| As new RNA viruses and their new variants are found continually, more and more attention is being paid on the adaptive variants of RNA viruses. In our present study, Cucumber mosaic virus (CMV) satellite RNA (satRNA), a subgenomic RNA, is used as an excellent model for studing variation and evolution of RNA virus. Artificial mutations of satRNAs were constructed by using various molecular means, combined by using transcription in vitro and recombination in vitro techniques. The results are shown as below:Two CMV isolates, HZ03P09 and HZ03P10, obtained from Capsicum annuum in Hangzhou, Zhejiang, were identified as associated satRNA by double-stranded RNA analysis. After cloning and sequencing, it was found that satC336 isolated from HZ03P09, comprised 336 nucleotides, and satC382 from HZ03P10 comprised 382 nucleotides. When the sequences of the two satRNAs compared with previously reported sequences of 45 CMV satRNAs, obtained from different part of the world. In certain degree, the similarity among satRNAs sequences was found being related with the geological origination, yet not with the host origin and its size. According to the sequence similarity, variations occur mostly in the middle region of the sequences of satRNAs and the hypervariable regions are the same in all the satRNAs, while 80 bases in the 5' end and 200 bases in the 3' end are conservative relatively. The largest CMV satRNA is supposed to be 425nt, and the current small satellite RNA almost approaches to minimal size. This suggests that the CMV satellite RNA has possibility to be artificially manipulated.Different blends of isolates of CMV and satRNAs, were compared with each other to test the selective support of CMV on satRNA by recombining in vitro. Two satRNA-free CMV isolates, CNa and CFq, were used to co-inoculate with satRNA-satY369 (369nt) onto Nicotiana glutinosa. We found that only CNa could act as a helper virus for the replication of this satRNA. Four satRNAs, such as satY369, satY385, satC336 and satC382, were recombined with CNa respectively. Results indicated that CNa was unable to support the replication of satC336, however, supported the replication and systemic accumulation of other three strains. In addition, we combined CNa with the mixture of satY369 and satY385, and tested their co-existence on host plants. Host inoculation tests showed that satY369 could maintain stable co-existence with CNa on N. glutinosa, as well as other fourAbstractsystemic hosts, but satY384 did not. Therefore, it could be concluded that the selection between CMV strains and satRNAs was not only determined by the strains of helper virus, but also by the sequences and structures of satRNAs, which was dependent for its replication on the helper virus.To investigate the relationship between satellite RNA and helper virus, satY369 was transcribed in vitro and inoculated onto N. glotinosa plants together with CNa to form a pseudorecombinant virus (NY). The both isolates were inoculated onto different host plants individually, followed by examining plant symptoms. Disease symptoms induced by CMV were attenuated by satY369 in most host plants, especially in Cucurbita pepo. By dot blot hybridization, relative RNA loadings (RRL) of both genomic RNA3 and satellite RNA from the systemically infected host tissues were quantitatively measured. Under the condition of 26, CNa and NY were inoculated onto six lands of host plants and examined 5d, l0d, 15d, 20d, 25d, 30d post inoculation (dpi), respectively. On N. glutinosa, the RRLs of both genomic RNA3 and satellite RNA showed declining trends during the tested period, and the RRL of genomic RNA3 of CNa was higher than that of NY. On 10 dpi, CMV genomic RNA3 and satellite RNA were different in host fitness. The RRLs of genomic RNA3 hi CNa and NY differed obviously among the hosts, but they were the same in the order of Lycopersicon esculentum>N. physanodes>N. glutinosa>C. pepo>C. moschata>Datura stramonium. Meanwhile, the RRL of satellite RNA was in the order of N. physanodes>N. glutinosa>L esculentum >D. st...
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