Origin, evolution and molecular epidemiology of papaya ringspot virus
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(2) 1. ABST RACT. 2. Papaya r ingspot viru s (P RSV) is the mo st impor tant virus affecting papay a and c ucurbit. 3. plants in trop ical and sub tropic al ar eas. PRSV is d ivided into two groups: th e W type,. 4. which in fects pap aya (Caric a p apaya) and p lants b elonging to th e Cucurbitac eae. 5. family, and th e P typ e, whic h on ly infects papaya. Th e study of molecular variation of. 6. this virus is of gr eat relevanc e, esp ecially to d evelop tr an sgen ic r esistanc e. In order to. 7. have a b etter under standing about the var iation, orig in, evolution and epidemiology of. 8. this virus it is necessary to con struc t a robu st p hylogenetic infer enc e. Moreover, it is. 9. essentia l to include a co mpr ehensive collectio n of sequ enc es of poor ly studied r egions,. 10. especially Sou th America. For th is purpose, th e seq uen ces of the Cap sid Pro tein (CP). 11. have been u sed. For th is gene, a phylogen etic approac h u sing a molecu lar clock is. 12. presented , includ ing seq uen ces reported pr eviously fro m differen t p arts of th e wor ld. 13. and new ones ob tain ed in Colo mbia. In ord er to c alibr ate the tree, the known d ates of. 14. collec tion fro m differ en t isolates were u sed. Th e advantag e o f th is method is th at. 15. diverg enc e times can b e accur ately calculated. Th ese dates can b e u sed to fur th er. 16. defin e th e or igin and ep idemio logy of P RSV. Our analysis shows that app ar ently,. 17. movement of th e virus b etw een region s contributes to th e ob served population. 18. variation. Th e hypo th esis o f th e or igin of P RSV- P b iotype fro m P RSV-W is r evised ,. 19. becau se evid enc e for th e oppo site process is found in sev eral in stances.. 20 21. Keywor ds: P apaya r ing spot virus, Relaxed Molecu lar Clock, Bayesian Phylogeny. 22 23 24 25 26 2.
(3) 1. INTROD UCTION. 2. Papaya r ingspot viru s (P RSV; Family Po tyvirid ae, gen u s Po tyvirus) is an important. 3. pathogen of p ap aya and cucurb its (Purc ifu ll et al., 1984). PRS V p ar tic les are flexuou s. 4. filaments of 780 x 12nm with a sing le stranded RNA g eno me of positive polarity with. 5. ca. 10.000 nucleo tid es th at encode a poly protein w ithin a single ORF (De La Rosa and. 6. Lastr a, 1983). P lants in fected w ith P RSV d evelo p sympto ms r anging fro m mo saic,. 7. chloro sis and distor tion of leaves, to ring spot and str eaking on fruits and w ater-so aked. 8. oily streaks on stem and petioles (Purc ifull et al., 1984). The viru s is tr ansmitted no n-. 9. persistently by sever al spec ies o f aphids (Purc ifull et al., 1984), a process requir ing the. 10. coat pro tein (CP) and th e helper co mponent (HC-Pro) (Maia, Haenn i, and Bernardi,. 11. 1996; Pirone, 1991; Piro ne and Blanc, 1996; W ang et al., 1998).. 12. PRSV iso lates ar e divided in to two groups: the P and W biotyp es (Purcifull et al., 1984).. 13. PRSV-W and PRS V-P can infec t plan ts in th e Cucurbitaceae family , ho wever on ly PRSV-. 14. P naturally infec ts p apaya (Caric a p apaya) (Gonsalves, 1998; Purcifull et al., 1984).. 15. Both isolates ar e serolo g ically indistingu ish able (Purcifull et al., 1984) an d. 16. exper imentally c an in fect Chenopod ium qu inoa and C. amar an ticolo r, produc ing loc al. 17. lesio n s (Gon salves, 1998).. 18. PRSV-P h as b een isolated in pap aya-producing areas inc ludin g Afr ica, th e C aribb ean,. 19. Austr ali a, Tropical A sia, Latin A merica and the Sou th P ac ific (Tennan t et al., 1994).. 20. Due to its large d istr ibu tion , this virus is known as th e major limiting fac tor for p apaya. 21. productio n throughout th e trop ics and subtrop ics (Gon salves, 1998). This inc lud es. 22. Colo mbia, wh ere the disease produc ed by PRSV-P has been r ecorded in several. 23. Depar tments, since 1952 (S ánch ez de Luqu e and Mar tínez López, 1998) and it is. 24. consider ed as the mo st important disease of p apay a in th is coun try (Páez, 2003).. 25. Control o f th e d isease in Colombia inc ludes cu ltural practice method s such as iso lation. 26. of infected cu ltivars and removal of young infected tr ees (Páez, 2003). The u se of the 3.
