Identification And Molecular Characterization Of Viruses Infecting Kiwifruit

Our project group found that kiwifruit virus disease appeared widespread in recent years during our field survey. In order to acertain the species of virus, the harmful characteristics and molecular features. Actinidia virus A(Ac VA) was firstly detected in kiwifruit grown in China by Reverse transcription PCR(RT-PCR) with the primers designed or synthesized according to the reported virus gene sequence. Through comparing the sequence of gene amplification products, we determined the molecular features and variation characteristic of Ac VA. In addition, we identified a new species of virus in the Emaravirus by deep sequencing of small RNA in a kiwifruit sample and analyzed its genome structure. This study got the following results:1. In 2013 to 2014, leaf samples were collected from 151 plants of six kiwifruit species and some unknown species in six provinces, including Hubei, Anhui, Shanxi, Zhejiang, Jiangxi and Yunnan. Most of sampled trees showed chlorotic mottle or ringspots, vein yellowing and mosic symptoms. Those kiwifruit samples were tested for the presence of Ac VA and Ac VB by Reverse transcription PCR(RT-PCR). Results revealed that all six Actinidia species tested were positive to one or two viruses, and the average infection frequencies of Ac VA and Ac VB in kiwifruit samples were 15.2% and 17.9%, respectively.2. Three sets of primers were used for the amplification fragments from ORF, ORF4 and ORF5 of Ac VA genome. Sequence analyzes showed that the virus isolates were molecularly divergent and the 269-bp fragment of ORF1 from different isolates shared nucleotide similarities of 77.3-99.3%. The 597-bp ORF4 fragment of different isolates shared nucleotide similarities of 88.1-99.8% with each other, and 88.4-90.6% nt and 94.2-100% aa similarities with the corresponding sequence of the New Zealand isolate TP7-93 A. The 283-bp ORF5 fragment of different isolates shared 89.0-100% nt similarity, and 90.1-94% nt and 94.6-96.8% aa similarities with the corresponding sequence of the New Zealand isolate TP7-93 A. In phylogenetic trees based on nucleotide sequences of the three fragments, all Ac VA isolates separated into two or three clades.3. ORF5 fragment of Ac VB were amplified from 3 and 20 Ac VBisolates by using primer sets Ac VB5F/ Viti3’R and Ac VB5F/Ac VB5 R. The ORF5 fragment of 20 Ac VB isolates amplified using primer set Ac VB5F/5R had a size of 342-bp or 338-bp. All those isolates shared nucleotide similarities of 81-100% with each other and 83.9-99.7% nt and 79.8-100% aa similarity with the corresponding sequence of the New Zealand isolate TP7-93 B, and clustered into three clades in the phylogenetic tree constructed basing on nucleotide sequences of the fragment.4. A novel virus, named as Actinidia chlorisis ringspot-associated virus( ACRa V) was identified by deep sequencing of small RNA in a kiwifruit sample Ac HN-6. The complete genome of the virus was determined. The genome of the virus consists of five negative single strained RNAs. The sizes of RNA1-5 are 7061 nt, 2267 nt,1678 nt, 1664 nt and 1476 nt, with one open reading frame(ORFS) in each of RNAs. ORF1, ORF2, ORF3 and RNA4 encode a RNA-dependent RNA polymerase(P1: Rd Rp) of 2303 amino acids(aa), a glycoprotein precursor(P2: GP) of 653 aa, a nucleocapsid protein(P3: CP) consisting of 310 aa and a protein of 379 aa(P4), which might be involved in virus cell-to-cell movement. RNA5 encoding a protein(P5) consisting of 226 aa, which function remains unknown. ACRa V has similar genome structure as that of viruses in the genus Emaravirus. ORFs1-4 of ACRa V share the highest aa similarities of 64.6%, 48.3%, 55.6% and 51.9% with the corresponding ORFs of Redbud yellow ringspot virus(RYRV). The viral P5 shared similarities of less than as 23% with corresponding proteins of viruses in the genus Emaravirus. In the phylogenetic trees based on amino acid sequences of P1, P2 and P3 from ACRa V and all known emaraviruses, and of proteins from representative members of most genera in the family Bunyaviridae, ACRa V clustered together with emaraviruses with a high bootstrap value, thus confirming a close phylogenetic relationship between these viruses.5. Two sets of primers were designed in the sequences of ORF1 and ORF3 of Ac CRa V. In total, out of 146 assayed samples collected from seven provinces, 36 tested positive to ACRa V, accounting for 24.6%.