A, Roumagnac P, Botha G, Maslamoney S, Schwab T, Kelz Z, Kumar V, Murrell B (2021) RDP5: a computer program for analyzing recombination in, and removing signals of recombination from, nucleotide sequence datasets. Virus Evol 7:veaa087. https://doi.org/10.1093/ve/veaa087 Muhire BM, Varsani A, Martin DP (2014) SDT: a virus classification tool based on pairwise sequence alignment and identity calculation. PLOS ONE 9:e108277. https://doi.org/10.1371/journal.pone.0108277 Nabeshima T, Abe J (2021) High-throughput sequencing indicates novel varicosavirus,emaravirus, and deltapartitivirus infections in Vitis coignetiae . Viruses 13:827. https://doi.org/10.3390/v13050827 Nagata T, Inoue-Nagata AK, Prins M, Goldbach R, Peters D (2000) Impeded thrips transmission of defective tomato spotted wilt virus isolates. Phytopathology 90:454–459. https://doi.org/10.1094/PHYTO.2000.90.5.454 Oh BG, Byun H-S, Ju H-J, Yoon J-Y (2023) First report of perilla mosaic emaravirus infecting Perilla frutescens in South Korea. Plant Dis 107:2269. https://doi.org/10.1094/PDIS-09-22-2035-PDN Rehanek M, Karlin DG, Bandte M, Al Kubrusli R, Nourinejhad Zarghani S, Candresse T, Büttner C, von Bargen S (2022) The complex world of emaraviruses—challenges, insights, and prospects. Forests 13:1868. https://doi.org/10.3390/f13111868 Saitou N, Nei M (1987) The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4:406–425. https://doi.org/10.1093/oxfordjournals.molbev.a040454 Shimomoto Y, Okada T, Ikeda K, Tatara A, Hasegawa Y, Yanagisawa H, Takeyama S, Hayashi K, Yano K, Morita Y, Kubota K (2022) Japanese star anise ringspot-associated virus is a distinct emaravirus transmitted by the eriophyid mite (the family Diptilomiopidae). J Gen Plant Pathol 88:69–80. https://doi.org/10.1007/s10327-021-01038-1 Sin S-H, McNulty BC, Kennedy GG, Moyer JW (2005) Viral genetic determinants for thrips transmission of tomato spotted wilt virus. Proc Natl Acad Sci U S A 102:5168–5173. https://doi.org/10.1073/pnas.0407354102 Suzuki T, Kadono F, Kagiwada S, Tatara A (2018) Overwintering ecology and reproductive diapause condition of Shevtchenkella sp. (Acari: Eriophyidae). Annu Rep Kanto-Tosan Plant Prot Soc 65:125–129 (in Japanese with English abstract) Takeyama S, Suzuki R, Kohyama M, Chiaki Y, Toda S, Kubota K (2022) Genetic diversity of Japanese isolates of pear chlorotic leaf spot-associated virus. Jpn J Phytopathol 88:1–11 (in Japanese with English abstract). https://doi.org/10.3186/jjphytopath.88.1 Tamura K, Stecher G, Kumar S (2021) MEGA11: molecular evolutionary genetics analysis version 11. Mol Biol Evol 38:3022–3027. https://doi.org/10.1093/molbev/msab120 Tatineni S, McMechan AJ, Wosula EN, Wegulo SN, Graybosch RA, French R, Hein GL (2014) An eriophyid mite-transmitted plant virus contains eight genomic RNA segments with unusual heterogeneity in the nucleocapsid protein. J Virol 88:11834–11845. https://doi.org/10.1128/JVI.01901-14 Thompson JD, Higgins DG, Gibson TJ (1994) CLUSTALW: improving the sensitivity of progressive multiple sequence alignment through weighing, position specific gap penalties and weight matrix choice. Nucleic Acids Res 22:673. Verchot J, Herath V, Urrutia CD, Gayral M, Lyle K, Shires MK, Ong K, Byrne D (2020) Development of a reverse genetic system for studying rose rosette virus in whole plants. Mol Plant Microbe Interact 10:1209–1221. https://doi.org/10.1094/MPMI-04-20-0094-R Verkleij FN, Peters D (1983) Characterization of a defective form of tomato spotted wilt virus. J Gen Virol 64:677–686. https://doi.org/10.1099/0022-1317-64-3-677 Supplementary Files SupplementaryTableandFigureRevised240821.pptx Cite Share Download PDF Status: Published Journal Publication published 08 Oct, 2024 Read the published version in Journal of General Plant Pathology → Version 1 posted Reviewers agreed at journal 25 Aug, 2024 Reviewers invited by journal 25 Aug, 2024 Editor assigned by journal 21 Aug, 2024 First submitted to journal 20 Aug, 2024 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. 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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-4818253","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":343004020,"identity":"06668aea-9872-4f76-bd5d-5154c8d7b4d7","order_by":0,"name":"Kenji Kubota","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA10lEQVRIie3QPwuCQBjH8Z8c3BS4ngTnW7gQej2GUIs2O1g4OUmu9jJ6ByeBLdVbSGgPp3CKlIKgwT9bxH2Xg4MPz90DqFQ/GMOoOWwOkhXvO9mDSNgWqCOGkVn4IR0ZqZddy2C+SCJCyworDu3UPmbMlo6QueulOSHbGAcLONuthDN3yiT1vfCS7AmQz0Ic21/4Ig9/YdZT+pFxQ7LItcWLBN3EiG+OOG3mk11NtFhIi3b9hR28rPDvjslrgspfc511bOwrsQdlxyECWAN6PIyoVCrV3/cE0lhFYeCjXPwAAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0002-6324-0891","institution":"Institute for Plant Protection, NARO","correspondingAuthor":true,"prefix":"","firstName":"Kenji","middleName":"","lastName":"Kubota","suffix":""},{"id":343004021,"identity":"03dad206-1898-44e4-bc45-fef8fc15742c","order_by":1,"name":"Yuya Chiaki","email":"","orcid":"","institution":"Institute for Plant Protection, NARO","correspondingAuthor":false,"prefix":"","firstName":"Yuya","middleName":"","lastName":"Chiaki","suffix":""}],"badges":[],"createdAt":"2024-07-28 23:35:56","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4818253/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4818253/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s10327-024-01203-2","type":"published","date":"2024-10-08T15:57:32+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":63113108,"identity":"d7605145-866e-4acd-aa18-40b5fdbd3ccb","added_by":"auto","created_at":"2024-08-23 09:16:09","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":151104,"visible":true,"origin":"","legend":"\u003cp\u003eNeighbor-joining phylogenetic trees of partial nucleotide sequences of RNAs 1–4 of field isolates of perilla mosaic virus. Segments (a) RNA1 (786 nt), (b) RNA2 (661 nt), (c) RNA3a and 3b (520 nt), and (d) RNA4 (878 nt) are displayed. The scale bars represent the number of nucleotide substitutions per site. The number at each node indicates a bootstrap percentage based on 1000 pseudoreplicates (only \u0026gt;70% are shown). The colors of the dots correspond to the prefectures where each isolate was collected. Clades containing isolates collected from Western Japan (i.e., Oita and Kochi prefectures) or including ones from Eastern Japan (i.e., Ibaraki prefecture) are highlighted in green and orange, respectively. From the isolate names, prefixes indicating prefecture namesare omitted (e.g.,Kochi_Nankoku_2011 \u0026gt; Nankoku_2011) (see Table 1). In the color matrices, each colored cell represents a percentage identity score between two sequences (one indicated horizontally to the left and the other vertically at the bottom).\u003c/p\u003e","description":"","filename":"FiguresRevised2408211.png","url":"https://assets-eu.researchsquare.com/files/rs-4818253/v1/5b5f691c6645881d00efa197.png"},{"id":63113105,"identity":"4a297822-00c4-4c03-be3e-0f9978346c47","added_by":"auto","created_at":"2024-08-23 09:16:08","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":78619,"visible":true,"origin":"","legend":"\u003cp\u003eNeighbor-joining phylogenetic trees of near-complete nucleotide sequences of field isolates of perilla mosaic virus. Segments (a) RNA1 (7131 nt), (b) RNA2 (2048 nt), (c) RNA3a and 3b (1036 and 1034 nt), (d) RNA4 (1250 nt), (e) RNA5 (1022 nt), (f) RNAs 6a, 6b, and 6c (981, 931, and 971 nt, respectively), and (g) RNA7 (954 nt) are presented. The scale bars represent the number of nucleotide substitutions per site. The number at each node indicates a bootstrap percentage based on 1000 pseudoreplicates (only \u0026gt;70% are shown). The dots and boxes are colored in the same way as in Figure 1.