(4) 1. attenu ated isolate HA 5- 1 developed in Hawaii (Yeh and Gon salves, 1984) h as also. 2. been used, show ing a low efficiency o f pro tec tion (Páez, 2003). Fur th er more, variou s. 3. studies in differ ent coun tries wh ere the attenu ated iso late or tran sg enic pro tec tion. 4. were not useful, have shown th at r esistanc e d epends on the lev el of RNA similar ity. 5. betw een the tran sg ene or mild str ain and the inco ming virus (Gonsalves, 1998;. 6. Tenn ant et al., 1994; Tripathi et al., 2008). Studies ev alu ating th e viability of virus mild. 7. strain s for cro ss-pro tection h ave also been achieved for P RSV-W, showing similar. 8. results (Rezende and P acheco, 1998; Rezende et al., 1994). Ther efor e to obtain an. 9. ad equ ate protec tion , viru s populations in each country mu st b e investig ated to eith er. 10. find a loc al attenuated viru s or to furth er und er stand the loc al virus v ariability. 11. (Gon salves, 1998; S ilva-Ro sales et al. , 2000; Trip athi et al., 2008).. 12. The molec ular variabili ty and p hylogenetic r elatio n ship s o f PRSV wer e investigated by. 13. Baten son et al. (2002), inc luding sequ enc es fro m I ndochin a, Jap an, Australia, United. 14. States, Br asil, Mexico and Pu erto Rico. The gen eral conclu sion w as th at P RS V. 15. populations show high variability, and that variability w as r elated to the geographic al. 16. origin of the str ains. More importan tly , eviden ce of th e origin of P RS V-P fro m P RSV-W,. 17. previously propo sed Batenson et al., 1994 eith er by mutation or r eco mbination, could. 18. not b e clearly resolved. Accord ing to th is, in order to incr ease th e knowledg e on th e. 19. origin , evolu tion and epidemiolo gy of P RS V, it is n ecessary to obtain mor e sequ enc es. 20. fro m d if feren t coun tries, esp ec ially fro m r egion s th at wer e missing in pr eviou s stud ies. 21. such as Cen tr al and Sou th Amer ica (Bateson et al., 2002).. 22. To h elp r eso lve these issues, w e examined th e evolu tion ary dynamic s o f PRS V. 23. includ ing rec ently published sequences fro m d ifferent countries and n ew sequences. 24. fro m P RS V-P ob tain ed in Colomb ia . For this p urpo se, nu merous sequ enc es of the. 25. Capsid Protein (CP) wer e employed and a phylog enetic appro ach using a relaxed. 26. molecular clock is pr esen ted. In or der to c alibrate the tree, th e known d ates of 4.
