Genome Determination And Characterization Of Two Novel Viruses Infecting Rosaceae Plants

The rosaceous plants are important horticultural crops, and they have been severely damaged by virus diseases. Identification of virus species responsible for the viral disease poses significance for its control as it provides basic information for designing control stategy. In recent years, we have found two virus-like diseases on rose and peach plants in Wuhan. As high-throughput deep sequencing has been strongly developed in recent years as a power tool for known and novel virus identification, we adopted this tool to identify the viruses related to the two disease in this study, discovering two novel viruses. The main results are as follows:1. The wild rose sample showing rosette symptom co-infected by four virusesA wild rose sample showing wild rose leaf rosette disease was subjected to deep sequencing with Illumina platform. Deep sequencing of viral-derived si RNA revealed that four viruses, of which three are known viruses: Blackberry chlorotic ringspot virus(BCRV), Prunus necrotic ringspot virus(PNRSV), Apple stem grooving virus(ASGV) and one is a novel virus, provisionally named ‘rose leaf rosetteassociated virus’(RLRa V). Among them, the full-length genome sequences of PRNSV, BCRV and ASGV can be assembled from their si RNAs and that share a high identities(>96%) with sequences reported previously. 97%(ASGV), 96%-97%(BCRV), 97%-98%(PNRSV) rsepectively. This is the first report of Closterovirus and Capillovirus viruses infected wild rose.2. Genome cloning and genomic organization analysis of RLRa V, and its correlation with wild rose rosette diseaseThe full genomic c DNAs were determined by clonging and sequencing the RT-PCR amplified products overlapped-covering the full genome using primers based on the contigs obtained, and the 5′ and 3′ terminal regions, suggesting that the genome is 17653 nucleotides in length(Gen Bank Accession No.KJ748003). Phylogenetic analysis clearly placed RLRa V alongside members of the genus Closterovirus, family Closteroviridae. Genome organization of RLRa V RNA showed 13 Open reading frames(Open reading frames, ORFs). Except ORF1 and the quintuple gene block, most of the ORFs showed no significant similarities with known viral proteins, but, instead, had detectable identities to fungal or bacterial proteins. In addition, some proteins, including L2, Mt, HSP70 h and CPh, show amino acid insertions and deletions in their conserved motifs when compared with those of the members of the corresponding families. Analysis based on genome organization of RLRa V RNA, RLRa V seems to be the most evolution compared with those of the members of the corresponding genus.Twenty wild rose samples from different Wuhan regions were collected for RT-PCR identification of RLRa V, ASGV, PNRSV and BCRV. The results indicated that RLRa V has a highest infection rate than the other three viruses. In addition, Most of the RLRa V-infected samples were not co-infected by BCRV, ASGV or PRNSV, but showed WRLRD or similar symptoms, thus suggesting that RLRa V is most likely to be responsible for WRLRD.3. The peach sample with fruit cracking symptom co-infected by two viroids and three virusesA peach leaves sample with fruit cracking symptom was subjected to deep sequencing with Illumina platform. Deep sequencing of viral-derived si RNA revealed that two viroids and three viruses, of which the viroids are Hop stunt viroid(HSVd), Peach latent mosaic viroid(PLMVd) and viruses are Apple chlorotic leafspot virus(ACLSV), Plum bark necrosis stem pitting- associated virus(PBNSPa V) and a novel virus, provisionally named ‘peach cracking fruit associated virus’(PCFa V).4. Cloning and identification of Genomic organization of PCFa V RNAOverlapping reverse transcription-polymerase chain reaction(RT-PCR) products covering the full genome were produced using primers based on the contigs obtained, together with sequencing the 5′ and 3′ terminal regions, the complete genome was determined. Phylogenetic analysis clearly placed PCFa V alongside members of the genus Fabavirus, family Comoviridea but with a far genetic distance with other genus members. Viral genome of PCFa V is composed of two strands of RNA molecules, with sizes of 6309 and 3837 nt, respectively(excluding ploy A tail). It was concluded that the polyprotein translated from RNA-1 could be processed into five mature proteins: protease cofactor(Co-pro), putative helicase(Hel), genome-linked protein(VPg), and RNA dpendent RNA polymerase(Rd Rp), respectively; and the polyprotein encoded by RNA-2 could be processed into movement protein(MP) and two coat proteins(LCP and SCP). The deduced proteins have low similarities with reported viral protein, with amino acid similarities less than 51%. To our knowledge, this is the first report fabavirus infecting peach, and its correlation with the symptoms needs further study.