First Report of Grapevine Virus F in Grapevine in China

Grapevine virus F(GVF), a member of the genus Vitivirus of the family Betaflexiviridae, was first described to infect grapevines in USA (Al Rwahnih et al. 2012). Since then, GVF has been reported from South African, Greece, Iran and Australia (Molenaar et al. 2015; Sabaghian et al. 2018; Panailidou et al. 2019; Wu et al. 2023). GVF and other several vitiviruses are associated with rugose wood (RW), which is one of the most important viral diseases. At present, five vitiviruses including grapevine viruses A, B, E, L and T have been detected in China vineyards, but GVF has not been reported in China so far. In 2023, during a study of the virome of China grapevines, one symptomless sample of grapevine cultivar ‘MERDE’ was collected from Taigu grapevine nursery in shanxi, China. Total RNAs were extracted using RNAprep Pure Plant Plus Kit (DP441, TIANGEN BIOTECH, Beijing), and the ribosomal RNA were removed by the Epicentre Ribo-Zero rRNA Removal Kit (Epicentre, Madison, WI, USA). The ribosomal RNA-depleted RNAs were then used to construct a cDNA library using a TruSeq RNA Sample Prep Kit (Illumina, San Diego, CA, USA), which was sequenced on an Illumina NovaSeq 6000 platform (Biomarker Biology Technology), resulting in 35,068,409 paired-end clean reads (150 nt × 2). Reads mapping to the grapevine genome (GenBank accession no PN40024) were removed using hisat2 2.1.0 software. The unmapped reads were de novo assembled using the rnaviralSPAdes method in the SPAdes v3.15.3 software with default parameters and analyzed through BLASTn and BLASTx analysis, which revealed the present of contig sequences representing 8 grapevine-infecting viruses and viroids. Among them, the five contigs of GVF were 183-4671 nt in length and shared 86.6%-96.72% nt identity with a GVF isolate AUD46129 (JX105428) with 99.7% coverage. To further confirm the infection of GVF, we designed two pairs of primers, GVF-1a/1b (5’- CGCCCAGGCTTCTGTCGGTGAAC-3’/5’-ACACCACTCATCCGGCTAAGCCG-3’) and GVF2a/2b (5’-CGAGTGATAGAGCGCGGGTTCCG-3’/5’- CGGTCCTTGAAGCGAAGCGCGAC-3’), which were used to amplify the 363-bp and 372-bp fragments for the partial sequences of Orf4-5 and partial sequences of Orf2-3 in the GVF genome, respectively. The amplified PCR products were cloned and sequenced and the two sequences (PV022101and PV022105) showed 96.7% and 92.2% nt identity with the GVF isolate AUD46129, respectively. Furthermore, 431 grapevine samples of 73 cultivars were collected from 21 provinces and tested by RT-PCR using the above primer pairs. The results showed that 0.7% (3/431) of the samples tested positive, including one ‘Xiabai’ grapevine (Shanxi province), one ‘Pinot gris’ grapevine (Shanxi), and one ‘Liao23’ (Liaoning). The PCR products from three GVF-infected grapevines were cloned and sequenced. The partial sequences of the Orf4-5 (PV022102-04) showed 95.9% to 96.4% nt identity with the isolate AUD46129, and the partial sequences of Orf2-3 (PV022106 and PV022107) showed 92.7% and 93.0% nt identity, respectively. To our best knowledge, this is the first report of GVF in grapevines in China, extending the information on its geographical distribution.