Studies On A Novel Virus And A Small Circular RNA Identified From Mulberry Trees

Recently, an novel isometric virus and a new small circular RNA were identified from mulberry leaves showing symptoms of mulberry mosaic leaf roll (MMLR) disease. To illuminate the biological and molecular biological properties of this novel virus and the new small circular RNA, the main research contents in this paper include:1. The genomic nucleotide sequences of the novel virus were determined by RT-PCR employing anchor random primers and5’,3’-RACE’(rapid-amplification of cDNA ends) technique. The characteristic of the viral genome organization was analyzed and the classification status of the virus was defined. Further studies include:producting the antiserum against the isometric virus and setting up the detection method using the viral antiserum. Based on the nucleotide sequence of the novel viral genome, a pairs of specific primer was designed to detect the novel virus in the mulberry fields in the different geographical regions by RT-PCR. The detection results will discover the geographical distribution of the novel virus and the relationship between the novel virus and the occurrence of MMLR disease.2. We will continue the studies on mulberry small circular RNA (mscRNA) found in the leaves with symptoms of MMLR disease in the previous study, clone the full length nucleotides sequence of mscRNA and analyze its molecular structure properties. The mscRNA infectivity, which will reveal the relationship between mscRNA and MMLR disease, was examined by the back-inoculation assay of mscRNA extracted following LiCl method and infectious mscRNA clone. A rapid and reliable detection method on nucleic acid level was established to detect the presence or absence of mscRNA in the trees of MMLR disease, these results will present the relationships between mscRNA and MMLR disease incidence, symptoms. The results were as follows:1. An isometric virus,28-30nm in diameter, was observed in semi-purified virus preparations from leaves of MMLR disease by transmission electron microscope. The whole genome sequence of the isometric viruses was sequenced, the genome consists of two positive sense single-stranded RNA (ssRNA). The genomic RNAs (RNA1and RNA2) contain a poly (A) tail at the3’-terminal. The full length of RNA1and RNA2was7183nt and3742nt excluding poly(A) tail, respectively. RNA1and RNA2contained a single large open reading frame (ORF), respectively. RNA1encoded a polyprotein (P1) of2,102amino acids, corresponding to molecular weight235.1kDa. RNA2encoded a polyprotein (P2) of1,093amino acids, corresponding to molecular weight120.5kDa. Phylogenetic tree based on amino acid sequence of P1indicated that this virus belonged to the genera Nepovirus. The amino acid sequence of P1contained the characteristic motifs A to C of the putative helicase (Hel), the conserved domain of viral genome-linked protein (VPg), and consensus motifs I to VIII conserved in the RNA dependent RNA polymerases (RdRp) of positive-strand RNA viruses. In addition, it contained catalytic triad of H, E/D, and C conserved in cysteine proteinases (C-Pro). The coat protein (CP) and movement protein (MP) were contained in the RNA2-encoded polyprotein P2. The CP was located at the C-terminal of P2and the MP was located upstream of the CP. The cleavage site between MP and CP was putative to be553E/V554by the prediction of NetPicoRNA1.0Server and estimation of Western blotting. The E/V cleavage site was a new cleavage site of3CL(pro). The deduced molecular weight of the CP was approx.59kD. The conserved nepovirus CP motif, such as FxFYGR motif, was located in the C-terminus of the CP. In addition, the P2also contained the consensus motif LPL conserved in the MP of nepoviruses and comoviruses. The virus is proposed to be a new species of the genus Nepovirus, subgroup A on the basis of phylogenetic analysis of P1, nucleic acid sequence length of RNA2, charactertic of3’-UTR (untranslated region) and5’-UTR between RNA1and RNA2, and species demarcation criteria (<75%identity in CP amino acid sequence) in the genus Nepovirus listed in Ninth report of the ICTV. In despite of several attempts, we failed to back-inoculate the crude sap of MMLR disease into not only mulberry seedlings, but also cowpea (Vigna sinensis) and Nicotiana benthamiana. A causal relationship between this virus and MMLR has not been established. The new virus is tentatively named as Mulberry Mosaic Leaf Roll-associated Virus (MMLRaV) since it was identified firstly from the leaves of MMLR disease.The detection results by RT-PCR showed that58of total61mulberry leaf samples with symptoms of MMLR disease collected from Huzhou, Zhejiang province, Hai’an and Zhenjiang, Jiangsu province were detected to infect MMLRaV, giving an infection rate of95%in average. These results suggested that all or majority of mulberry trees with the symptoms of MMLR disease were infected by MMLRaV. MMLRaV distributed widespreadly in the mulberry fields where MMLR disease occurred. MMLRaV had a close relationship with MMLR disease.2. Zhejiang isolate of mscRNA (mscRNA-zj) consist of365nucleotides with G+C content of51.12%. BLAST analysis of the nucleotide sequence indicated that mscRNA-zj sequence shared99%identities with mulberry small circular viroid-like RNA1isolate201110151(JQ809700) and mulberry small circular viroid-like RNA1(EU547491), but it has reverse polarity with that of two sequences. The secondary structure of lowest free energy predicted by mfold is a branched conformation with65.7%paired nucleotides and with lowest free energy of-601.6kJ/mol at37℃. The secondary structure of mscRNA was similar to that of Avsunviroidae and circular satellite RNA of some plant viruses. The mscRNA possess a hammerhead ribozyme structure in genomic strand but not in anti-genomic strand. The cleavage site of mscRNA during replication was predicted according character of the hammerhead ribozyme. mscRNA presented sequence diversity in different geographical regions. Back-inoculation assay of mscRNA extracted following LiCl method and infectious mscRNA clones indicated that mscRNA could replicate neither in the natural host-mulberry, nor in the differential host-tomato, which suggested that mscRNA was most likely a circular satellite RNA of an unknown mulberry virus, but not a viroid. One-tube one-step RT-PCR method was established and the detective results indicated that mscRNA was not associated with symptom and occurrence of MMLR disease.In conclusion, MMLRaV is a novel member of the genus Nepovirus, subgroup A. MMLRaV has a close relationship with MMLR disease. mscRNA is most likely a satellite RNA of a unknown mulberry virus, but not a viroid. mscRNA is associated with neither the occurrence of MMLR disease, nor the symptoms expression degree of the disease.