Metagenome Sequencing Of Viruses Infecting Kiwifruit And Molecular Characterization Of Three Newly Identified Viruses

Kiwifruit(Actinidia spp.)is a perennial vine plant.China is the largest producer of kiwifruit with both great growing area and yield worldwide.Viral diseases of kiwifruit cultivated in China were found to be very common during our field investigation and have seriously affected kiwifruit production.Compared with the research progress of viral diseases of some other important fruit trees,the study on viral disease of kiwifruit started late and progress are very limited.In this study,by using Metagenome sequencing,viruses of kiwifruit grown in some provinces and cities of China were identified,and the molecular characteristics,genetic diversity and infection status of three newly identified viruses were studied,the main results are listed as follows:(1)Six kiwifruit samples(including two single samples and four pooled samples)showing obvious viral disease-like symptoms were used for r RNA-depleted RNA-seq analyses.After de novo assembling for RNA-seq derived reads and blast analyses against the NCBI databases,11 plant viruses belonging four families were identified.Of these viruses,eight viruses Actinidia virus 1(Ac V-1),Actinidia chlorotic ringspots associated virus(Ac CRa V),Actinidia seed-borne latent virus(ASb LV),Actinidia virus A(Ac VA),Actinidia virus B(Ac VB),Apple stem grooving virus(ASGV),Citrus leaf blotch virus(CLBV)and Actinidia emaravirus 2(Ac EV-2)were known to infect kiwifruit as reported previously,and three viruses Apple stem pitting virus(ASPV),Tea plant line pattern virus(TPLPV)and Apple latent sephical virus(ALSV)have not been reported to infect kiwifruit.Sequence analysis for RNA-seq derived contigs matching to the genomes of these 11 viruses showed that the populations of Ac V-1,ASb LV,ALSV and TPLPV had high molecular diversity.The occurrence frequncies of 11 viruses in 69 individual samples were determined by RT-PCR using virus specific primers.Results showed that 30 samples were free for 11 viruses mentioned above,and 14 samples were infected by single virus,25 samples were co-infected by two or more viruses.(2)AcV-1,a tentative member of the family Closteroviridae,was firstly molecular characterized from A.chinensis in New Zealand in 2018.Two full length and two near full genome sequences of Ac V-1 isolates were determined by RT-PCR using specific primers.The genome structure of these Chinese Ac V-1 variants is identical to that of isolate K75,and consisted of 12 ORFs.Analyses of these sequences together with the NGS-derived contig sequences(>10000 nt)revealed high molecular diversities of Ac V-1 population,with the highest sequence variation occurring at the 5′ UTR,ORF1 a,ORF2 and ORF3,and the available variants clustered into three phylogenetic clades.For the first time,our study revealed the different domain compositions in the viral ORF1 a and molecular recombination events among Ac V-1 variants.Leaf samples of 249 kiwifruit plants of five kiwifruit species and unknown Actinidia spp.in ten different provinces and cities were subjected to RT-PCR detection of Ac V-1,and the detection frequency of Ac V-1 was 31.7%.(3)ASbLV,a new member of genus Prunevirus in the family Betaflexiviridae,was firstly reported in 2018.In this study,the complete genomic sequences of six ASb LV variants were determined by RT-PCR using primers designed based on the sequences derived from the RNA-seq.The genome structure of these variants is the same as that of reported isolate ASb LV-01227,and consists of four ORFs.However,the length of ORF1 and the domains contained in the replicase encoded in ORF1 had some differences among different ASb LV variants.The multiple sequence alignments and phylogenetic analyses of the viral genomic sequences and amino acid sequences of four proteins showed that ASb LV had high molecular diversity and the determined ASb LV variants clustered into three phylogenetic clades.RT-PCR detection for ASb LV indicated that the viral detection frequency in 77 kiwifruit samples was 22.1%.(4)ALSV is a member of genus Cheravirus in the family Secoviridae.The virus was firstly identified from an apple seedling grown in Japan.Until now,only one complete genome sequence of ALSV isolate Fukushima is available.In this sudy,the full-length genomic sequences of two ALSV variants infecting kiwifruit were determined by RT-PCR using virus specific primers.Both variants had the same genomic structure as that of ALSVFukushima.Their genomes is composed of two positive single-stranded RNAs(RNA1 and RNA2),which respectively encoded a large polypeptide.The genomic RNA1 and RNA2 sequences of the two kiwifruit variants ALSV-CQ6 and ALSV-CQ8 showed 95.0-98.0% identities with each other,and 73.0-78.0% nt identities with the RNA1 and RNA2 of isolate Fukushima,suggesting that the two kiwifruit variants had great genetic distance to the ALSV apple isolate Fukushima and ALSV variants infecting kiwifruit belong to a novel molecular variant of ALSV.RT-PCR detection for 77 kiwifruit samples showed that the occurence frequency of ALSV was 9.1%.This is the first for the identification and the determination of genomic sequences of ALSV from kiwifruit.The obtained results of this study confirmed the species and distribution of viruses infecting kiwifruit grown in China and can enrich kiwifruit virus species and genome sequence informations,which will provide important basis for further clarifying taxonomy positions,pathogenic characteristics and establishing efficient molecular detection methods of these viruses,and furtherly provide scientific information for the control of viral disease of kiwifruit.