\u003c/p\u003e","description":"","filename":"FiguresRevised2408212.png","url":"https://assets-eu.researchsquare.com/files/rs-4818253/v1/8b048c85373b7301220c6b51.png"},{"id":66597155,"identity":"11af74ae-f172-499f-99eb-018d7312f006","added_by":"auto","created_at":"2024-10-14 16:07:47","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":799938,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4818253/v1/099dd361-cc49-476c-9f17-6cd943647fec.pdf"},{"id":63113107,"identity":"ad2ae303-c633-4c47-a7bd-35a19128007c","added_by":"auto","created_at":"2024-08-23 09:16:08","extension":"pptx","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":666304,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTableandFigureRevised240821.pptx","url":"https://assets-eu.researchsquare.com/files/rs-4818253/v1/19000d0e246bbb1488d89113.pptx"}],"financialInterests":"","formattedTitle":"Nucleotide sequence survey of perilla mosaic virus isolates in Japan unveils complex genetic structure and conserved RNA segments encoding homologous protein groups","fulltext":[{"header":"Introduction","content":"\u003cp\u003eFimoviruses, also referred to as emaraviruses, are plant-infecting, multipartite, negative-sense RNA viruses, and are members of the \u003cem\u003eFimoviridae\u003c/em\u003e family, consisting of only the \u003cem\u003eEmaravirus\u003c/em\u003e genus. (Rehanek et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). The number of species in the \u003cem\u003eFimoviridae\u003c/em\u003e family has been increasing due to the continuous discovery of new species, reaching up to 32 species (Digiaro et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Kuhn et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). While not all fimoviruses are transmitted this way, some have been shown to be transmitted by specific eriophyid mites (families Eriophydae and Diptilomiopidae) (Rehanek et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eFimoviruses are related to plant-infecting viruses in the \u003cem\u003eclass Bunyaviricetes\u003c/em\u003e, which encompasses orthotospoviruses (family \u003cem\u003eTospoviridae\u003c/em\u003e, order \u003cem\u003eElliovirales\u003c/em\u003e), fimoviruses (\u003cem\u003eElliovirales\u003c/em\u003e), and tenuiviruses (family \u003cem\u003ePhenuiviridae\u003c/em\u003e, order \u003cem\u003eHareavirales\u003c/em\u003e) (Kormelink et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Unlike orthotospoviruses and tenuiviruses, fimoviruses\u0026rsquo; genome varies in RNA segments (five to ten) depending on species, and RNA segments are primarily monocistronic, not ambisense. Among fimoviruses segments, RNA1 to RNA4 encode proteins P1; RNA-dependent RNA polymerase (RdRp), P2; glycoprotein precursor (GPP), P3; nucleocapsid protein (NP), and P4; movement protein (MP), respectively. While each fimovirus harbors additional protein-encoding RNA segments (e.g., RNA5 encoding protein P5), their amino acid sequences are less conserved, and their functions remain unclear (Rehanek et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). However, some fimovirus proteins share amino acid sequence homology among different species [e.g., P5s of rose rosette virus (RRV), fig mosaic virus (FMV), etc.] [summarized by Rehanek et al. (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2022\u003c/span\u003e)], suggesting they play crucial roles in virus infection and transmission. Another unique fimovirus genome feature is the presence of RNA segments encoding likely redundant proteins. High Plains wheat mosaic virus (HPWMoV) was the first reported to harbor two RNA3 variants, both encoding putative NP (P3) with ~\u0026thinsp;80% amino acid sequence identities (Tatineni et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2014\u003c/span\u003e), followed by the discovery of three other fimoviruses encoding two P3s (Buzkan et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Kubota et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2020\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2021c\u003c/span\u003e). As FMV encodes only one P3, enabling full ribonucleoprotein complex composition (Izhaki-Tavor et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), the reason for some fimoviruses encoding two NP variants remains unclear. Moreover, some fimoviruses possess two to four homologous proteins; e.g., P6, P7, P8a, and P8b of raspberry leaf blotch virus (Lu et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2015\u003c/span\u003e) or P5a, P5b, and P7 of Pistachia virus B (PiVB) (Buzkan et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). At least eight fimoviruses harbor RNAs encoding homologous protein groups (Rehanek et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Although the functions and reasons for these homologous proteins\u0026rsquo; presence are unknown, they likely hold biological significance and importance.\u003c/p\u003e \u003cp\u003eFimoviruses possess enveloped virions, also known as double-membrane-bound bodies (DMBs) (Rehanek et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Similar to the role of glycoproteins (G\u003csub\u003eN\u003c/sub\u003e/G\u003csub\u003eC\u003c/sub\u003e) of orthotospoviruses in enveloped virion formation (Bahat et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), fimovirus GPs are presumed to be responsible for DMB formation (Rehanek et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Among fimoviruses, perilla mosaic virus (PerMV, the species \u003cem\u003eEmaravirus perillae\u003c/em\u003e)has the most RNA segments, totaling ten; RNAs 1, 2, 3a, 3b, 4, 5, 6a, 6b, 6c, and 7 (Kubota et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Proteins P1 to P4 are putative RdRp, GPP, NP, and MP, respectively. Proteins P3a and P3b are putative NPs, sharing 83.0% amino acid sequence identity. While redundancy in RNA3 has been observed in some other fimoviruses [HPWMoV, PiVB, and chrysanthemum mosaic-associated virus (ChMaV)] (Buzkan et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Kubota et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2021c\u003c/span\u003e; Tatineni et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2014\u003c/span\u003e), its biological significance remains unclear. PerMV P5 contains a domain homologous to Glu2-Pro, a glutamic protease encoded by a sadwavirus, and Glu-2 Pro is shared by some other fimovirus proteins (Rehanek et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). P6a\u0026ndash;c of PerMV are proteins with molecular masses of 26.8\u0026ndash;30.2 kDa, sharing amino acid sequence identities of 24.4\u0026ndash;62.3% with each other (Kubota et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). PerMV P7, a 28.5 kDa protein, shares weak sequence homology (Rehanak et al. 2022). However, the molecular functions of P5, P6s, and P7 of PerMV remain elusive to date.\u003c/p\u003e \u003cp\u003ePerMV was first identified in glasshouse grown shiso (\u003cem\u003ePerilla frutescence\u003c/em\u003e L.) plants exhibiting mosaic symptoms in Kochi Prefecture, Japan (Kubota et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). It has also been found in other shiso production areas in Aichi, Ibaraki, Oita, and Ehime prefectures (Ehime Plant Protection Office \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). PerMV is effectively transmitted by the perilla rust mite (PRM), \u003cem\u003eAculops thymi\u003c/em\u003e (Nalepa 1889) (previously referred to as \u003cem\u003eShevtchenkella\u003c/em\u003e sp.) (Kadono et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Kubota et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), and PRM has also been discovered in cultivated or wild-grown shiso plants in Kochi, Aichi, and Oita prefectures (Suzuki et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2018\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn Japan, six fimoviruses have been reported: PerMV, FMV (\u003cem\u003eEmaravirus fici\u003c/em\u003e), pear chlorotic leaf spot-associated virus (PCLSaV, \u003cem\u003eEmaravirus pyri\u003c/em\u003e), ChMaV (\u003cem\u003eEmaravirus chrysanthemi\u003c/em\u003e), Vitis emaravirus (VEV, \u003cem\u003eEmaravirus