(5) 1. collec tion fro m differ en t isolates were u sed. Th e advantag e o f th is method is th at. 2. diverg enc e times can b e accur ately calculated and hen ce the vir al diver sity acro ss time. 3. and sp ac e can b e studied. This typ e o f an alysis has b een perfor med with div erse kinds. 4. of viru ses, and h as b een proved to be help ful to solv e epidemiologic al issues (Ramb au t. 5. et al., 2008).. 6. MATERIAL S AND METHODS. 7. Virus isolates. 8. Virus isolates used in th is study wer e co l ec ted fro m p apaya field s in Colo mbia. The. 9. iso lates wer e collected betw een 2008 and 2009 in the fo llowing Dep artments:. 10. Casan ar e, Santander, Valle d el Cauca, Risar alda, C ald as and Cundin amarca (Figure 1).. 11. W henever possible, every location was georeferenced with th e Mag ellan SporTr akTM or. 12. 12XL Gar minT M GPS. Th e names an d origin of the isolates ar e li sted in Supp lementary. 13. Table 1.. 14. Young p apaya leaves showing or no t sy mp toms o f PRSV infec tion w ere collected .. 15. These samp les w er e plac ed in p lastic b ags with an absorb en t slig htly moist pap er towel. 16. sligh tly moist. To pr eserve the samp les un til th e day o f its processing plastic bags w er e. 17. kept o pen on ic e. Once in the labor atory, samples wer e mac erated in liq uid nitrogen. 18. with ster ile mortars and pistils. Fin ally, all samp les wer e stor ed at -80°C until RNA. 19. extraction.. 20. RNA extraction, cDNA synthesis and PCR. 21. In order to avoid RN ases, all surfac es w er e clean ed w ith RNase away (Molecular. 22. BioProduc ts, San Diego, California, USA). Total RNA w as extr acted from mac erated. 23. tissu e using the Concer tTM Plan t RNA Reag ent (Invitrogen, California, USA) according. 24. to manu facturer ’s instruc tion s. The r esulting p ell et was r esusp end ed in 20µl of sterile. 25. water tr eated with Diethilpirocarbon ate (DEPC; 0,1%v/v). Four µ l of each extrac ted. 26. RNA w as den atur ed by h eating at 65°C for 10 minutes. Denatur ed RNA was subj ec ted 5.
(6) 1. to elec trophor esis in a d enatur ing 1.2% ag aro se- for mald ehyde gel, using a con stan t. 2. voltag e of 70V for 25 minu tes.. 3. cDNA was synthesized from total RNA u sing th e co mplementary primer 04- 04 (Chin et. 4. al., 2007). For th is purpo se either the Sup erScr ipt TM I II RT/Taq mix Platinum®. 5. (Invitrog en) or th e iScrip tT M Select c DNA Synth esis Kit (Biorad) w ere used . To avoid. 6. RNA d egr ad atio n, 2µ l of RN asin® P lus RNase Inhib itor (Promega) were add ed to all of. 7. th e r eaction mixtur es mentioned above.. 8. Subsequ ently, double-str and ed DNA fr agments of the CP and HC-Pro gen es wer e. 9. gener ated. For th e CP g ene, th e fo llow ing primer s wer e u sed: 04-04 and 04-02 (Chin. 10. et al, 2007) or MB11M (mod ified fro m Bateson et al, 1994; r emoving 15 nuc leotides. 11. not r elev ant for this study). PC R cond ition s inc lud ed an in it ial denaturation cycle of 2. 12. min at 94 °C follo wed by 39 cycles of den atur ation for 30 s at 94 °C, ann ealing for 30 s. 13. at 46.5°C and exten sion for 30 s at 72 °C, with a final exten sion of 6 min at 72 °C. For. 14. th e amp lification of the HC-Pro gen e, the primers FHCP- 566 and RHCP-566 were u sed.. 15. The PCR amplif ic ation sc heme w as the same as d escr ibed for merly for the CP g ene. 16. excep t for th e annealing temper ature (56°C for th e HC-Pro g ene). A ll primer. 17. infor mation is shown in Figure 2.. 18. Both g en es w er e amplified using either Go Taq® Green Master Mix (Promega) or a mix. 19. of Pfu DNA poly mer ase (Fer mentas) and F ermentas Taq DNA po lymerase. For th e CP. 20. gene 3µl of c DNA and 0.5µ l of each pr imer (10µM) w ere u sed in a fin al volu me of 25µ l.. 21. For HC-Pro gene, 3µ l of cDNA w er e also u sed in a 20 µl r eaction mixture con tain ing 1X. 22. Buffer , 2.5mM MgSO 4 , 0.2mM d NTP s, 0.2µ M Pr imers, 0,25U o f Taq,, 1U of P fu and 5%. 23. DMSO. Amplified produc ts w er e an aly zed in 1% ag aro se g el elec trophor esis in TAE. 24. buffer.. 25 6.
