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Molecular characterization of a novel amalgavirus infecting Lilium spp. in China

February 2023

DOI:10.21203/rs.3.rs-2546133/v1

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Authors:

Mengji Cao

Citrus Research Institute

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A novel plant virus with a double-stranded (ds) RNA genome was detected in Lilium spp. in China by high-throughput sequencing, tentatively named “Lily amalgavirus 2” (LAV2). The genomic RNA of LAV2 is 3432 nucleotides (nt) and encodes a large fusion protein with 1053 amino acids (aa). It encodes two open reading frames (ORFs). The two ORFs putatively encode a ‘1 + 2’ fusion protein generated by a ‘+1’ programmed ribosomal frameshift (PRF). ORF1 encodes a 386-aa protein with unknown function, ORF2 overlaps ORF1 by 350 nt and encodes a 783-aa protein consists of a conserved sequence, which is RNA-dependent RNA polymerase (RdRp). The ‘+1’ ribosomal frameshifting motif sequence, UUU_CGN, which was highly conserved among known Amalgaviruses , was also found in LAV2. Sequence analysis suggested that it shared 46.04%-51.59% complete nucleotide sequence identity with those members of the genus Amalgavirus and had the highest identity (51.59%) to Lily amalgavirus 1 (accession no. OM782323). Phylogenetic analysis based on the RdRp amino acid sequences showed that LAV2 clustered with Amalgavirus. Overall, our data suggest that LAV2 is a new member of the genus Amalgavirus .

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Page 1/8

Molecular characterization of a novel amalgavirus

infecting Lilium spp. in China

Zhihao Yuan

Huazhong Agricultural University College of Plant Science and Technology

Zhenfeng Li

Huazhong Agricultural University College of Plant Science and Technology

Yuexia Lu

Huazhong Agricultural University College of Plant Science and Technology

Mengji Cao

Southwest University

Ni Hong

Huazhong Agricultural University College of Plant Science and Technology

Guoping Wang

Huazhong Agricultural University College of Plant Science and Technology https://orcid.org/0000-

0003-0681-4630

Li Cai (  caili@mail.hzau.edu.cn )

Huazhong Agriculture University https://orcid.org/0000-0003-2409-9722

Research Article

Keywords:

Posted Date: February 9th, 2023

DOI: https://doi.org/10.21203/rs.3.rs-2546133/v1

License:   This work is licensed under a Creative Commons Attribution 4.0 International License. 

Read Full License

Version of Record: A version of this preprint was published at Archives of Virology on June 14th, 2023.

See the published version at https://doi.org/10.1007/s00705-023-05806-6.

Page 2/8

Abstract

A novel plant virus with a double-stranded (ds) RNA genome was detected in

Lilium

spp. in China by high-

throughput sequencing, tentatively named “Lily amalgavirus 2” (LAV2). The genomic RNA of LAV2 is

3432 nucleotides (nt) and encodes a large fusion protein with 1053 amino acids (aa). It encodes two

open reading frames (ORFs). The two ORFs putatively encode a ‘1 + 2’ fusion protein generated by a ‘+1’

programmed ribosomal frameshift (PRF). ORF1 encodes a 386-aa protein with unknown function, ORF2

overlaps ORF1 by 350 nt and encodes a 783-aa protein consists of a conserved sequence, which is RNA-

dependent RNA polymerase (RdRp). The ‘+1’ ribosomal frameshifting motif sequence, UUU_CGN, which

was highly conserved among known

Amalgaviruses

, was also found in LAV2. Sequence analysis

suggested that it shared 46.04%-51.59% complete nucleotide sequence identity with those members of

the genus

Amalgavirus

and had the highest identity (51.59%) to Lily amalgavirus 1 (accession no.

OM782323). Phylogenetic analysis based on the RdRp amino acid sequences showed that LAV2

clustered with

Amalgavirus.