vitis\u003c/em\u003e), and Japanese star anise ringspot-associated virus (JSARaV, \u003cem\u003eEmaravirus illicii\u003c/em\u003e) (Ishikawa et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Kubota et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2021a\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2021b\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2021c\u003c/span\u003e; Nabeshima and Abe \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Shimomoto et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Takeyama et al. (\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) reported that the nucleotide sequences of full-length ORFs encoded by RNAs 1 to 5 of 16 PCLSaV isolates collected from the Tohoku to Kyushu regions in Japan showed significantly lower genetic diversity compared to three Chinese isolates (Liu et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). This suggests that PCLSaV might have been introduced from China and spread only recently in Japan. Chlorotic leaf spot symptoms of pear trees, presumably caused by PCLSaV infection, have been noticed only after ~\u0026thinsp;2010 in Japan. In contrast, mosaic diseases on shiso crops caused by PerMV occurred in the late 1980s and around 2000 in Aichi and Kochi prefectures, respectively (Kubota et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Therefore, investigating the genetic diversity of PerMV isolates in Japan provides significant insight into the origin and distribution of PerMV. The first objective of this study is to overview the genetic diversity of PerMV populations in Japan by comparing nucleotide sequences of RNAs 1, 2, 3a, 3b, and 4 of 21 field isolates of PerMV collected from Ibaraki, Aichi, Kochi, and Oita prefectures from 2011 to 2015.\u003c/p\u003e \u003cp\u003eDuring our above investigation, an occurrence of PerMV in South Korea was reported. The RNA segments of the South Korean isolate (PerMV-IS) shared nucleotide sequence identities of 92.09\u0026ndash;97.37% with the Japanese isolate, Kochi_Nankoku_2011 (Oh et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). However, the nucleotide sequences of PerMV-IS were reported only for seven segments (each of RNAs 1 to 7), raising a question about whether PerMV indeed possesses 10 segments as we previously reported (Kubota et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). The complete nucleotide sequences of PerMV in Japan were determined only for one isolate, i.e., Kochi_Nankoku_2011, which harbors two RNA3s (RNA3a and 3b) and three RNA6s (RNA6a\u0026ndash;c). These RNAs and proteins may share redundant functions and be dispensable for infection and transmission cycles; hence, isolates lacking these RNAs might exist. To clarify this possibility, we further attempted to amplify and determine the near-complete nucleotide sequences of all RNA segments of selected isolates in Ibaraki, Aichi, and Oita prefectures and also compared them to those of the previously identified Kochi and South Korean isolates.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eCollection of virus isolates and sequencing\u003c/h2\u003e \u003cp\u003eLeaves of green varieties of shiso plants exhibiting typical mosaic symptoms were collected from production greenhouses, an experimental greenhouse, or home gardens in Kochi, Oita, Aichi, and Ibaraki prefectures in Japan between 2011 and 2015 (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, Supplementary Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). Total RNA was extracted from the collected leaves using Trizol Reagent (ThermoFisher Scientific, Waltham, MA, USA), as previously described (Kubota et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). To obtain cDNAs of the PerMV segments, the total RNAs underwent reverse transcription using PrimeScript Reverse Transcriptase (TakaraBio, Shiga, Japan) with primers complementary to the 11-nt conserved 5\u0026prime; and 3\u0026prime; termini of RNA segments of PerMV, essentially as previously described (Kubota et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Using these cDNAs as templates, attempts were made to amplify the cDNA of partial nucleotide sequences of RNAs 1, 2, 3a, 3b, and 4 of all the collected isolates by PCR, followed by direct sequencing with primers shown in Supplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e. Additionally, for a total of four selected isolates (two from Ibaraki and one each from Aichi and Oita prefectures), near full-length sequences of all ten segments (hereafter, near-complete sequence) were determined by RT-PCR amplification followed by direct sequencing with previously reported primers (Kubota et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). PCR amplification was conducted using Tks Gflex DNA Polymerase (TakaraBio) or KOD One PCR Master Mix (Toyobo, Osaka, Japan). The amplified products underwent purification using ExoSAP-IT (ThermoFisher) and were then subjected to a direct sequencing reaction utilizing a BigDye Terminator v3.1 Cycle Sequencing Kit (ThermoFisher). The primers indicated in Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e, along with those previously outlined by Kubota et al. (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), were employed to determine partial or near-complete sequences. The sequencing process was executed using an ABI 3130xl Genetic Analyzer (Applied Biosystems, Waltham, MA, U.S.A.).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eCollection of field isolates of perilla mosaic virus utilized in the study\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePlace (Condition)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eYear\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eIsolate\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eRNA\u003c/p\u003e \u003cp\u003eamplified\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eSequence\u003c/p\u003e \u003cp\u003edetermined\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eAccession\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eReference\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNankoku, Kochi, Japan (Production greenhouse)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e2011\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eKochi_Nankoku_2011\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1\u0026ndash;7\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eComplete\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eNC_078367\u0026ndash;NC_078376\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eKubota et al. (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2020\u003c/span\u003e)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eKochi_Nankoku_2011_2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1, 3a, 3b, 4\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003ePartial\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eLC830810\u0026ndash;LC830813\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eThis study\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e2015\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eKochi_Nankoku_2015_1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1\u0026ndash;4\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003ePartial\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eLC776290\u0026ndash;LC776294\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eThis study\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eKochi_Nankoku_2015_2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1, 3a, 3b, 4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003ePartial\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eLC776295\u0026ndash;LC776298\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eThis study\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eOita, Oita, Japan\u003c/p\u003e \u003cp\u003e(Production