(7) 1. Cloning and Sequencing. 2. PCR fr ag men ts wer e purified fro m the agarose gel with th e W izard® SV Gel and PCR. 3. Clean-Up System (Promega). Th e pur ified PCR produc ts wer e polyad enilated in a 10µl. 4. fin al volu me r eac tio n con tain ing Fer mentas Taq DNA poly mer ase (5U), Taq DNA Buffer. 5. (10X), MgCl2 (25mM) and dATP (0,2mM). The poly ad enilated produc ts w er e cloned into. 6. pGEM®-T (Promega) accord ing to manufac tur er’s instr uction s.. 7. Comp eten t c ell s of E sch erich ia coli DH5α w er e pr epar ed by a series of washes as. 8. previously d escr ibed (Maniatis, Fritsch, an d Sambrook, 1982). Forty µl aliquots of. 9. th ese cells w ere wer e main tain ed at - 80°C, and 1.5µl of th e vec tor lig ation w as. 10. electropor ated in th ese c ells at 1.25V in a MicroPu lserTM (Bio rad) elec tropor ator .. 11. Tran sfor med cells were cultured in plates with LB med ium su pplemen ted with. 12. ampicill in (100µg/ml) and X-gal (20µg/ml). White colonies were selected to perfor m. 13. PCR w ith the Un iver sal M-13 primer s, u sin g Go Taq Gr een Master Mix (Promega). After. 14. visualization o f th e amp lified produc ts in a 1% ag aro se g el, th e PCR frag ments were. 15. gel-p urified with the Clean-Up System describ ed above. The purified products w ere. 16. sequ enc ed at Macrog en (Kor ea). A total o f thr ee clon es per viru s isolate per gen e wer e. 17. sequ enc ed in bo th direc tion s (3’ and 5’).. 18. Sequence analysis. 19. Nucleotid e sequ enc es for th e CP and HC-Pro g enes for oth er PRS V isolates w er e. 20. retrieved fro m G enb ank (http://www.ncbi.nlm.nih.gov/Genb ank/). Th e in for mation. 21. regarding these PRSV isolates is listed in Supp lementary Tab le 2.. 22. The quality o f th e ob tained sequ enc es was assessed in BioEdit (Hall, 1999), with wh ich. 23. th e DNA sequ encing chro matogr aph s w er e an alyzed. A Vec Screen filter was. 24. perfor med in ord er to c lean sequences of vec tor contamin ation (availab le at. 25. http://www.ncbi.nlm.nih.gov/VecS creen/VecScreen.h tml). Subsequently, sequences. 26. were assembled in S equ encherTM (G en e Codes Corpor ation) and align ed using th e 7.
(8) 1. MUSCLE algorith m (Edg ar, 2004) in th e EMBL-EBI web p age. 2. (http://www.ebi.ac.uk/Tools/mu scle/). A r eco mbin ation analysis b etw een and with in. 3. groups of sequences (i.e., Coun tries) w as p erformed in Reco mb iTES T (P igan eau,. 4. Gardn er, and Eyr e-W alker, 2004), u sing th e Maximum Ch i-squar e test (S mith, 1992).. 5. On th e o ther hand, a to tal of 13 genomes o f P RS V-P and PRSV-W h ad b een co mpletely. 6. sequ enc ed (AY231130, S 46722, DQ374153, DQ374152, DQ340771, DQ340769,. 7. DQ340770, AY027810, X97251, AY162218, AY010722, EF017707, X67673). Th ese 13. 8. sequ enc es w ere also aligned w ith MUSCLE as d escrib ed pr eviously with the CP and HC-. 9. Pro sequ enc es o btained fro m Colo mbia. Th e n ucleotid e d istan ce was calculated using. 10. MEGA (Tamur a et al., 2007), and the nuc leotide diver sity (π), th e Segreg ant Sites (S),. 11. th e W atterson’s θ (θW), Taj ima’s D (Tajima, 1989) and th e propor tio n of non-. 12. synonymous to synonymou s sub stitutions (ω = dN/dS), w er e imp lemented u sing. 13. DNAsp (Rozas and Rozas, 1995).. 14. Phylogenetic analyses. 15. The Maximu m Likelihood (ML) analysis w as imp lemen ted using PAUP*. 16. (Swofford , 2002). Th e nuc leotid e sub stitu tion model was obtain ed with ModelTest3.7. 17. (Posada and Crandall, 1998), following the Akaik e Infor mation Criteria (AIC) (Akaike,. 18. 1974; Akaike, Petrov, and Csaki, 1973). Th e branc h support was calculated with a. 19. 1000 replicates boo tstr ap. This an aly sis w as perfor med for the sequences of CP and. 20. HC-Pro, and th e ob tain ed topologies wer e compar ed with a Shimod air a-Haseg awa. 21. statistical test (Shimod air a an d Hasegaw a, 1999). Potato viru s Y (P VY, Accessio n. 22. nu mber AY884985) was used as ou tgroup.. 23. A phylogeny und er a relaxed-c lock mod el w as con structed for th e CP gen e u sing. 24. BEAST v 1.4.8 (Dru mmo nd et al., 2006). In this Bayesian phylog enetic in ference, an. 25. Uncorrelated Lognor mal mo lecular c lock model was chosen and the tree prior w as the. 26. model of Con stan t S ize. Th e known dates o f co l ec tio n were u sed as calibration poin ts. 8. 4.0b10.
