Analysis Of The Genetic Diversity Of Citrus Tristeza Virus In China And Identification Of The Epitopes In The Coat Protein

Citrus tristeza virus is the causal agent of citrus tristeza disease, which has been one of the most important diseases affecting citrus growth and industry. CTV, a member of the genus Closterovirus in the family Closteroviridae, possesses a number of distinct and characterized strains and genotypes. Up to now, there is still incomplete understanding of the characterization method of CTV genotype, and the CTV genotype composition in China. CTV has two main structural proteins, major coat protein (CP) and minor coat protein (CPm), and both of them participate in multiple biological functions. Further study on the genetic variation and antigenic characteristics of CPm and CP will provide important information on the CTV evolution history and possible important structural or functional domains in two structural proteins. In this study, the genotype composition of CTV isolates from some regions in China, genetic structure of CTV population in China, and epitopes in CP are studied in depth. The main results are listed as follows:1. Genotype study of CTV isolates from ChinaTotally10CTV-infected citrus samples collected from Hubei and Jiangxi provinces were used for genotype study. By using RT-PCR method with11multiple molecular markers (MMMs)(VTK17, VTPOL, VT-5’, T3K17, T30K17, T30POL, T30-5’, T36K17, T36POL, T36-5’, and B165-LPro II), combined with sequencing of the products and phylogenetic analysis. Our results revealed four known genotypes (VT, T36, T30and T3) in Chinese CTV population, and two genotypes RB and B165were identified for the first time in China. In addition, only three isolates N4, S4and BX were singly infected, while other seven isolates were infected with mixtures of two or more genotypes. The results indicated that the MMMs in5’region were much more specific with the corresponding genotype, while MMMs in POL and K17regions were nonspecific, and could not discriminate between VT and T3, VT and T30, and T30and RB. Multiple alignments of deduced amino acid sequences in K17, POL, and LPro II regions showed genotype-specific amino acids in CTV isolates. Although the sequences of S45-VTPOL clustered with reference isolate T36, the similarity of nucleotide sequence between them was only89.7%. Similarly, the sequences of B15-LPro Ⅱ and B16-LProⅡ clustering with B165showed only70.0-81.8%nt and67.1-76.6%aa identity with the reference isolate B165. Recombination analysis showed no recombinant events in these sequences, suggesting novel genotypes existing in CTV population in China. Our results also found that that the genotype identification results showed somewhat inconsistency when comparing the RT-PCR and sequence analysis results, thus, genotype assignment of CTV cannot be based solely on the amplification profiles of MMMs, and sequencing of MMMs is required.2. Genetic diversity and evolution characteristics of CP and CPm genesTotally85citrus leaf or branch samples (including six CTV positive samples from Hubei province) collected from four main citrus growing provinces (Hunan, Sichuan, Jiangxi, and Hubei) were used in this study, and most of samples from Sichuan showed decline symptoms. RT-PCR results showed that the total detection rate of CTV is32.8%(26/79), while that of CTV from samples in Sichuan was100%(11/11). By using the primer sets CPm (F) and T36-CP (R) with RT-PCR,32CTV-infected samples were further used for the cloning of an approximately1540-bp fragment consisting of partial P61, complete CPm, CP and the internal non-coding region. The fragment sequences from29isolates were obtained. Sequence analysis revealed intra-and inter-isolate sequence diversity. Phylogenetic analysis of the whole fragment nucleotide sequences revealed five known groups (RB, T30, T36, HA, and VT), and one new group VI solely consist of Chinese CTV isolates. Most of isolates from Hunan province belonged to group RB, and one popular and unique CTV subgroup representated by aphid transmissible isolate AT-1in the group VT was found in China, and this subgroup had unique aa insertion in the C-terminus end of P61, here designated as AT-1. The CPm-and CP-based phylograms shared similar tree topologies with fragment-based phylogram except for some groups. In CPm-based phylogram, isolates represented by the mild strain T30formed a subgroup RB-d, which has low genetic distance ranging from0.024±0.005to0.037±0.005with other three subgroups in the group RB. Recombination analyses of the1540-bp sequences revealed CPm gene as the new recombination hot regions across CTV genome, and25recombination