greenhouse)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e2013\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eOita_Oita_2013_1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1\u0026ndash;7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eNear-complete\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eLC830770\u0026ndash;LC830779\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eThis study\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e2015\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eOita_Oita_2015_1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1\u0026ndash;4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003ePartial\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eLC776304\u0026ndash;LC776308\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eThis study\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eOita_Oita_2015_2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1\u0026ndash;4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003ePartial\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eLC776309\u0026ndash;LC776313\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eThis study\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eOita_Oita_2015_3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1, 3a, 3b, 4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003ePartial\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eLC776314\u0026ndash;LC776317\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eThis study\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eOita_Oita_2015_4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1\u0026ndash;4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003ePartial\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eLC776318\u0026ndash;LC776322\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eThis study\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eToyohashi, Aichi, Japan\u003c/p\u003e \u003cp\u003e(Production greenhouse)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e2013\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAichi_Toyohashi_2013_1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1\u0026ndash;4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003ePartial\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eLC776323\u0026ndash;LC776327\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eThis study\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAichi_Toyohashi_2013_2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1\u0026ndash;7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eNear-complete\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eLC830780\u0026ndash;LC830789\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eThis study\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e2015\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAichi_Toyohashi_2015_1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1\u0026ndash;4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003ePartial\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eLC776333\u0026ndash;LC776337\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eThis study\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAichi_Toyohashi_2015_2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1, 3a, 3b, 4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003ePartial\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eLC776338\u0026ndash;LC776341\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eThis study\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eUshiku, Ibaraki, Japan\u003c/p\u003e \u003cp\u003e(Experimental greenhouse)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e2013\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eIbaraki_Ushiku_2013_1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1\u0026ndash;4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003ePartial\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eLC776347\u0026ndash;LC776351\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eThis study\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eIbaraki_Ushiku_2013_2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1\u0026ndash;4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003ePartial\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eLC776352\u0026ndash;LC776356\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eThis study\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNamegata, Ibaraki, Japan\u003c/p\u003e \u003cp\u003e(Production greenhouse)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e2014\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eIbaraki_Namegata_2014_1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1\u0026ndash;7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eNear complete\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eLC830800\u0026ndash;LC830809\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eThis study\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e2015\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eIbaraki_Namegata_2015_1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1\u0026ndash;4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003ePartial\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eLC776367\u0026ndash;LC776371\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eThis study\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eIbaraki_Namegata_2015_2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1, 3a, 3b, 4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003ePartial\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eLC776372\u0026ndash;LC776375\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eThis study\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMiho, Ibaraki, Japan (Home garden, wild grown)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e2013\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eIbaraki_Miho_2013_1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1\u0026ndash;7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eNear-complete\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eLC830790\u0026ndash;LC830799\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eThis study\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e2015\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eIbaraki_Miho_2015_1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1\u0026ndash;4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003ePartial\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eLC776357\u0026ndash;LC776361\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eThis study\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eTsukuba, Ibaraki, Japan (Flower bed)\u003c/p\u003e \u003cp\u003e(Home garden, wild grown)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e2015\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eIbaraki_Tsukuba_2015_1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1\u0026ndash;4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003ePartial\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eLC776376\u0026ndash;LC776380\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eThis study\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eIbaraki_Tsukuba_2015_2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1\u0026ndash;4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003ePartial\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eLC833855, LC776381\u0026ndash;2, LC833856, LC776383\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eThis study\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIseo-myeon, South Korea\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e2021\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eIS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1\u0026ndash;7e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eNear-complete\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eLC721296\u0026ndash;LC721302\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eOh et al. (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2023\u003c/span\u003e)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"7\"\u003e\u003csup\u003ea\u003c/sup\u003e Isolates sharing with same place names (e.g., Kochi_Nankoku) were collected in the same greenhouse or a home garden, except that Ibaraki_Tukuba_2015_1 and Ibaraki_Tsukuba_2015_2.