(9) 1. An arbitr ary d ate was set in cases where th e collec tion dates wer e unknown. Th e. 2. substitution mod el w as G TR with g amma and invarian t sites as a model for site. 3. heterogen eity, with 4 g amma categories. The leng th o f th e MCMC w as 20.000.000 with. 4. a scr een every 1.000th g ener ation. The converg enc e o f th is an aly sis was stu died with. 5. Trac er v 1.4 (Avaible at h ttp://tree.b io.ed.ac .uk/so ftware/trac er/), in ord er to examin e. 6. th e robu stness of the ob tain ed model.. 7. RESULT S. 8. Sample collection. 9. Typical sy mp toms o f PRS V w er e ob served in Colo mbian p apaya field s, includ ing. 10. reduc tion in foliar ar ea, mo saic, distor tion , and chloro sis , as w ell as fruit ring spot. 11. (Figure 3).. 12. For th e amp lific ation of the CP g en e, th e primer pair 04- 02 and 04- 04 did no t produce. 13. an amplicon in many cases. Th e best r esult w as ob tain ed with pr imer s 04-02 an d. 14. MB11M. A to tal o f n ine n ew sequences of the CP g en e were obtained . N ever theless,. 15. virus mechanical inoculatio n in indic ator p lants (i.e. in Ch enopodium) needs to b e. 16. perfor med to disc ard the pr esen ce of th e viru s in the o th er co llec tio n samples.. 17. Sequence analysis. 18. Sequences from Colomb ia n samples, and 118 additional sequ enc es obtained in. 19. GenBank of PRSV-P and PRS V-W from differen t parts of th e wor ld, w ere aligned using. 20. MUSCLE. Th e recombin atio n an aly sis showed two po ssible events of r eco mbination. 21. (p<0,05), both involving as r eco mb inan t and donors Ind ian isolates (data not shown).. 22. The r eco mbinan t sequence, Ind ia_UPLK, h as as a major p arent th e isolate In dia_UP V.. 23. The other case of recombin atio n is the Ind ia_W B, having as a major par ent the iso late. 24. India_HP.. 25. The nuc leotide diver sity (π) ranged fro m π =0,01102 (Jamaic an isolates) to π =0,07977. 26. (Indian isolates) followed by π=0,07177 (Mexic an iso lates). For the 15 sequ enc es o f 9.
(10) 1. each CP and HC-Pro, HC-Pro w as found to be mor e d iver se (π =0,15277) th an CP. 2. (π=0,10718). For both g en es, th e observed value of Taj ima’s D within group s and in. 3. th e over all align ment w as significan tly n eg ativ e, indicating th at th ey are not und er. 4. neutral evolu tio n. I n ord er to id entify whic h selec tion forc e is ac ting on each gen e, the. 5. proportion of non synonymous/synonymou s substitutions (ω) was calcu lated, obtaining. 6. for bo th g en es a ω greater than 1, ind icating positive selection (Supp lementary Tab le. 7. 3).. 8. Phylogenetic analysis. 9. A phylogeny of th e total CP sequenc es was con stru cted u sing an uncorr elated. 10. LogNormal Molecular Clock and th e resulting topology situated P RSV viru s origin. 11. approximately b etween 75 and 150 years ago (Figur e 4). In concord anc e with previou s. 12. reports (Bateson et al., 2002), the orig in o f th e virus is p laced in Sou th Asia, more. 13. precisely in India. Ho wev er, th is basal clad e is for med on ly by isolates o f PRSV-P and. 14. no isolates o f PRSV-W are found th ere. Moreov er, Asian isolates are grouped in to two. 15. clu ster s. One group is monophyletic and includ es eastern iso lates (Chin a, Th ailand,. 16. Vietnam, Jap an, Taiw an and Korea). Th e o ther is the paraphyletic and basal group of. 17. South Asia, includ ing iso lates fro m I ndia and Bang ladesh . This group is the c losest. 18. ancestor of th e A mer icas -Au str ali a mo nophyletic grou p. I n th e A mericas-Au str alia. 19. group, Jamaic a and Australia are monophyletic in con trast with th e Mexic an isolates. 20. th at appear to h ave several orig ins. Ind eed, one of the Mexic an isolates is b asal to the. 21. Austr ali an clade (Figure 4, black arrow).. 22. Regar ding th e Colombian iso lates o f P RSV, the on es from Valle d el Cauc a,. 23. Cundinamarc a and S an tand er (Figure 1) grouped tog ether in a monophyletic clade. 24. (Figure 4, pink arrow). However, isolates from Casan are w er e found with in the. 25. Venezuela clade (Figur e 4, p ink and or ang e), show ing two differ ent orig ins for the. 26. Colo mbian isolates of P RSV-P. 10.
