彩色海芋黃化斑點病毒雙極性基因體 M RNA與負極性基因體 L RNA 與其他Tospovirus屬之病毒比較

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ABSTRACT Calla lily chlorotic spot virus (CCSV) has been classified as a distinct tospovirus belonging to Watermelon silver mottle virus (WSMoV) serogroup. To further characterize the relationships of CCSV with members of WSMoV serogroup, the nucleotide sequences of M RNA and L RNA of CCSV were determined by cloning and sequencing the cDNA fragments amplified by reverse transcription-polymerase chain reaction (RT-PCR). The complete sequence of CCSV M RNA contains 4,704 nucleotides in length. The CCSV M RNA encodes a nonstructural (NSm) protein of 309 amino acids in the viral strand, and a GN/GC protein precursor of 1,123 amino acids in the viral complementary strand. The NSm protein of CCSV shares amino acid identities of 37-39% with those of TSWV and INSV, 57-72% with those of MYSV, IYSV, WSMoV, CaCV and PBNV. In additon, the GN/GC protein precursor of CCSV shares amino acid identities 32-34% with those of TSWV and INSV, 64-73% with that of MYSV, CaCV, WSMoV, and PBNV. Our results indicate that both NSm protein and GN/GC protein of CCSV share higher amino aicd identities with those of WSMoV serogroup tospoviruses. The complete sequence of CCSV L RNA contains 8,911 nucleotides in length. The L RNA is of negative polarity, encoding L protein in the viral complementary strand with a predicted Mr of 332 kDa. L protein of CCSV shares low amino acid identities of 43-45% with those of TSWV and INSV; but it shares higher amino acid identities of 74-77% with those of MYSV, CaCV, PBNV, and WSMoV. Comparison of the deduced L protein of CCSV with that of the other members of the family Bunyaviridae indicated that its amino acid sequence include six conserved motifs A, B, C, D, E, and F of RNA-dependent RNA polymerases (RdRp). Additional conserved regions among tospoviral regions 1, 2, 3, 4, 5, and 6 were also identified. A phylogenetic cladogram of the RdRp indicated that CCSV belongs to the WSMoV serogroup of the genus Tospovirus and has a closer relationship with the viruses of genus Bunyavirus than with those of the genera Phlebovirus and Hantavirus. Multiple nucleotide alignments of L RNAs of CCSV with those WSMoV, PBNV, CaCV, MYSV, TSWV and INSV showed that there are several L RNA conserved regions at the nucleotide level among WSMoV serogroup or all tospoviruses. Five L RNA conserved regions a, b, c, d, and e containing 21 conserved nucleotides were suggested as an artificial micro RNA targeting sites for construction of transgenic crops with broad-spectrum resistance against tospoviruses. The degenerate primer pairs t2740/t3920 and tNSm410/tNSm870 were designed from the conserved regions of L RNA of WSMoV, PBNV, CaCV, CCSV, MYSV, TSWV, INSV and the conserved regions of NSm ORFs of WSMoV, PBNV, CaCV, CCSV, MYSV, TSWV, TCSV, GRSV, INSV, respectively, for amplification of the targeted regions of tospoviruses. The positions of degenerate primer t2740 was located in RdRp conserved region 2, and t3920 in conserved motif F. Tospoviruses with different serogroups or serotypes: WSMoV, CCSV, CaCV, MYSV, TSWV, GRSV, INSV, IYSV, and TYRV were detected by using the primer pairs. The amplified product obtained from PCFV-infected N. benthamiana was formed with size less than 0.46 kbp by using tNSm410/tNSm870. This may due to PCFV has no serological relationship with other tospoviruses. Our results indicated that degenerate primer pair tNSm410/tNSm870 have a better ability for broad-spectrum detection of tospoviruses from field crops. Degenerate primer pair WG1/WG960 were designed from the conserved regions of GNGC ORFs of WSMoV, PBSV, CaCV, CCSV, MYSV, and IYSV for amplification of a 0.96 kbp GNGC fragment of WSMoV, IYSV serogroup tospoviruses, or MYSV by RT-PCR. No cDNA products were obtained from TSWV, INSV, GRSV, or PCFV-infected plants of N. benthamiana. Degenerate primer pair WG1/WG960 have a potential to be used as a diagnosis tool to differentiate Asian type tospoviruses from European type tospoviruses.