Identification Of A Filamentous DsRNA Virus And Study On Its Molecular And Biological Characteristics

Colletotrichum camelliae Massee,the causal agent of brown blight disease of tea,is a popular pathogen attacking tea all over the word,causing leaf blight,shoot death,and fragility,and resulting in big loss of crop yield and quality of tea trees.In this study,C.camelliae strains LT-3-1 and DP-3-1 were obtained from tea leaves showing tea brown blight symptoms.Compared with standard C.camelliae strain DP-3-1,C.camelliae strain LT-3-1 showed abnormal colony morphologies including lack of mycelium rings,pigments being nonuniform and lower growth rate.Importantly,LT-3-1 strain exhibited weaker virulence on non-wounded tea leaves.Extraction of dsRNA from strains LT-3-1 and DP-3-1 showed that the latter has eight unique genomic dsRNA segments,with sizes of 2444,2253,2012,1299,1122,1085,1053 and 990 bp,respectively.Each contains a single(for dsRNA1 to 4 and dsRNA6 and 7)or two(for dsRNA 5 and 8)open reading frames(ORF)on one of the dsRNA strands.The ORFs were named according to the dsRNA on which they are located,e.g.,ORF 5-1 is the first ORF on dsRNA 5.The ORF1,3 and 4 was predicted to encode a RNA-dependentRNA polymerase(RdRp),Methyltransferase(MET)and coat protein(CP),respectively.However,the functions of the putative protein encoded by other ORFs were unknown.Sequence similarity alignment with known viruses and phylogenetic analysis based on RdRps revealed that the dsRNAs are genomic components of a novel dsRNA virus,and we named it as Colletotrichum camelliae filamentous virus 1(CcFV1)considering the particle morphology(see below).The virus particles were purified by ultra-centrifugation using stepwise sucrose gradients.The eight genomic dsRNA segments were recovered from the 20% to 30% sucrose fractions,with the same sizes of the dsRNAs extracted from the mycelium.SDSPAGE protein analysis of the gradient fractions corresponding to the sucrose gradient revealed that there is a protein with a size of 31 kDa(named P31);peptide mass fingerprinting(PMF)analysis of the P31 show it is the CP protein encoded by dsRNA4.Transmission electron microscopy(TEM)observation of the purified virus particles ranging from the 20% to 30% sucrose fractions showed that there are filamentous particles(lengths 127.1-4661.6 nm,outer widths 11.9-17.7 nm).To further confirm that the dsRNA are encapsidated in filamentous virus-like particles,polyclonal antibody against the CP protein was prepared by injecting 2-3 weeks old of BALB/C mice with the purified CP protein.Indirect enzyme-linked immunosorbent assays analyses revealed that the polyclonal antibody had a titer of an approximately 128,000-fold dilution,with the best fit at a dilution of 2000-fold.Western blot analyses showed that the prepared antibody can specifically recognized CP protein.Immuno-capture electron microscopy(ISEM)observation showed that filamentous virus-like particles were clearly decorated by the polyclonal antibody(1: 2000 or 1: 8000).The results confirmed that the observed filamentous particles are indeed the viral particles of CcFV-1.Forty-seven subisolates were generated from the conidial colonies of strain LT-3-1,and identified the virus presence with electrophoretic analysis of the dsRNA and RT-PCR amplification.The results showed that CcFV-1 was eliminated from all the isolates,having a virus elimination rate of 100%.Protoplasts prepared from the CcFV-1-free strain LT-3-1D2 and DP-3-1 were transfected with purified CcFV-1 dsRNAs(1 to 8 together)extracted from mycelia,and indexed by electrophoretic analysis of dsRNA in the regenerated colonies.The results indicated that the virus was successfully transfected in 10/94 and 3/129 subisolates,respectively.The dsRNA,protein and viral particle of CcFV1-transfected subisolates were extracted same to those in strain LT-3-1,but not in the virus-free strains.Compared with the CcFV1-free strain,the CcFV1-transfected subisolates showed the morphologies significantly different with the starting CcFV1-free strain including increase of the black pigments,disappear of the hyphal rings,reduced growth rates(5.4 mm/d versus 6.3-6.5)and virulence(5.4 versus 6.1-14.9 mm lesion lengths).Ultrathin hyphal sections for strains LT-3-1 and LT-3-1D2 were observed under TEM,indicating that the cells of hyphal compartments of strain LT-3-1 showed an abnormal syndrome,including formation of big membranous vesicle structures,reduction of woronin bodys and vacuoles compared with the CcFV-1 free strain LT-3-1D2.