(11) 1. For th e co mpar ison o f th e CP and HC-Pro g en es, ML phylogen etic tr ees wer e. 2. construc ted. A h euristic search of the ML analysis for CP-gen e gener ated an op timal. 3. tree und er th e GTR+I+G model (Figur e 5.A), and for HC-Pro-gen e th e g ener ated tr ee. 4. was u nder the TI M+I +G model (Figur e 5.B). Both tr ees show ed Brazil and Hawaii as. 5. monophyletic group s. However, on ly the HC-Pro tr ee cou ld r esolv e th e r elationsh ip s of. 6. th e Taiw an isolates, wh ereas th e CP tr ee r esolved those for Thailand. Although for. 7. both c ases, th ese br anch es were not h ighly suppor ted. N either tr ee could reso lve the. 8. relationsh ips of Colo mbian and Mexican isolates; how ever th ese wer e group ed w it h. 9. Brazilian and Hawaiian isolates. According to the SH test, for topo lo gy co mp arison,. 10. th ese topolog ies differed significan tly from each o ther (p < 0.05).. 11. DISC USSION. 12. The phylogeny construc ted und er a relaxed molecular clock showed a supported. 13. topology, with th e exception of a few poorly supported c lades (Figur e 4, br anch es in. 14. red). How ever , th e r ang e o f dates app ear s to b e c alculated accur ately. For example,. 15. PRSV-W was fir st descr ibed in Austr alia 31 years ago (Gr eber, 1978), date th at is. 16. includ ed in the r ang e o f 30 to 45 year s ago calcu lated in this study to b e th e or igin of. 17. th at clad e. For the c ases of Colo mb ia and Haw aii, PRSV-P w as fir st d escrib ed around. 18. 50 years ago (Jensen, 1949; S ánch ez d e Luqu e and Mar tínez Lóp ez, 1998) and in. 19. Venezuela it was reported 60 year s ago (Muller, 1941). In th e constr ucted phylogeny,. 20. th e d ates for these grou ps w er e placed between 5 to 45 year s ago. The deviation of. 21. th ese obtain ed d ates and those repor ted, may be du e to the use of r elatively r ec en t. 22. sequ enc es (th e sequ enc es included fro m Colo mbia ar e fro m the last two years), wh ich. 23. can lead to under estimation of div ergence times. Mor eover th e collection dates of. 24. Hawaii wh ere unknown, wh ich c an also influence the estimation of d ivergenc e d ates.. 25. For th e Mexic an iso lates, collected during a wid er time range (fro m 1994 to 2000), the. 26. estimated date of origin is b etw een 65 to 45 year s ago, a range that cover s the first 11.