Overall, our data suggest that LAV2 is a new member of the genus

Amalgavirus

Full Text

Lily (

Lilium

spp.) is a perennial herb belongs to the genus

Lilium

of the family Liliaceae, with ornamental

value, medicinal value and edible value, it has become a prevalent economic crop in the oricultural

industry worldwide [1].To date, there are more than 20 viruses infecting lily reported in the world, among

these viruses, lily mottle virus (LMoV), lily symptomless virus (LSV), and cucumber mosaic virus (CMV)

are the main viruses infecting lily plants, and often show mixed infection [2, 3]. Lily amalgavirus 1 (LAV1),

lily yellow mosaic virus (LYMV), prunus necrotic ringspot virus (PNRSV), CMV, LMoV, LSV, plantago

asiatica mosaic virus (PlAMV), shallot yellow stripe virus (SYSV) and arabis mosaic virus (ArMV) have

been reported to infect lily plants in China [4-8].

The family of

Amalgaviridae

currently comprises ten virus species , which are blueberry latent virus

(BBLV) [9], rhododendron virus A (RHV-A) [10], southern tomato virus (STV) [11], vicia cryptic virus M

(VCV-M) [12], allium cepa amalgavirus 1 (AcAV1), allium cepa amalgavirus 2 (AcAV2) [13], spinach

amalgavirus 1 (SpAV1) [14], zoostera marina amalgavirus 1 (ZmAV1) and zoostera marina amalgavirus 2

(ZmAV2) [15] in the genus of

Amalgavirus

and zygosaccharomyces bailii virus Z (ZbV-Z) [16] in the

genus of

Zybavirus

.The plant amalgaviruses have small dsRNA genomes (3316-3453 bp) that

encompass two partially overlapping long open reading frames (ORFs), with downstream ORF2

overlapping ORF1 in the ‘+1’ frame. The ORF1 encodes a protein with unknown function. The ORF2

encodes RNA-dependent RNA polymerase (RdRp) that is translated through a ‘+1’ programmed ribosomal

frameshifting (PRF) strategy and the product is actually a part of the fusion protein encoded by ORF

‘1+2’[9, 12, 13, 15, 16].

In September 2020, leaves of a lily plant showing mosaic, yellowing and leaf deformation symptoms

were collected from Kunming, Yunnan province. Total RNA was extracted and then subjected to Illumina

Page 3/8

NovaSeq6000 sequencing (Berry Genomics Corporation, Beijing, China). A total of 63,790,206 high

quality clean reads were obtained after trimming and used for contig assembly. The assembled contigs

(n=62,755) were searched using Basic Local Alignment Search Tool (BLAST) at GenBank. In total, 60

contigs showed similarity to sequences from plant viruses by BLASTx analysis (NCBI), and three contigs

showed similarity to amalgaviruses. Reverse transcription-polymerase chain reaction (RT-PCR) ultimately

detected eight lily viruses in the sample, of which a novel amalgavirus virus, tentatively named “Lily

amalgavirus 2” (LAV2), was selected for further characterization. To obtain the full-length genome

sequence of LAV2, total RNA was extracted using a SteadyPure Plant RNA Extraction Kit (Accurate

Biotechnology, Hunan, China) and treated with DNase I for 15 min at room temperature.

Escherichia coli

(

E. coli

) poly (A) polymerase (Beyotime, Shanghai, China) was used to add a poly (A) tail to the 3’ end of

the LAV2 molecules. The rst-strand cDNAs were synthesized using EVO M-MLV reverse transcriptase

(Accurate Biotechnology, Hunan, China) with random hexamer primers. Firstly, the terminal sequences

were determined by Rapid Amplication of cDNA Ends (RACE) using a SMARTer RACE 5'/3'Kit (Clontech,

USA) with 5'and 3'primers (Supplementary Table 1). Then a full-length primer pair was designed and

used to amplify the full genome of LAV2 by RT-PCR (Fig. 1A). The target RT-PCR products were puried

and inserted into the vector using One step ZTOPO-Blunt/TA Zero Background Fast Cloning Kit

(ZOMANBIO, Beijing, China) and sequenced by Sangon Biotech Co., Ltd (Shanghai, China).

