Genomic Analysis of a Novel Torradovirus "Rehmannia Torradovirus Virus": Two Distinct Variants Infecting Rehmannia glutinosa

Abstract

Rehmannia glutinosa, a crucial medicinal plant native to China, is extensively cultivated across East Asia. We used high-throughput sequencing to identify viruses infecting R. glutinosa with mosaic, leaf yellowing, and necrotic symptoms. A novel Torradovirus, which we tentatively named "Rehmannia torradovirus virus" (ReTV), was identified. The complete sequences were obtained through reverse-transcription polymerase chain reaction (RT-PCR), 5' and 3' rapid amplification of cDNA ends, and Sanger sequencing. The amino acid sequence alignment between the ReTV-52 isolate and known Torradovirus species in the Pro-Pol and coat protein regions were 51.3-73.3% and 37.1-68.1%, respectively. Meanwhile, the amino acid sequence alignment between the ReTV-8 isolate and known Torradovirus species in the Pro-Pol and coat protein regions were 52.7-72.8% and 36.8-67.5%, respectively. The sequence analysis classified ten ReTV strains into two variants. The ReTV-52 genome has two RNA segments of 6939 and 4569 nucleotides, while that of ReTV-8 consists of two RNA segments containing 6889 and 4662 nucleotides. Sequence comparisons and phylogenetic analysis showed ReTV strains clustered within the Torradovirus, exhibiting the closet relation to the squash chlorotic leaf spot virus. The RT-PCR results showed a 100% ReTV detection rate in all 60 R. glutinosa samples. Therefore, ReTV should be classified as a novel Torradovirus species. ReTV is potentially dangerous to R. glutinosa, and necessitating monitoring this virus in the field.

Keywords: Rehmannia glutinosa; Rehmannia torradovirus virus; Torradoviruses; high-throughput sequencing; phylogenetic relationship; sequence comparison.

Figure 1

Genome organization of the rehmannia torradovirus virus (ReTV) showing relative positions of ORFs and their expression products. (a) rehmannia torradovirus virus-52, (b) rehmannia torradovirus virus-8. The RNA1 indicated the positions of sequences encoding conserved protein domains (HEL, Pro, and RDRP), while the RNA2 indicate the putative cleavage sites for the MP and CPs. The molecular weight predicted for each protein is reported above the boxes. RNA1 and RNA2 have indicated the start and stop positions of each virus segment in the viral genome organization.

Figure 2

Cleavage sites of RNA2-ORF2. (a) RNA2-ORF2 MP/CP1 cleavage site, (b) RNA2-ORF2 CP1/CP2 cleavage site, and (c) RNA2-ORF2 CP2/CP3 cleavage site. Putative conserved glutamine (Q) at the −1 position of the cleavage site is highlighted in red. Identical bases are highlighted in yellow. ORF, open reading frame.

Figure 3

Recombination analysis of ReTV-41 isolates using the recombination detection program RDP4.1 Dark gray regions represent a 95% breakpoint confidence interval, light gray region indicates a 99% breakpoint confidence interval, while the pink region highlights a tract of sequence with a recombination origin. ReTV, rehmannia torradovirus virus.

Figure 4

Phylogenetic analysis of ReTV and representative members of the family Torradovirus based on the amino acid sequence of (a) the Pro-Pol regions and (b) the CP-containing regions. The phylogenetic trees were constructed using the maximum likelihood method with 1000 bootstrap replications. Red dots: Sequences obtained in this study, TI, tomato-infecting, NTI, non-tomato-infecting. Motherwort yellow mottle virus (MYMoV), Codonopsis torradovirus A (CoTVA), lettuce necrotic leaf curl virus (LNLCV), carrot torradovirus 1 (CaTV1), tomato marchitez virus (ToMarV), tomato chocolate spot virus (ToCSV), tomato torrado virus (ToTV), cassava torrado-like virus (CsTLV), squash chlorotic leaf spot virus (SCLSV), cherry rasp leaf virus (CRLV), apple latent spherical virus (ALSV), currant latent virus (CuLV), satsuma dwarf virus (SDV), lychnis mottle virus (LycMoV), and strawberry latent ringspot virus (SLRSV).

Figure 4

Phylogenetic analysis of ReTV and representative members of the family Torradovirus based on the amino acid sequence of (a) the Pro-Pol regions and (b) the CP-containing regions. The phylogenetic trees were constructed using the maximum likelihood method with 1000 bootstrap replications. Red dots: Sequences obtained in this study, TI, tomato-infecting, NTI, non-tomato-infecting. Motherwort yellow mottle virus (MYMoV), Codonopsis torradovirus A (CoTVA), lettuce necrotic leaf curl virus (LNLCV), carrot torradovirus 1 (CaTV1), tomato marchitez virus (ToMarV), tomato chocolate spot virus (ToCSV), tomato torrado virus (ToTV), cassava torrado-like virus (CsTLV), squash chlorotic leaf spot virus (SCLSV), cherry rasp leaf virus (CRLV), apple latent spherical virus (ALSV), currant latent virus (CuLV), satsuma dwarf virus (SDV), lychnis mottle virus (LycMoV), and strawberry latent ringspot virus (SLRSV).