Genome Sequencing Of Cucumber Green Mottle Mosaic Virus And Sugarcane Mosaic Virus And Their Population Genetic Structure Analyses

Cucumber green mottle mosaic virus (CGMMV) and Sugarcane mosaic virus (SCMV) are two important viruses which can infect many crops. They widely distribute and severely harm to crops, threating the development of our national agriculture. This paper mainly focuses on the study of genome sequencing, population genetic structure analyses of CGMMV and SCMV, as well as preparation of monoclonal antibodies (MAbs) against SCMV. The research results are as follows:1. Genome sequencing of 7 CGMMV Zhejiang isolates and population genetic structure analysis of CGMMVIn this study, we surveyed CGMMV infection in 762 cucurbitaceous crop samples,4 batch of cucurbitaceous crop seed samples and 60 field weed samples from different areas in Zhejiang Province with the dot enzyme-linked immunosorbent assay (dot-ELISA) using a CGMMV specific monoclonal antibody. The results showed that CGMMV infection was detected in cucurbit plants and their seeds, watermelon plants and their grafted plants, and melon plants. In addition, CGMMV infection was also detected in field weeds, such as Cardamine hirsuta L. and Alternanthera philoxeroid.es (Mart.) Griseb. That is the first report that CGMMV can infect weeds. According to geography distributions and hosts of CGMMV isolates from Zhejiang Province, the complete genome of seven CGMMV Zhejiang isolates including 4 isolates from watermelon plants,2 isolates from grafted watermelon plants and 1 isolate from melon plant were cloned and sequenced. Genomes of Taizhou watermelon isolate and Jiande watermelon isolate were 6 425 bp and 6 423 bp in length, respectively. Genomes of two Dongyang watermelon isolates were 6 423 bp and 6 425 bp in length, respectively. Genomes of Xiaoshan grafted watermelon and Dongyang grafted watermelon isolates were 6 423 bp and 6 424 bp in length, respectively. Genome of Jiande melon isolate was 6 423 bp in length. Identities analysis showed that the nucleotide identities of seven complete genome sequences ranged 99.2～100%. Phylogenetic analysis of seven CGMMV isolates as well as other 24 CGMMV isolates from the GenBank database showed that CGMMV isolates could be grouped into two distinct monophyletic clades according to geography distribution, i.e. Asia isolates for subgroup I and European isolates for subgroup Ⅱ, indicating that the population diversification of CGMMV isolates could be affected by geographical distribution. Site variability rate analysis of CGMMV found that the overall variability rate was below 8% and mainly ranged 2-5%, indicating that CGMMV sequence was more conservative. Base substitution types analysis of CGMMV showed that the mutational bias was existed in transitions (A←→G and C←→T) over transversions (A←→C, A←→T, G←→C and G←→T). The majority variability occurred in CGMMV genome resulted in non-synonymous substitutions and the variability rate of some sites was up to 30% because of the mutational bias. Selection constraint analysis of CGMMV ORPs showed that strong negative selection acted on the replication-associated proteins, which was similar with the phenomenon occurred in other plant RNA viruses. Finally, potential recombination analysis showed that isolate Ec as a recombinant was in a low degree of confidence with different recombination analysis programs.2. Genome sequencing of SCMV Yunan isolate and population genetic structure analysis of SCMVSCMV infection was identified in a field maize sample from Yunan with RT-PCR, and then its complete genome were cloned and sequenced. The result showed that the genome of the sequenced isolate (named SCMV-YN) was 9 598 bp in length excluding the 3’-terminal poly (A) tail. Predicted genomic structure showed that the characteristic RNA helicase domain V1215LLLEPTRPL1224 changed to V1215LLIEPTRPL1224 