Genomic Characterization and Molecular Detection of Rehmannia Allexivirus Virus, a Novel Allexivirus Infecting Rehmannia glutinosa

Abstract

Rehmannia glutinosa is one of the most important medicinal plants in China and is affected by viral diseases. In this study, a new virus tentatively named Rehmannia Allexivirus virus (ReAV) was identified through high-throughput sequencing, reverse-transcription polymerase chain reaction (RT-PCR), and Sanger sequencing. The complete genome length was 7297 nt and it contained five open reading frames (ORFs) encoding replicase, triple gene block 1(TGB1), TGB2, TGB3, and coat protein (CP). The replicase and CP presented nucleotide homology ranges of 59.9-65.2% and 47.5-55.5% between the nine ReAV isolates and the other 12 species of the genus Allexivirus. In the nine isolates, ReAV-20 and ReAV-31 isolates showed breakpoints in the replicase and CP regions, respectively. The other isolates shared 87.2-96.5% nt with the whole genome nucleotide identity. The phylogenetic tree showed that seven ReAV isolates based on replicase, CP, and whole genome sequences were clustered in the same branch and were related to the genus Allexivirus. The ReAV detection rates for 60 R. glutinosa samples were 73.3-81.7% through RT-PCR using primers targeting the replicase or CP genes. These results demonstrate that ReAV is the dominant virus in R. glutinosa. This study provides important evidence for understanding viruses infecting R. glutinosa and for establishing efficient strategies to prevent viral spread.

Keywords: Allexivirus; ReAV; genomic sequence; molecular variation; phylogenetic relationship.

Figure 1

Genome organization of rehmannia allexivirus virus isolate (ReAV-59) showing relative positions of ORFs and their expression products (a), and the positions fragments amplified through RT-PCR and 5′ RACE and 3′RACE on six contigs (b).

Figure 2

Genome organization of Rehmannia allexivirus virus isolates (ReAV-20 and ReAV-31) showing relative positions of the open reading frames (ORFs) and their expression products ((a) ReAV-20, (b) ReAV-31).

Figure 3

Recombination analysis of ReAV-58 isolate using the recombination detection program RDP4.1. Dark gray regions: 95% break point confidence interval; light gray region: 99% break point confidence interval; purple region: sites excluded from analysis; pink region: tract of sequence with a recombination origin.

Figure 4

Phylogenetic analysis of ReAV and representative members of the family Alphaflexiviridae based on the nucleotide sequences of their replicase (a), CP (b), and whole genome (c). The phylogenetic trees were constructed using a neighbor-joining algorithm with 1000 bootstrap replications. Red dots: Sequences obtained in this study, AI: Allium-infecting allexiviruses, NAI: non-Allium-infecting allexiviruses. Alfalfa virus S (AVS), Arachis pintoi virus (ApV), blackberry virus E (BVE), garlic virus A (GarV-A), garlic virus B (GarV-B), garlic virus C (GarV-C), garlic virus D (GarV-D), garlic virus E (GarV-E), garlic virus X (GarV-X), shallot virus X (ShVX), vanilla latent virus (VLV), Senna severe yellow mosaic virus (SSYMV), Botrytis virus X (BotVX), Lolium latent virus (LoLV), citrus yellow mottle-associated virus (CiYMaV), citrus yellow vein clearing virus (CYVCV), Indian citrus ringspot virus (ICRSV), donkey orchid symptomless virus (DOSV), Alstroemeria virus X (AlsVX), Allium virus X (AVX), and Cactus virus X (CVX).

Figure 4

Phylogenetic analysis of ReAV and representative members of the family Alphaflexiviridae based on the nucleotide sequences of their replicase (a), CP (b), and whole genome (c). The phylogenetic trees were constructed using a neighbor-joining algorithm with 1000 bootstrap replications. Red dots: Sequences obtained in this study, AI: Allium-infecting allexiviruses, NAI: non-Allium-infecting allexiviruses. Alfalfa virus S (AVS), Arachis pintoi virus (ApV), blackberry virus E (BVE), garlic virus A (GarV-A), garlic virus B (GarV-B), garlic virus C (GarV-C), garlic virus D (GarV-D), garlic virus E (GarV-E), garlic virus X (GarV-X), shallot virus X (ShVX), vanilla latent virus (VLV), Senna severe yellow mosaic virus (SSYMV), Botrytis virus X (BotVX), Lolium latent virus (LoLV), citrus yellow mottle-associated virus (CiYMaV), citrus yellow vein clearing virus (CYVCV), Indian citrus ringspot virus (ICRSV), donkey orchid symptomless virus (DOSV), Alstroemeria virus X (AlsVX), Allium virus X (AVX), and Cactus virus X (CVX).

Figure 5

(a) Phylogenetic tree based on PCR products of ReAV-rep-1F/1R amplification. (b) Phylogenetic tree based on PCR products of ReAV-rep-2F/2R amplification. (c) Phylogenetic tree based on PCR products of ReAV-cp-1F/1R amplification. (d) Phylogenetic tree based on PCR products of ReAV-cp-2F/2R amplification.