\u003c/td\u003e\u003c/tr\u003e \u003ctr\u003e\u003ctd colspan=\"7\"\u003e\u003csup\u003eb\u003c/sup\u003e RNAs 1, 2, 3a, 3b, 4, 5, 6a, 6b, 6c, and 7.\u003c/td\u003e\u003c/tr\u003e \u003ctr\u003e\u003ctd colspan=\"7\"\u003e\u003csup\u003ec\u003c/sup\u003e Among RNAs 1, 2, 3a, 3b, and 4, RNA2 was not amplified by RT-PCR.\u003c/td\u003e\u003c/tr\u003e \u003ctr\u003e\u003ctd colspan=\"7\"\u003e\u003csup\u003ed\u003c/sup\u003e RNAs 1, 2, 3a, 3b, and 4.\u003c/td\u003e\u003c/tr\u003e \u003ctr\u003e\u003ctd colspan=\"7\"\u003e\u003csup\u003ee\u003c/sup\u003e Only RNAs 1, 2, 3, 4, 5, 6, and 7 were reported for the isolate IS.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eSequence analyses\u003c/h2\u003e \u003cp\u003eNucleotide sequence reads were assembled using CodonCode Aligner Software (CodonCode Corporation, Centerville, MA, USA). The analysis utilized a total of 23 isolates, including the previously reported Kochi_Nankoku_2011 (Kubota et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; NC_078364\u0026ndash;NC_078373) and IS (Oh et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; LC721296\u0026ndash;LC721303). Nucleotide and amino acid sequence identities were calculated using SDTv1.2 (Muhire et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2014\u003c/span\u003e), with the alignment option set to ClustalW (Thompson et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e1994\u003c/span\u003e). Nucleotide sequences were aligned using ClustalW, as implemented in MEGA11 (Tamura et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), and phylogenetic trees were reconstructed using the neighbor-joining method (Saitou and Nei \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e1987\u003c/span\u003e) with Kimura\u0026rsquo;s two parameter model (Kimura \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e1980\u003c/span\u003e). The phylogeny was tested by performing 1,000 bootstrap pseudoreplicates. Recombination analysis of isolates with nearly complete sequences (five Japanese and one South Korean isolates) was conducted using RDP5 (Martin et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eGenetic diversities of the Japanese isolates based on partial RNA1\u0026ndash;RNA4 sequences\u003c/h2\u003e \u003cp\u003eOut of the 21 newly collected isolates, 17 yielded partial nucleotide sequences of RNAs 1, 2, 3a, 3b, and 4. Full-length RT-PCR products for RNA2 were not detected from five isolates (Oita_Oita_2015_3, Kochi_Nankoku_2011_2, Kochi_Nankoku_2015_2, Aichi_Toyohashi_2015_2, and Ibaraki_Namegata_2015_2). Further attempts to amplify the regions nts 948\u0026ndash;1369 or nts 1258\u0026ndash;1714 using two other primer sets shown in Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e yielded consistent results across the 21 tested isolates (positive, 16; negative, 5).\u003c/p\u003e \u003cp\u003eThe lengths of the obtained partial nucleotide sequences used for the subsequent analysis are as follows: RNA1, 786 nt (corresponding to nts 3375\u0026ndash;4112 of Kochi_Nankoku_2011); RNA2, 661 or 658 nt (nts1064\u0026ndash;1724); RNA3a, 520 nt (nts 430\u0026ndash;949); RNA3b, 520 nt (nts 429\u0026ndash;948); RNA4, 878 nt (nts 391\u0026ndash;1268) (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). RNA2 of 14 isolates was 661 nt, while the isolates Aichi_Toyobashi_2013_1 and Aichi_Toyohashi_2013_2 harbored a common 3-nt deletion, leading to a deletion of the aspartic acid residue at aa 204 of P2. Nucleotide sequence identities among the Japanese isolates were 93.7\u0026ndash;100% for RNA1, 91.1\u0026ndash;100% for RNA2, 92.5\u0026ndash;100% for RNA3a, 91.7\u0026ndash;100% for RNA3b, and 93.7\u0026ndash;100% for RNA4.\u003c/p\u003e \u003cp\u003ePhylogenetic analysis of the partial nucleotide sequences of RNAs 1\u0026ndash;4 from the PerMV isolates revealed that the segments of the PerMV isolates were divided into two clades (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The sequences of RNA1 fell into one clade consisting of isolates collected from Ibaraki and Aichi prefectures, and another clade composed of isolates from Kochi and Oita prefectures (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). The isolate IS from South Korea grouped with the latter, although it appeared distantly related to the Japanese isolates. However, RNA2 displayed a different phylogenetic pattern; one clade consisted of isolates only from Ibaraki, and another was composed of isolates from Aichi, Kochi, and Oita prefectures (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb). The phylogeny of RNA3a and RNA3b was similar to that of RNA2 and RNA1, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec), except that RNA3a of the isolate Aichi_Toyohashi_2015_1 fell into the clade with the isolates from Ibaraki. The phylogeny of RNA4 was similar to that of RNA2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eNear-complete nucleotide sequences of the four isolates\u003c/h2\u003e \u003cp\u003eWe determined the near-complete nucleotide sequences of all segments from four selected isolates, namely Oita_Oita_2013_1, Aichi_Toyohashi_2013_2, Ibaraki_Miho_2013_1, and Ibaraki_Namegata_2014_1, collected from distant areas in Japan. PCR amplification from the cDNAs of these isolates generated products of corresponding sizes for each segment (data not shown), and their nucleotide sequences were determined through direct sequencing. The lengths of the nucleotide sequences used for alignment were as follows: RNA1, 7131 nt (corresponding to nts 115\u0026ndash;7245 of Kochi_Nankoku_2011); RNA2, 2048 nt (nts 23\u0026ndash;2070); RNA3a, 1036 nt (nts 23\u0026ndash;1058); RNA3b, 1034 nt (nts 23\u0026ndash;1056); RNA4, 1250 nt (nts 23\u0026ndash;1272), RNA5, 1022 nt (nts 23\u0026ndash;1044); RNA6a, 981 nt (nts 126\u0026ndash;1106); RNA6b, 931 nt (nts 145\u0026ndash;1075); RNA6c, 971 nt (nts 58\u0026ndash;1028); RNA7, 954 nt (nts 23\u0026ndash;976) (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). All sequenced segments encoded a single protein, sharing amino acid sequence identities with proteins P1 to P7 of Kochi_Nankoku_2011 (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Notably, the segments encoding homologous proteins (i.e., RNAs 3a/b and RNAs 6a\u0026ndash;c/7) were present in all isolates, regardless of collection area, year of collection, or position in phylogenetic clades. Among the proteins, P4 was the most highly conserved (\u0026gt;\u0026thinsp;99.4%), while P6c was the least conserved (85.0\u0026ndash;98.3%). Generally, two isolates from Ibaraki showed lower identities to Kochi_Nankoku_2011 compared to those from Oita and Aichi, but P1 and P3b of Aichi_Toyohashi_2013_2 showed lower identities. Consistent with these results, phylogenetic analysis using near-complete nucleotide sequences indicated that each segment divided into two groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e), as observed in the partial RNA1 to RNA4. RNA3 and RNA6 of the isolate IS fell into each clade together with RNA3a and RNA6b of the Japanese isolates, respectively. No recombination event was detected in all segments of the five Japanese isolates.