First Report of Vitis Cryptic Virus from Grapevines in China

Vitis cryptic virus (VCV) was recently identified on wild Vitis coignetiae in Japan in 2021 and was tentatively classified as a new member of the genus Deltapartitivirus, which is consistent with the two-segmented genome encoding RdRp and CP (Nabeshima et al. 2021). In June 2020, a grapevine cv. Jinhuanghou in a vineyard exhibiting chlorotic mottling was collected in Xingcheng, Liaoning province of China. Total RNA was extracted using RNAprep Pure Plant Plus Kit (DP441, TIANGEN BIOTECH, Beijing), and the ribosomal RNA was removed by the Epicentre Ribo-Zero rRNA Removal Kit (Epicentre, Madison, WI, U.S.A.). The ribosomal RNA-depleted RNA was then used to construct a cDNA library using a TruSeq RNA Sample Prep Kit (Illumina, San Diego, CA, U.S.A.), which was sequenced on an Illumina NovaSeq 6000 platform (Biomarker Biology Technology), resulting in 60,208,348 paired-end clean reads (150 nt × 2). Reads mapping to the grapevine genome (PN40024 assembly 12X) were removed by hierarchical indexing using hisat2 2.1.0 software (Kim et al. 2019). The unmapped reads were de novo assembled into 116,809 contigs using the rnaviralSPAdes method in the SPAdes v3.15.3 software with default parameters (Prjibelski et al. 2020) and analyzed through BLAST analysis. Two viruses and two viroids were identified: VCV (two contigs), grapevine emaravirus A (GEVA; five contigs), grapevine yellow speckle viroid 1 (GYSVd1; one contig), and hop stunt viroid (HSVd; one contig). The two contigs of VCV had lengths of 1,575 nt and 1,563 nt and shared 95% and 90% nt identity with RNA1 and RNA2 genomes of the VCV isolate H1 (GenBank accession nos. LC602838 and LC602839) with 99% and 96% coverage, respectively. To further confirm the infection of VCV, we designed two pairs of primers, VCV-RP1a/1b (5′-TGGTCGAGAAGTTACTATACTCG-3′/5′-AGACCACAATATTGCTTTGGCTC-3′) and VCV-CP1a/1b (5′-TTACGAAGTCCGCACTATTGC-3′/5′-AGCATACGGATAGCTCCTGAC-3′), which were used to amplify the 297-bp and 279-bp fragments in the RdRp and CP genes encoded by RNA1 and RNA2 genomes of VCV, respectively. The amplified PCR products were cloned and sequenced and the two sequences (OM460075-76) showed 93% and 91% nt identity with the genomic segments of the VCV isolate H1, respectively. The graft transmissibility of VCV was assessed in July 2021 by grafting the VCV-infected grapevine buds onto 2-year-old VCV-free ‘Beta’ grapevine seedlings with four replicates; the leaves of the first bud below the grafting site showed chlorotic mottling symptoms and tested positive for VCV 2 months after grafting. To further determine the incidence and distribution of VCV in China, 470 grapevine samples of 71 cultivars were collected from 21 provinces and tested by RT-PCR using primers VCV-RP1a/1b and VCV-CP1a/1b. The results showed that 2.6% (12/470) of the samples tested positive with both primers, including 10 ‘Jinhuanghou’ grapevines (Jilin province), 1 ‘Zuoyouhong’ (Jilin province), and 1 ‘Куртсет’ grapevine (Liaoning province). This is the second report of VCV in the world, and it confirms the graft transmissibility of VCV for the first time. Given the VCV infectivity in the two important cultivars in Jilin province and strong graft transmissibility, it is necessary to further study its pathogenicity and its effect on grapes. Unveiling the presence of VCV in China contributes to understanding the occurrence of the virus and developing management measures should they become necessary.