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

doi:10.21203/rs.3.rs-4818253/v1

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

https://doi.org/10.21203/rs.3.rs-4818253/v1

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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

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.

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).

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. 1a). 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. 1b). The phylogeny of RNA3a and RNA3b was similar to that of RNA2 and RNA1, respectively (Fig. 1c), 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. 1d).

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.

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.

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.

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

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Abstract

Figures

Introduction

Materials and methods

Collection of virus isolates and sequencing

Sequence analyses

Results

Genetic diversities of the Japanese isolates based on partial RNA1–RNA4 sequences

Near-complete nucleotide sequences of the four isolates

Discussion

Declarations

List of Supplementary files

Compliance with Ethical Standards

Acknowledgments

References

Supplementary Files

Status:

Version 1