events occurring in CPm were detected, while only two recombination events occurred in the CP gene. Our results revealed frequent gene flow between subpopulations from China and other countries, and the CTV subpopulations of CP gene worldwide were in a state of increasing. Analysis of synonymous and nonsynonymous substitutions indicated that both CPm and CP genes are under strong negative selections, and positively selected sites at the position9in CPm, and the positions of31,41, and68in CP were identified in CTV for the first time. Our results suggested that gene flow, recombination, and negative selection could contribute to the complex CTV population in China.3. Cloning and sequence analysis of the complete genomic sequence of AT-1Isolate CTV-AT-1was obtained by aphid transmission, and the complete genomic sequence of AT-1was cloned, sequenced, and deposited in the GenBank with an accession number JQ061137. The AT-1had19,252nt. Hinf I/RFLP analysis of CP gene combined with sequence analysis showed that AT-1was single infection and belonged to the prevalent Hinf/RFLP group III and VT genotype. Complete genome sequence analysis showed that the AT-1had the highest identity to the aphid-transmissible sub-isolate T318A (96.8%) and clustered together in whole genome-based phylogram, while the lowest identity to the isolates Qaha and Mexico (80.91%) belonging to the group T36. Sequence analysis of each gene and two UTRs revealed that AT-1had highest identity to representative isolates VT (98.1%) and B165(98.9%) in5’-UTR and3’-UTR, respectively. In addition, AT-1showed highest identity irrespective of nt and aa level with isolate B165in continuous nine genes except for p6in3’terminal, while in ORF1a and RdRp regions in5’terminal, AT-1had highest nucleotide identity with VT, and amino acid identity with T30and T3, respectively. Recombination analysis further demonstrates that AT-1was a recombinant from two parents VT and B165, and the breaking point was located in the region between RdRp and p33.4. Epitope mapping and site-directed mutagenesis of coat protein of CTVTen monoclonal antibodies were selected randomly to map the epitopes in the coat protein of isolate CTV-S4. By using truncated coat proteins and Western blotting method, we identified three epitope regions recognized by seven monoclonal antibodies. Of them, MAb5and6recognized the epitope I region (48LGTQQNAALNRDLFLT63), MAb3and 7recognized the epitope III region (114LSDKLWTDVVFN125), and MAb1,4, and10recognized the epitope II region (97DDDSTGIT104), which was a novel epitope. The epitope was further confirmed by using synthesized oligopeptides and ELISA method, and the deletion of two amino acids (T104and1103) in the C-terminus of epitope II has significant effect on its reaction with these three MAbs. Multiple alignments of deduced amino acid sequences of CP gene showed that the epitope II region is much conserved in CTV isolates belonging to different groups. Site-directed mutagenesis analysis showed that the natural variations in this region could not abolish the reaction between MAbs and the epitope II, while I103M and S100T variation reduced the reaction signals significantly.5. Identification of the critical amino acids in the interaction between CTV-HB1coat protein and monoclonal antibodies and epitope structure analysisThe reactivity of those10monoclonal antibodies with the crude extracts and expressed coat protein of isolate CTV-HB1was evaluated by Western blotting method. Results showed that five monoclonal antibodies (MAb1,4,5,6and10) could not recognize the CP of CTV-HB1. Multiple alignments of deduced aa sequences of CP gene showed conserved and the same aa site D98in all CTV isolates except for HB1(G98). When the D98in S4-CP was replaced with G98, MAbs1,4, and10could not recognize the mutated epitope II, suggesting the important role of D98in the recognization of CP by these three MAbs. In addition, the interaction between HB1-CP and these three MAbs was recovered when G98in HB1-CP was replaced with D98, indicating that the presence of G98in CP of HB1is crucial for its interaction with them. Further comparative analysis of predicted epitope and structure of coat protein between isolates CTV-S4and CTV-HB1suggested that the less exposed surface area of G98in HB1CP might result in the negative reaction with these MAbs. Our results also suggested that the conformational differences in the epitope I (48LGTQQNAALNRDLFLT63) between the CPs of isolates S4and HB1might contribute to the different reactions of two isolates to MAbs5and6, although the amino acid sequences in the epitope I region was the same between them.