LAV2 was 3,432 nucleotides (nt) in length (Supplementary File S3), and deposited into GenBank under the

accession number ON715004. The 5' and 3' untranslated regions (UTRs) of LAV2 consist of 164 nt and

105 nt, respectively. The secondary structure of the terminal sequences of the LAV2 genome was

predicted using the online software RNAfold (http://rna.tbi.univie.ac.at/cgi-

bin/RNAWebSuite/RNAfold.cgi). It reveals the presence of putative stem-loop or hairpin-like structures in

the 5' and 3' UTRs (Fig. 1B). The ORFs were predicted using the ORF Finder website

(https://www.ncbi.nlm.nih.gov/ornder/). LAV2 encodes two ORFs, ORF1 encodes a 387-aa protein with

unknown function, and a fusion protein encoded by ORF ‘1+2’. The ORF2 encodes part of the fusion

protein consists of a conserved sequence, which is RNA-dependent RNA polymerase (RdRp) of 784 aa,

and ORF2 overlaps ORF1 by 349 nt (Fig. 1C). The ‘+1’ ribosomal frameshifting motif sequence UUU_CGN,

which is highly conserved among known amalgaviruses, was also found in LAV2. LAV2 contains a

conserved RdRp domain at aa 333-445 in ORF2, which was identied in a search of the Conserved

Domain Database (CDD) of the NCBI database. A multiple sequence alignment was made using

CLUSTAL X [17], and domain motifs were identied using GeneDoc software [18]. A multiple alignment of

the RdRp encoded by LAV2 and those of ten members of the genus

Amalgavirus

showed that the LAV2

RdRp contains all seven conserved motifs (I to VII) (Fig. 2A), these results were consistent with LAV1 [4].

To investigate the taxonomic status of LAV2, phylogenetic analysis was performed using the RdRp aa

sequences of LAV2 and other selected viruses of the families

Amlgaviridae

Partitiviridae

and

Totiviridae

Maximum likelihood phylogenies were inferred under the model automatically selected by IQ-TREE for

1000 ultrafast bootstraps [19, 20]. Phylogenetic analysis based on the RdRp aa sequence showed that

LAV2 clustered into a group with members of the genus

Amalgavirus

, and LAV2 formed a separate

branch in the genus

Amalgavirus

of the family

Amlgaviridae

(Fig. 2B). Amino acids sequence comparison

Page 4/8

showed that the ORF1 and ORF ‘1+2’ sequences of LAV2 had the highest identity (22.56% and 44.78%,

respectively) to that of LAV1, and ORF1 had 16.97%-21.88% identity, ORF ‘1+2’ had 33.46%-44.15%

identity to the other members of the genus

Amalgavirus

.Completenucleotide sequencecomparison

showed that LAV2 had 46.04%-51.59% identity to those members of the genus

Amalgavirus

and had the

highest identity (51.59%) to that of LAV1 (Supplementary Table 2).

To investigate the occurrence of LAV2, a total of 48 leaf samples of symptomatic lily plants were

collected from Yunnan, Jiangxi, Zhejiang, Hunan, Guangdong, and Shanghai in China. These samples

were screened by RT-PCR amplication using the specic detection primer pair LAV2-F/R (Supplementary

Table 1) targeting a 422 nt fragment. The amplicons with expected size were detected from 10 of 48

samples but not from the negative control. However, no association between virus-like symptoms in lily

plants and LAV2 was found. Overall, our data suggest that LAV2 isolated from

Lilium

spp. in China is a

novel virus of the genus

Amalgavirus

Declarations

Acknowledgements This study was supported by the National Key Research and Development Program

of China (2019YFD1001800).

Author contributions Z.H. Yuan and L. Cai designed the experiments and wrote the manuscript. Z.F. Li

and Y.X. Lu contributed to sample preparation. M.J. Cao contributed to data analysis. H. Ni, G.P. Wang

and L. Cai contributed to critically revising of the manuscript. All authors read and approved the nal

manuscript.

Conict of interest All authors declare that they have no conicts of interest regarding this article.

Ethical approval This article does not contain any experiments with human participants or animals

performed by any of the authors.

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Figures

Figure 1

A The 5' and 3' RACE of LAV2 were performed using a SMARTer RACE 5'/3' Kit. The larger partial genomic

product was amplied by RT-PCR using the primer sets. H2O was used as a negative control. M, GL DNA

Marker 5000. B The predicted secondary structures of the 5' and 3' UTRs of LAV2. C Schematic

representation of the genomic organization of LAV2 isolated from China lily.