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eComparison of amino acid sequence identities between Kochi_Nankoku_2011 and five other perilla mosaic virus isolates\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"11\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c11\" colnum=\"11\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colspan=\"11\" nameend=\"c11\" namest=\"c1\"\u003e \u003cp\u003eKochi_Nankoku_2011\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eIsolate\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eP1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eP2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eP3a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eP3b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eP4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eP5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eP6a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eP6b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eP6c\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003eP7\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eOita_Oita_2013_1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e99.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e98.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e99.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e99.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e98.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e98.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e98.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e98.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e99.6\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAichi_Toyohashi_2013_2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e97.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e99.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e99.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e96.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e96.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e99.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e97.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e99.2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIbaraki_Miho_2013_1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e97.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e98.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e95.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e94.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e99.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e90.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e93.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e89.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e85.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e98.4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIbaraki_Namegata_2014_1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e97.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e96.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e94.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e96.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e99.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e90.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e93.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e91.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e85.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e98.4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e99.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e98.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e92.9\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e88.0\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e93.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e62.1\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e96.1\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e25.3\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e97.2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"11\"\u003e\u003csup\u003ea\u003c/sup\u003e Amino acid sequence identities between P3 of the IS isolate of PerMV (LC721296) and P3a and P3b of Kochi_Nankoku_2011.\u003c/td\u003e\u003c/tr\u003e \u003ctr\u003e\u003ctd colspan=\"11\"\u003e\u003csup\u003eb\u003c/sup\u003e Amino acid sequence identities between P6 of the IS isolate of PerMV (LC721301) and P6a, P6b, and P6c of Kochi_Nankoku_2011.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this study, we collected a total of 21 PerMV isolates from four prefectures in Japan between 2011 and 2015. We determined the partial nucleotide sequences of RNA1-RNA4 and the near-complete genome sequences of four selected isolates.\u003c/p\u003e \u003cp\u003eThe partial nucleotide sequences of RNA1\u0026ndash;RNA4 from the Japanese isolates shared more than 90% identity. However, phylogenetic analysis revealed a clear division into two clades: one containing segments of isolates from Western Japan (Kochi and Oita prefectures), and another containing isolates from Eastern Japan (Ibaraki prefecture) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Isolates from Aichi consisted of RNA1 and RNA3b from Eastern Japan and the others from Western Japan. This suggests that the PerMV population in Japan is genetically diverse, with at least two groups. Regarding the isolates in Aichi, they could be reassortants of isolates from Eastern and Western Japan. Alternatively, Aichi, located near the center of origin of PerMV, could be the point from which they diversified and spread to Eastern and Western regions in Japan. The latter scenario may be more likely, as occurrences of the mosaic disease, presumably caused by PerMV, were first noticed in Aichi prefecture as early as 1980s, followed by Kochi around 2000 (Kubota et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). However, further investigation into the phylogenetic diversities of the other segments and isolates in other regions is needed to elucidate the PerMV genetic structure in Japan. In contrast to the very low genetic diversity observed in 16 PCLSaV isolates collected from 13 prefectures in Japan (Takeyama et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), the PerMV isolates in four prefectures exhibited much higher genetic diversities. This could result from significant differences in the origin, spread, and distribution of these viruses.\u003c/p\u003e \u003cp\u003eAn interesting discovery in our survey was that PerMV isolates, likely lacking RNA2, were found in all four prefectures (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Although the three RNA2-specific primer sets used for RT-PCR were designed based on the Kochi_Nankoku_2011 isolate, the failure to detect RNA2 could not be attributed to sequence mismatches, as such RNA2-negative isolates were also found in Kochi and Oita prefectures. If these isolates indeed lack RNA2, they would not express GPs, suggesting that GPs are dispensable for systemic infection in shiso plants.\u003c/p\u003e \u003cp\u003eElliovirus GPs on the surface of enveloped virions play pivotal roles in virus entry, virion assembly, and vector transmission (Hulswit et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Mutants of tomato spotted wilt virus (TSWV), an orthotospovirus lacking enveloped virions and thrips-borne transmissibility, can be obtained through repeated mechanical passages (de Oliveira Resende et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e1991\u003c/span\u003e; Ie \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e1982\u003c/span\u003e; Nagata et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2000\u003c/span\u003e; Verkleij and Peters \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e1983\u003c/span\u003e). These mutants harbor nucleotide substitutions or deletions in the GP ORF of the M RNA (Sin et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2005\u003c/span\u003e), demonstrating that orthotospovirus GPs are dispensable for systemic infection in plants after they invade plant cells. Since the M RNA encodes not only GPs but also NSm,which is required for cell-to-cell movement (Feng et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), TSWV mutants lacking the M RNA should no longer infect systemically. Unlike orthotospoviruses, fimovirus RNA2 encodes only GP, and the MP is encoded by RNA4. Thus, mutations, deletion of partial sequence, or complete loss of RNA2 