in RNA helicase CI and the prokaryotic lipoprotein motif I2554LAFNYTLLSC2564 mutated to I2554IAFNYAMLSS2564 in the C-terminal of NIb of isolate SCMV-YN. The identities analysis between isolate SCMV-YN and other 28 SCMV isolates from the GenBank database found that SCMV-YN shared a highest identity of 95.2% with SCMV-BD8 from Hebei, maize. Among these isolates, P1 was the most variable, while CI and PIPO were much more conserved compared with other ORFs. Phylogenetic analysis based on complete genome level of 29 SCMV isolates showed that SCMV isolates could be clustered into two large clades, i.e. group Ⅰ and Ⅱ, which can be further divided into two small clades:subgroup ⅠA (Zea mays) and subgroup IB (Zea mays/Sugarcane) for group Ⅰ, subgroup ⅡA (Zea mays/Sugarcane) and subgroup ⅡB (Sugarcane) for group Ⅱ. Among these isolates, the effect geography distribution played on genetic relationship of isolates had been attenuated, but, the effect of host origin still existed. Site variability rate analysis of SCMV found that the variability rate ranged 15-20%, indicating that a high mutation existed in SCMV genome. Base substitution types analysis of SCMV showed that mutational bias was similar with other RNA viruses, existing a bias in transitions (A(?)G and C(?)T) over transversions (A(?)C, A(?)T, G(?)C and G(?)T). The genetic differentiation and gene flow analysis revealed that significant genetic differentiation between isolates of group Ⅰ and group Ⅱ existed in all ORFs. Meanwhile, frequent gene flow was detected in P3, NIa-VPg, NIa-Pro and NIb-replicase ORFs. Selection constraint analysis showed that negative selection acted on all ORFs of SCMV and the CI ORF had the strongest negative selection. Recombination analysis showed that of these 29 isolates,7 isolates had experienced multiple potential recombination events in a high confidence with different programs. Except P3 ORF, all ORF regions of SCMV genome were detected recombination events.3. MAb against SCMV and its applicationThe purified virions of isolate SCMV-YN were used as the immunogen to produce MAbs. Via cell fusion, cell culture, antibody detection and cell cloning,9 hybridoma cell lines (6A1,6A12,10B11,12A10,15C12,19F3,21C12,21H3 and 22A2) secreting sensitive and specific murine MAbs against SCMV were obtained. And then their ascitic fluids containing MAbs were prepared. The titers of ascitic fluids of 9 MAbs ranged 10-6 to 10-7by indirect-ELISA, and all MAbs were isotyped as IgG1, kappa light chain. Western blot analysis indicated that MAbs 6A1,21H3 and 22A2 could react with CP of SCMV-YN and Beijing isolates, indicating that they could specifically recognize common antigenic determinant, while MAbs 10B11, 12A10,15C12,19F3 and 21C12 could only react with CP of SCMV-YN isolate, indicating that these MAbs could recognize specific antigenic determinant in coat protein of SCMV-YN isolate. MAb 6A12 could not recognize any protein of SCMV.Base on result of the dot-ELISA, we speculated that it likely recognized the conformational antigenic determinant. At the meantime, the dot enzyme-linked immunosorbent assay (dot-ELISA) was set up using these 9 MAbs for analyzing their characteristics. The specificity analyses of MAbs revealed that MAbs 6A1,21H3 and 22A2 had a positive response with SCMV-YN and Beijing isolates, while MAbs 6A12,10B11,12A10,15C12,19F3 and 21C12 only had a positive response with SCMV-YN isolate, indicating that they could be applied to identification and detection of SCMV-YN isolate. The sensitivity analysis by the dot-ELISA revealed that MAbs 6A1,15C12,10B11 and 21H3 were the most sensitive, and their sensitivities for detecting infected leaves were 1:10 240 (w/v, g/mL). Detection sensitivities of MAbs 12A10 and 19F3 were 1:5 120 (w/v, g/mL), and MAbs 21C12 and 22A2 were 1:2 560 (w/v, g/mL). Detection sensitivities of MAb 6A12 also reached 1:1280 (w/v, g/mL).