Page 7/8

Figure 2

A Sequence alignment of LAV2 RdRp motifs with those of selected members of the genus

Amalgavirus

Horizontal black lines above the sequence alignment indicate the conserved motifs I to VII. Shaded areas

represent identical aa residues. A red star indicates LAV2. B Phylogenetic analysis of LAV2 and other

selected viruses based on the aa sequences of ORF2 (RdRp). Sequences of ORF2 translation products

were aligned using MAFFT and then subjected to phylogenetic analysis using IQ-TREE. The tree is

Page 8/8

displayed as a rectangular phylogram rooted on the branch to the family

Amalgaviridae

Partitiviridae

and

Totiviridae

members. Branch support values are shown in %. The scale bar indicates a genetic distance of

0.6 aa substitutions per site. A red star indicates LAV2.

Supplementary Files

This is a list of supplementary les associated with this preprint. Click to download.

ElectronicSupplementaryles.docx

ResearchGate has not been able to resolve any citations for this publication.

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J Microbiol Biotechnol

Complete genome sequences of three new plant RNA viruses, Spinach deltapartitivirus 1 (SpDPV1), Spinach amalgavirus 1 (SpAV1), and Spinach latent virus (SpLV), were identified from a spinach (Spinacia oleracea) transcriptome dataset. The RNA-dependent RNA polymerases (RdRps) of SpDPV1, SpAV1, and SpLV showed 72%, 53%, and 93% amino acid sequence identities with the homologous RdRp of the most closely related virus in each case, respectively, suggesting that SpDPV1 and SpAV1 were novel viruses. Sequence similarity and phylogenetic analyses revealed that SpDPV1 belonged to the genus Deltapartitivirus of the family Partitiviridae, SpAV1 to the genus Amalgavirus of the family Amalgaviridae, and SpLV to the genus Ilarvirus of the family Bromoviridae. Based on the demarcation criteria, SpDPV1 and SpAV1 are considered as novel species of the genera Deltapartitivirus and Amalgavirus, respectively. This is the first report of these two viruses from spinach.

Nucleotide sequence of Zygosaccharomyces bailii virus Z: Evidence for +1 programmed ribosomal frameshifting and for assignment to family Amalgaviridae

Article

Mar 2016

Zygosaccharomyces bailii virus Z (ZbV-Z) is a monosegmented dsRNA virus that infects the yeast Zygosaccharomyces bailii and remains unclassified to date despite its discovery >20years ago. The previously reported nucleotide sequence of ZbV-Z (GenBank AF224490) encompasses two nonoverlapping long ORFs: upstream ORF1 encoding the putative coat protein and downstream ORF2 encoding the RNA-dependent RNA polymerase (RdRp). The lack of overlap between these ORFs raises the question of how the downstream ORF is translated. After examining the previous sequence of ZbV-Z, we predicted that it contains at least one sequencing error to explain the nonoverlapping ORFs, and hence we redetermined the nucleotide sequence of ZbV-Z, derived from the same isolate of Z. bailii as previously studied, to address this prediction. The key finding from our new sequence, which includes several insertions, deletions, and substitutions relative to the previous one, is that ORF2 in fact overlaps ORF1 in the +1 frame. Moreover, a proposed sequence motif for +1 programmed ribosomal frameshifting, previously noted in influenza A viruses, plant amalgaviruses, and others, is also present in the newly identified ORF1-ORF2 overlap region of ZbV-Z. Phylogenetic analyses provided evidence that ZbV-Z represents a distinct taxon most closely related to plant amalgaviruses (genus Amalgavirus, family Amalgaviridae). We conclude that ZbV-Z is the prototype of a new species, Zygosaccharomyces bailii virus Z, which we propose to assign as type species of a new genus of monosegmented dsRNA mycoviruses in family Amalgaviridae. Comparisons involving other unclassified mycoviruses with RdRps apparently related to those of plant amalgaviruses, and having either mono- or bisegmented dsRNA genomes, are also discussed.

Molecular characterization of a novel amalgavirus infecting Lilium spp. in China

Abstract and Figures

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