might not affect systemic infection ability, suggesting the potential existence of fimovirus isolates lacking RNA2, given that GPs are unnecessary for replication and cell-to-cell and long-distance movement in an infected plant. Furthermore, once such RNA2-lacking isolates emerge from a wild-type, i.e., RNA2-retaining isolate in an infected plant, they may replicate and move faster than the wild-type, although they would not be transmissible to another host by mites. To our knowledge, the presence of fimovirus isolates lacking RNA2 under natural conditions (i.e., not under experimental condition) has not been reported yet. Analogous to orthotospoviruses, it is also expected that fimovirus isolates lacking RNA2 would lose the enveloped virion and mite-borne transmissibility, making the occurrence of RNA2-lacking isolates unusual except under specific conditions. However, we have observed that RNA2-lacking PerMV isolates can be reproducibly obtained by maintaining PerMV in infected shiso plants without infestation of PRMs (Kubota et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Notably, the RNA2-lacking PerMV isolates were found only in production greenhouses, where insecticides were periodically applied (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Furthermore, Verchot et al. (\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) have reported that an agrobacteium-mediated infectious clone of RRV, that was composed only of the constructs of RNA1 (RdRp), RNA3 (NP), and RNA5 (whose ORF was replaced with that of a fluorescent protein), can infect in an agro-inoculated \u003cem\u003eNicotiana benthamiana\u003c/em\u003e leaf, indicating that RNA2 (GP) is dispensable at least for replication of fimovirus.\u003c/p\u003e \u003cp\u003eNear-complete nucleotide sequence analysis of the four isolates from Ibaraki, Aichi, and Oita prefectures, compared with previously reported isolates Kochi_Nankoku_2011 and IS, clearly demonstrated that RNAs encoding homologous proteins (i.e., RNA3ab and RNA6a\u0026ndash;c/7) were conserved in all five investigated Japanese isolates. Although Oh et al. (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) reported only seven RNA segments for the IS isolate, we cannot specify whether it indeed harbors only seven RNAs, or possesses a total of 10 segments, including unreported homologs of RNA3 and RNA6. Regardless, all five Japanese isolates, collected from different areas and belonging to genetically distinct clades (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e), commonly possessed ten RNAs, including ones encoding homologous proteins. This strongly supports that all ten PerMV proteins are indispensable for the virus\u0026rsquo;s infection cycle. Thus, understanding the molecular functions of these proteins is of great importance.\u003c/p\u003e \u003cp\u003eInvestigating the differences in biological properties, such as host range, symptomatology, and transmissibility, between isolates belonging to different clades could be of interest. Another intriguing question is whether reassortants can be obtained or if some genetic exclusion occurs when two isolates from different clades infect a plant.\u003c/p\u003e \u003cp\u003eSince the first identification of a fimovirus two decades ago (Benthack et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2005\u003c/span\u003e), the number of species in the \u003cem\u003eEmaravirus\u003c/em\u003e genus of the \u003cem\u003eFimoviridae\u003c/em\u003e family has rapidly expanded to 32, surpassing those of the \u003cem\u003eOrthotospovirus\u003c/em\u003e (26) and \u003cem\u003eTenuivirus\u003c/em\u003e (8) genera (Kuhn et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Fimoviruses appear to be more diverse than the other two; for instance, while the hosts of orthotospoviruses and tenuiviruses are primarily confined to herbaceous dicots and monocots, respectively, fimoviruses have a broader host range that includes herbaceous and woody dicots as well as monocots. Consequently, based on the high diversity in encoded proteins and genome sequences, the separation of the \u003cem\u003eEmaravirus\u003c/em\u003e genus into two or more genera has been proposed (Rehanek et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). The genome\u0026rsquo;s plasticity, largely due to its monocistronic nature, may be a key driver for the rapid diversification and evolutionary adaptation of fimoviruses.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eList of Supplementary files\u003c/h2\u003e \u003cp\u003eSupplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e: Primers list\u003c/p\u003e \u003cp\u003eSupplementary Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e: Geographical map\u003c/p\u003e \u003c/p\u003e\u003cp\u003e \u003ch2\u003eCompliance with Ethical Standards\u003c/h2\u003e \u003cp\u003eThis article does not contain any studies with human participants or animals performed by any of the authors.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eConflicts of interest\u003c/strong\u003e \u003cp\u003eThe authors declare that they have no conflict of interest.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eAcknowledgments\u003c/h2\u003e \u003cp\u003eThe authors are grateful to M. Yasunaga, T. Usugi, M. Kubota, T. Kawano, H. Tanaka, Y. Shimomoto, and K. Tanaka for the collection of shiso samples, and would like to thank reviewers for valuable comments that improved our manuscript. This work was partly supported by JSPS KAKENHI Grant Number JP21K05605.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eBahat Y, Alter J, Dessau M (2020) Crystal structure of tomato spotted wilt virus G\u003csub\u003eN\u003c/sub\u003e reveals a dimer complex formation and evolutionary link to animal-infecting viruses. Proc Natl Acad Sci U S A 117:26237\u0026ndash;26244. https://doi.org/10.1073/pnas.2004657117\u003c/li\u003e\n\u003cli\u003eBenthack W, Mielke N, B\u0026uuml;ttner C, M\u0026uuml;hlbach HP (2005) Double-stranded RNA pattern and partial sequence data indicate plant virus infection associated with the ringspot disease of European mountain ash (\u003cem\u003eSorbus aucuparia\u003c/em\u003e L.). 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XXVII Int Congr Entomol P0233 (Abstract)\u003c/li\u003e\n\u003cli\u003eKubota K, Chiaki Y, Yanagisawa H, Takeyama S, Suzuki R, Kohyama M, Horikawa T, Toda S, Kadono F (2021a) First report of pear chlorotic leaf spot-associated virus on Japanese and European pears in Japan and its detection from an eriophyid mite. Plant Dis 105:1234. https://doi.org/10.1094/PDIS-09-20-2035-PDN\u003c/li\u003e\n\u003cli\u003eKubota K, Chiaki Y, Yanagisawa H, Yamasaki J, Horikawa H, Tsunekawa K, Morita Y (2021b) Novel degenerate primer sets for the detection and identification of emaraviruses reveal new chrysanthemum species. J Virol Methods 288:113992. https://doi.org/10.1016/j.jviromet.2020.113992\u003c/li\u003e\n\u003cli\u003eKubota K, Usugi T, Tomitaka Y, Shimomoto Y, Takeuchi S, Kadono F, Yanagisawa H, Chiaki Y, Tsuda S (2020) Perilla mosaic virus is a highly divergent emaravirus transmitted by \u003cem\u003eShevtchenkella\u003c/em\u003e sp. (Acari: Eriophyidae). Phytopathology 110:1352\u0026ndash;1361. https://doi.org/10.1094/PHYTO-01-20-0013-R\u003c/li\u003e\n\u003cli\u003eKubota K, Yanagisawa H, Chiaki Y, Yamasaki J, Horikawa H, Tsunekawa K, Morita Y, Kadono F (2021c) Complete nucleotide sequence of chrysanthemum mosaic-associated virus, a novel emaravirus infecting chrysanthemum. Arch Virol 166:1241\u0026ndash;1245. https://doi.org/10.1007/s00705-021-04979-2\u003c/li\u003e\n\u003cli\u003eKuhn JH, Abe J, Adkins S, et al. (2023) Annual (2023) taxonomic update of RNA-directed RNA polymerase-encoding negative-sense RNA viruses (realm \u003cem\u003eRiboviria\u003c/em\u003e: kingdom \u003cem\u003eOrthornavirae\u003c/em\u003e: phylum \u003cem\u003eNegarnaviricota\u003c/em\u003e). J Gen Virol 104. https://doi.org/10.1099/jgv.0.001864\u003c/li\u003e\n\u003cli\u003eLiu