First Report of Actinidia Virus 1 Infecting Kiwifruit in China

The family Closteroviridae comprises important viruses infecting a wide range of economically important plant species, including several species of woody fruit trees (King et al. 2012). Recently, a virus tentatively named as actinidia virus 1 (AcV-1) was identified in Actinidia chinensis grown in Italy (Blouin et al. 2013, 2018). AcV-1 has a 18,848 nucleotide (nt) genome with an organization like that of members in the family Closteroviridae. During 2013 to 2017, severe symptoms of chlorotic leaf spots, vein yellowing, and leaf deformation were continuously observed on a kiwifruit (Actinidia arguta) tree (ID: G4) grown at Wuhan city, in Hubei province in central China. Leaves collected from the symptomatic tree were subjected to small RNA sequencing on an Illumina Genome Analyzer (Biomarker Biology Technology Company, Beijing, China) as described previously (Zheng et al. 2016). The obtained clean reads within the range of 18 to 26 nt were assembled into contigs using Velvet software. BLASTn and BLASTx searches against the NCBI GenBank database showed that some contigs had the closest matches to the sequences of actinidia virus A, actinidia virus B, and actinidia chlorotic ringspot-associated emaravirus. Additionally, nine contigs coded for proteins matching partial amino acid (aa) sequences of the polymerase, HSP70h, CPm, and CP encoded by AcV-1, and showed nucleotide sequence identities of 65 to 85% with the corresponding fragments of the AcV-1 genome (GenBank accession no. KX857665), indicating a possible presence of an AcV-1 genetic variant, tentatively named AcV-1ch, in the sample. Multiple alignment showed that the polymerase of AcV-1 identified in Italy and AcV-1ch had a similar 26-aa insert located immediately after the GDD motif as compared with some other viruses in the family Closteroviridae. Two sets of primers, hsp70F/hsp70R (5′-GACGATCATAGGTATTGACTACGG-3′/5′-CCWCCRCCRAARTCGTAAACTATRT-3′) and cpF/cpR (5′-TGAGCTRGGRATAGATGTTGC-3′/5′-TCTCTCAGGGTTMGGATGAGT-3′) targeting the HSP70 and CP genes of the virus were designed based on the sequences obtained from our deep sequencing data and the KX857665 sequence. Leaf samples showing chlorotic leaf spots or mottle were collected from 12 kiwifruit trees (ID: XW1-12) grown in Jiangxi province. Total RNAs extracted from the 12 leaf samples together with the sample G4 (harboring AcV-1ch) were subjected to reverse transcription polymerase chain reaction testing for AcV-1, according to the previously described protocol (Zheng et al. 2016), except for annealing for 30 s at 53 to 57°C. Target products of 606 bp (primers hsp70F/hsp70R) and 376 bp (primers cpF/cpR) were amplified from samples G4 and XW1, and samples G4, XW1, XW4, XW5, and XW12, respectively. The 606-bp HSP70 fragments from isolates AcV-1ch and XW1 (GenBank accession nos. MH545710 and MH545709) shared 82.7% nt identity with each other and 84.7 and 81.5% nt identities with the corresponding sequence of reported AcV-1. The 376-bp CP fragments from isolates XW1, XW4, XW5, XW12, and AcV-1ch (GenBank accession nos. MH545704 to MH545708) shared 80.9 to 98.9% nt identities with each other and 81.4 to 90.7% nt identities with the corresponding sequence of AcV-1. The result further confirmed AcV-1 infection in kiwifruit plants in China, in at least two provinces, Hubei and Jiangxi. To our knowledge, this is the first report of AcV-1 in China, and the first outside of Italy. Further extensive study will be necessary for understanding the viral infection status and effect on kiwifruit production in China.