Research On Molecular Characterization Of Two Viruses Infecting Actinidia

Kiwifruit(Actinidia spp.)is an economically important crop grown in China.Previous investigation have found the wide distribution of viral diseases infecting kiwifruit plants.To understand the viral pathogens and their molecular characteristics,conventional RT-PCR combined with small RNA(sRNA)and RNA sequencing were applied to molecular identification and characterization of Citrus leaf blotch virus(CLBV)and a novel virus in the family Closteroviridae.The main results are illustrated as follows.1.During 2014 to 2015,leaf samples were collected from 134 kiwifruit plants grown Hubei,Yunnan,Anhui,Guizhou,Shandong and Fujian provinces.Most of the sampled plants showed leaf yellow mottle,curling,mosaic,vein yellowing and other viral disease-like symptoms.RT-PCR using primer set CLBV1F/CLBV5R revealed that 15 plants were positive for CLBV,accounting for 11.3%.The target fragment is 425 bp of partial coat protein gene.The kiwifruit species positive for CLBV included Actinidia chinensis,Actinidia deliciosa and some hybrids or wild species.2.Primers for the amplification of the complete cp gene of CLBV were designed.The complete cp gene of five CLBV isolates was 1092 nucleotides(nts)in length and shared 86.6%-99.7%nt and 96.4%-99.4%amino acid(aa)sequence identities with each other,respectively.However,they shared only about 83%nt identity with a kiwifruit isolate M3-A reported in New Zealand,and 84%-86%identity with CLBV isolates infecting citrus and its relatives.In the phylogenetic tree basing on nucleotide sequences of their cp genes,CLBV isolates identified in the present study clustered into a group with New Zealand kiwifruit CLBV isolates except for isolate M3-A.3.The complete genome sequence of CLBV isolate AH 17 was sequenced.The genome of AH 17 consisted of 8797 nt(excluding Poly A)and had the same genomic structure as those of CLBV isolates reported previously.The genome of AH17 includes three open reading frame(ORFs),5 ’ terminal un-translation region(5’ UTR,71 nt)and 3’terminal un-translation region(3’ UTR,530 nt),ORF1,2 and 3 were 5958 nt,1089nt and 1092nt,coded replication,movement and coating proteins,and had 77.9%-82.8%、78.0%-84.2%and 84.6%-85%nt identities with the corresponding ORFs of other CLBV isolates.4.A novel virus were identified from a kiwifruit plant(ID,G4)by sRNA sequencing and RNA-seq combined with RT-PCR amplification.A genomic fragment of 15766 nts were sequenced.The virus has a genomic structure consists of 12 ORFs encoding functions known proteins RdRp、HSP70h、HSP90h、CPm and CP,and some other functions unknown proteins.The virus has the typical genomic features characterizing viruses in the family Closteroviridae.Proteins encoded by the virus usually show the relatively higher identities of 12.3%-50.5%with the corresponding protein of Persimmon virus B(PeVB),an undersigned member in the family Closteroviridae.In the phylogenetic tree constructed basing on amino acid sequences of these proteins,the novel virus is in the same cluster with PeVB and Blueberry virus A(BVA).Therefore,basing the genomic structure and phylogenetic relationships with the viruses in the family Closteroviridae,the virus is tentatively designed as a novel virus in the family Closteroviridae,and tentative name as Actinidia closterovirus A(ACVA).5.A primer set was designed basing on the obtained cp sequence of ACVA and used for the detection of virus in 65 kiwifruit samples.Results showed that 20 samples were positive for the virus,accounting for 30.8%.The amplification products from 15 samples were sequenced,and their amplified fragments were 375 bp,shared 81.6%-100%nt and 1.2%-100%aa identities.