H, Wang G, Yang Z, Wang Y, Zhang Z, Li L, Waqas M, Hong N, Liu H, Wang G, Hong N, Hong J, Zhang J, Xu L, Qi L (2020) Identification and characterization of a pear chlorotic leaf spot-associated virus, a novel emaravirus associated with a severe disease of pear trees in China. Plant Dis 104:2786\u0026ndash;2798. https://doi.org/10.1094/PDIS-01-20-0040-RE\u003c/li\u003e\n\u003cli\u003eLu Y, McGavin W, Cock PJA, Schnettler E, Yan F, Chen J, MacFarlane S (2015) Newly identified RNAs of raspberry leaf blotch virus encoding a related group of proteins. J Gen Virol 96:3432\u0026ndash;3439. https://doi.org/10.1099/jgv.0.000277\u003c/li\u003e\n\u003cli\u003eMartin DP, Varsani A, Roumagnac P, Botha G, Maslamoney S, Schwab T, Kelz Z, Kumar V, Murrell B (2021) RDP5: a computer program for analyzing recombination in, and removing signals of recombination from, nucleotide sequence datasets. Virus Evol 7:veaa087. https://doi.org/10.1093/ve/veaa087\u003c/li\u003e\n\u003cli\u003eMuhire BM, Varsani A, Martin DP (2014) SDT: a virus classification tool based on pairwise sequence alignment and identity calculation. PLOS ONE 9:e108277. https://doi.org/10.1371/journal.pone.0108277\u003c/li\u003e\n\u003cli\u003eNabeshima T, Abe J (2021) High-throughput sequencing indicates novel varicosavirus,emaravirus, and deltapartitivirus infections in \u003cem\u003eVitis coignetiae\u003c/em\u003e. Viruses 13:827. https://doi.org/10.3390/v13050827\u003c/li\u003e\n\u003cli\u003eNagata T, Inoue-Nagata AK, Prins M, Goldbach R, Peters D (2000) Impeded thrips transmission of defective tomato spotted wilt virus isolates. Phytopathology 90:454\u0026ndash;459. https://doi.org/10.1094/PHYTO.2000.90.5.454\u003c/li\u003e\n\u003cli\u003eOh BG, Byun H-S, Ju H-J, Yoon J-Y (2023) First report of perilla mosaic emaravirus infecting \u003cem\u003ePerilla frutescens\u003c/em\u003e in South Korea. Plant Dis 107:2269. https://doi.org/10.1094/PDIS-09-22-2035-PDN\u003c/li\u003e\n\u003cli\u003eRehanek M, Karlin DG, Bandte M, Al Kubrusli R, Nourinejhad Zarghani S, Candresse T, B\u0026uuml;ttner C, von Bargen S (2022) The complex world of emaraviruses\u0026mdash;challenges, insights, and prospects. Forests 13:1868. https://doi.org/10.3390/f13111868\u003c/li\u003e\n\u003cli\u003eSaitou N, Nei M (1987) The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4:406\u0026ndash;425. https://doi.org/10.1093/oxfordjournals.molbev.a040454\u003c/li\u003e\n\u003cli\u003eShimomoto Y, Okada T, Ikeda K, Tatara A, Hasegawa Y, Yanagisawa H, Takeyama S, Hayashi K, Yano K, Morita Y, Kubota K (2022) Japanese star anise ringspot-associated virus is a distinct emaravirus transmitted by the eriophyid mite (the family Diptilomiopidae). 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Jpn J Phytopathol 88:1\u0026ndash;11 (in Japanese with English abstract). https://doi.org/10.3186/jjphytopath.88.1\u003c/li\u003e\n\u003cli\u003eTamura K, Stecher G, Kumar S (2021) MEGA11: molecular evolutionary genetics analysis version 11. Mol Biol Evol 38:3022\u0026ndash;3027. https://doi.org/10.1093/molbev/msab120\u003c/li\u003e\n\u003cli\u003eTatineni S, McMechan AJ, Wosula EN, Wegulo SN, Graybosch RA, French R, Hein GL (2014) An eriophyid mite-transmitted plant virus contains eight genomic RNA segments with unusual heterogeneity in the nucleocapsid protein. J Virol 88:11834\u0026ndash;11845. https://doi.org/10.1128/JVI.01901-14\u003c/li\u003e\n\u003cli\u003eThompson JD, Higgins DG, Gibson TJ (1994) CLUSTALW: improving the sensitivity of progressive multiple sequence alignment through weighing, position specific gap penalties and weight matrix choice. Nucleic Acids Res 22:673.\u003c/li\u003e\n\u003cli\u003eVerchot J, Herath V, Urrutia CD, Gayral M, Lyle K, Shires MK, Ong K, Byrne D (2020) Development of a reverse genetic system for studying rose rosette virus in whole plants. Mol Plant Microbe Interact 10:1209\u0026ndash;1221. https://doi.org/10.1094/MPMI-04-20-0094-R\u003c/li\u003e\n\u003cli\u003eVerkleij FN, Peters D (1983) Characterization of a defective form of tomato spotted wilt virus. J Gen Virol 64:677\u0026ndash;686. https://doi.org/10.1099/0022-1317-64-3-677\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"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":"journal-of-general-plant-pathology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jgpp","sideBox":"Learn more about [Journal of General Plant Pathology](http://link.springer.com/journal/10327)","snPcode":"10327","submissionUrl":"https://www.editorialmanager.com/jgpp/default2.aspx","title":"Journal of General Plant Pathology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"fimovirus, emaravirus, genetic diversity, glycoprotein, homologous proteins","lastPublishedDoi":"10.21203/rs.3.rs-4818253/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4818253/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003ePerilla mosaic virus (PerMV) is a fimovirus that harbors ten RNA segments (RNAs 1, 2, 3a, 3b, 4, 5, 6a, 6b, 6c, and 7). The presence of PerMV was first reported in Kochi, followed by Ibaraki, Aichi, and Oita prefectures in Japan, and most recently from South Korea. To understand the genetic diversity of these PerMV isolates, partial nucleotide sequences of RNAs 1 to 4 from 21 Japanese isolates were determined. Phylogenetic analysis revealed that the segments of the isolates are divided into two clades: the Eastern clade (isolates in Ibaraki) and the Western clade (isolates in Kochi, Oita, and South Korea). Isolates in Aichi appeared to be reassortants of both; RNAs 1 and 3b belonged to the Eastern clade, while the others were of the Western clade. Isolates lacking RNA2, found in four prefectures, suggest RNA2 is not essential for plant infection. Near-complete nucleotide sequences from four isolates in Ibaraki, Aichi, and Oita were determined. They maintained ten RNA segments, including RNAs encoding proteins of P3s, P6s, and P7. This indicates that these proteins, encoded by an apparently redundant segment, are crucial for PerMV\u0026rsquo;s infection and transmission cycles.\u003c/p\u003e","manuscriptTitle":"Nucleotide sequence survey of perilla mosaic virus isolates in Japan unveils complex genetic structure and conserved RNA segments encoding homologous protein groups","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-08-23 09:16:04","doi":"10.21203/rs.3.rs-4818253/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"","date":"2024-08-25T22:39:58+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-08-25T22:33:07+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-08-21T06:21:32+00:00","index":"","fulltext":""},{"type":"submitted","content":"Journal of General Plant Pathology","date":"2024-08-21T02:15:38+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"journal-of-general-plant-pathology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jgpp","sideBox":"Learn more about [Journal of General Plant Pathology](http://link.springer.com/journal/10327)","snPcode":"10327","submissionUrl":"https://www.editorialmanager.com/jgpp/default2.aspx","title":"Journal of General Plant Pathology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"cbf700ce-de4a-49d9-a5c9-b0a5cd8549bf","owner":[],"postedDate":"August 23rd, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-10-14T16:01:16+00:00","versionOfRecord":{"articleIdentity":"rs-4818253","link":"https://doi.org/10.1007/s10327-024-01203-2","journal":{"identity":"journal-of-general-plant-pathology","isVorOnly":false,"title":"Journal of General Plant Pathology"},"publishedOn":"2024-10-08 15:57:32","publishedOnDateReadable":"October 8th, 2024"},"versionCreatedAt":"2024-08-23 09:16:04","video":"","vorDoi":"10.1007/s10327-024-01203-2","vorDoiUrl":"https://doi.org/10.1007/s10327-024-01203-2","workflowStages":[]},"version":"v1","identity":"rs-4818253","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4818253","identity":"rs-4818253","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}