Identification And Characterization Of A Novel Mandarivirus In Citrus

Citrus is of economic importance,which is widely cultivated in 140 countries for the production of fresh fruits and juice,as well as other processed goods.Citrus trees along with long-term cultivation are usually affected by one or several species of viruses,which is spread mainly by grafting,some also by vector such as insects or mites in the field,a few even by agricultural operations such as knives used for grafting,trimming and fruit-picking,seriously threatening the development of citrus industry.Since China‘s joining the World trade organization in 2000,the Chinese citrus industry has been facing potential risk brought by overseas virus diseases.Being part of a strategy for preventing the occurrence and spread of exotic and dangerous virus diseases in China‘s citrus production areas,we investigated the presence and spread in Pakistan of some of the viruses that impact citrus production and six samples,grafting of which onto Symons sweet orange(SSO)(Citrus sinensis)induce virus-like leaf symptoms of vein yellowing and mottle,were tested negative for all the targeted viruses.Therefore,to further investigate whether these samples were infected by some unknown virus or virus strain(s)undetectable by Reverse transcription-polymerase chain reaction(RT-PCR)methods used in the survey,High-throughput sequencing(HTS)of citrus transcriptome was performed and a novel virus identified.Here,the molecular,phylogenetic,biological,ultrastructural and serological features of this virus,supporting the proposal of classifying it as a novel species in the genus Madarivirus,were reported.Finally,Quantitative real-time PCR(qPCR)for CiYMaV detection was developed.The main results are summarized as follows:1.Discovery of a novel virus,Citrus yellow mottle-associated virus(CiYMaV).A total of 120 citrus samples were collected from different orchards of the Punjab Province,Pakistan,and used to graft-inoculate virus-free SSO seedlings.Leaf samples from all the grafted trees were collected and tested by RT-PCR using primers designed to specifically detect eight citrus viruses,including Citrus concave gum-associated virus(CCGaV),Citrus leaf blotch virus(CLBV),Citrus psorosis virus(CPsV),Citrus tatter leaf virus(CTLV),Citrus tristeza virus(CTV),Citrus yellow vein clearing virus(CYVCV),Citrus virus A(CiVA),Satsuma dwarf virus(SDV).Of these viruses only CTV and CYVCV were detected in some of the grafted trees,while ten samples were tested negative for the eight viruses,among which six(isolates PCV31,PCV49,PCV59,PCV62,PCV64,and PCV82)grafted to SSO trees displaying obvious leaf vein yellowing,which was accompanied by leaf mottling at a later stage.These six samples were further analyzed by specific PCR assay to detect potential infection of Candidatus Liberibacter asiaticus,which could induce leaf yellowing in citrus,but showed the negative results.Then,six libraries(PCV1–43,PCV44–93,PCV94–103,PCV104–112,PCV113–120 and PCV31)were built and further tested using HTS technique,respectively.Bioinformatics analyses showed that there were sqeunces of an potential new virus with the highest nt sequence identity with mandariviruses in the six libraries,besides CTV and CYVCV.Finally,the full genome sequence of the new virus was determined by cloning and sequencing of overlapping cDNA fragments generated by RT-PCR and Rapid amplification of cDNA ends(RACE).2.Analysis of molecular biological characteristics of CiYMaV,such as genome structure,protein function and phylogenetic development relationship.The genome of CiYMaV was composed of a positive-sense single-stranded RNA of 7 479 nt,with six Open reading frames(ORF).ORF1(nt 78–4 973)encoded a 1 631-aa putative Replicase protein(REP,184.2 kDa)with four domains typical of REP of members of the genus Mandarivirus: methyltransferase,AlkB oxygenase,helicase 1 and RNA-dependent RNA polymerase 2.A Triple gene block(TGB),comprising the overlapping ORF2(nt 4 981–5 658),ORF3(nt 5 636–5 965),and ORF4(nt 5 892–6 074),was seemingly responsible for the expression of three synergic proteins(TGB1–3)that may contribute to virus cell-to-cell and systematic movement in the plant as previously reported for other plant viruses,such as fovea-,carla-,and potexviruses.TGB1(255 aa)was a putative protein of 25.02 kDa,with a Hel-1 domain.TGB2(109 aa),a 12.4-kDa putative protein with the conserved domain Plant_vir_prot,was found to be related to the movement proteins of potex-,hordei-,and carlaviruses.TGB3(60 aa)was the smallest protein(6.4 kDa)of the three and contained a ?7kDa viral coat protein-like domain at aa 22–58.ORF5(nt 6 098–7 075)encoded a putative 35.8-kDa(325-aa)Coat protein(CP)that contains the pfam00286 domain(aa 140–278)of potexand carlavirus CPs.ORF6(nt 6 775–7 443)partially overlapped with ORF5 and encoded a hypothetical Nucleic acid-binding protein(NaBP,222 aa and 25.4 KDa).ORF1-ORF6 shared the maximum sequence identity of 82.73%,63.72%,68.47%,78.69%,75.15%,and 78.48% at nt level(73.31%,64.16%,71.21%,73.22%,72.90%,and 80.87% at aa level)with members of the genus Mandarivirus,respectively.Phylogenetic trees reconstructed with amino acid sequences of the REP,CP and nucleotide sequences of the whole genome of representative members of the families Alphaflexividae,Betaflexiviridae and CiYMaV,showed that CiYMaV and the two mandariviruses(CYVCV and ICRSV)clustered into a single clade.These results indicate that CiYMaV is a new species of the genus Mandarivirus.3.Relationship between CiYMa V and CYVCV in serology,virus particle morphology and biology.CiYMaV and CYVCV have both similarities and obvious differences.1.similarities:(1)observation of negatively stained leaf extracts from the CiYMaV-infected symptomatic SSO sample by TEM showed filamentous virus-like particles,similar to CYVCV,(2)samples infected by CiYMaV showed cross-reactivity with the 1E1 antibody of monoclonal antibodies against CYVCV;2.differences:(1)CYVCV induced vein clearing in Daidai sour orange(DSO)and Eureka lemon(EL),while CiYMaV was not associated with any leaf symptom in these hosts but with vein yellowing and mottling in SSO,whereas CYVCV did not induce any leaf symptom in this host,(2)samples infected by CiYMaV did not react with MAB 26A1 of monoclonal antibodies against CYVCV.These results strengthen the conclusion that CiYMaV is a novel species in the genus Mandarivirus.4.Development and application of qPCR detection system of CiYMaV.Five sets of specific primer pairs were designed for qPCR detection system based on the full genome sequence.Through a series of optimization of reaction conditions,one feasible pair of primers was selected and a specific qPCR detection system was established for the detection of CiYMaV.The system has the advantages of strong specificity,high sensitivity(10 times higher than the conventional RT-PCR),good repeatability,and simple operation,so it is suitable for rapid detection of field samples.It was applied to test 40 graft-inoculated CiYMaV samples,and compared with conventional PCR,it shortens the detection time.It has been approached to detect of 200 citrus samples from Yunnan province,China with negative results of CiYMaV.