(12) 1. report in this country in 1975 (Téliz-Or tiz et al., 1991). Ho wev er, as ther e appears to. 2. be multip le or igin s for th e Mexic an, Colomb ian and Venezuela isolates (non-. 3. monophyletic group s), th e c alculus for the orig in o f th is isolates c an be d eviated , as. 4. probably no every iso late has b een record ed. N evertheless it is noted th at th e fir st. 5. report of a d isease migh t also b e an underestimation o f th e d ate of orig in of a viru s in. 6. a given plac e.. 7. Previous r eports sustain ed that PRS V appears to b e or iginated in Asia (Baten son et al.,. 8. 2002), and th e present study also supports this hypothesis. This is also in accordance. 9. with the h igh nucleo tide diversity found in Asia, more spec if ic ally in Ind ia, th e b asal. 10. clade. Ho wev er, neither Ind ian nor Asian clad es ar e monophyletic. More inter esting, it. 11. has b een proposed that PRS V-P evolved fro m P RS V-W , but in this study ther e is. 12. evidence that the oppo site proc ess is also possible and th at it actually h as o ccurred. 13. many times. The fir st evidence that PRSV-P evolv ed from PRS V-W came fro m. 14. Austr ali an population s of th e virus (Bateson et al., 1994). As for this case, PRSV-W was. 15. fir st d escribed in Australia in 1978 (Gr eber, 1978) at least 20 year s b efor e PRSV-P. 16. appeared (Thomas and Dod man, 1993). W hen th at study w as c arr ied out, o nly few. 17. sequ enc es o f PRS V (all o f th em fro m Asia and USA), w er e availab le. Th e fin al r esult. 18. revealed that PRS V-P fro m Austr alia arose fro m an isolate of an Austr alian P RS V-W.. 19. However, th e inc lusion of more sequenc es fro m C entr al and South A merica sho wed. 20. th at an isolate of PRSV-P fro m Mexico is th e c losest ancestor to the PRSV-W (Figur e 4,. 21. black arrow). Moreov er, when mor e sequences of PRS V-W fro m Australia w ere. 22. includ ed , so me o f th em appear ed to have arisen from PRSV-P iso lates (Figur e 4). Later. 23. studies th at con firmed the hypothesis of the origin of PRS V-P fro m P RSV-W (Bateso n. 24. et al., 2002; Inou e-N ag ata et al., 2007), d id no t include th e sequ enc es of P RSV-P from. 25. India used in this study (Jain et al., 2004). When included in this and in Jain et al.. 12.
(13) 1. (2004) studies, th ese sequences ar e situated in th e most basal p art o f th e tr ee,. 2. show ing P RS V-P fro m India as the b asal ancestor eith er for P RS V-P than for PRS V-W.. 3. For th e Colo mbian c ase, the iso lates ob tain ed fro m Casan ar e did no t group tog ether. 4. with the isolates of the r est of Colo mb ia bu t with isolates fro m Venezuela (Figure 4,. 5. pink arrow). Of all th e depar tments wh ere the collectio ns wer e mad e, Casan are is the. 6. closest to Ven ezuela (F igur e 1), implying a probab le migr ation o f infected p lants. 7. betw een these two coun tries. This shows that the vir us movement is impor tant in. 8. sh aping the molecular population evolutio n of PRS V, as this is also ob served in the. 9. overall phylogeny with many non- monophyletic group s. Unfortun ately, migr ation. 10. an aly ses c annot be p er formed w it h th e CP gen e b ecau se it is und er positive selection,. 11. which can bias th is type of analysis. Becau se of this, a highly suppor ted phylogeny as. 12. th e on e construc ted her e, show s to be a good approach to in fer the migr ation patter ns. 13. for P RSV. Noneth eless, a b etter ap proach that can be don e with a c alibr ated phylo g eny. 14. is the fac t th at w ith this on e on e c an take in account evolutionary h istory no t o nly in. 15. sp ac e bu t also in time. For this, th e study of popu lation dynamic s of gen etic diversity in. 16. time (Dru mmond et al., 2005), has been propo sed for all genes of the virus genome.. 17. However on ly 14 co mplete geno me sequences have b een reported and only 4 on th em. 18. ar e fro m the W biotype. Anoth er good approach co uld be phylog eogr aphic. 19. The tr ee obtained w ith the HC-Pro g en e w as b etter at resolvin g the r elation ship s. 20. betw een the iso lates th an th e one u sing CP. Even if th e over all tr ee topology was very. 21. similar , th ey were significan tly d ifferen t. In a previou s study of P RSV-P isolates,. 22. neighbor joining trees of th e CI, 6K and HC-Pro proteins show ed a d iffer ent topo lo gy. 23. fro m the oth er five proteins of P RS V (Noa-Carr azana, Gon zález-d e-León, and Silva-. 24. Ro sales, 2007). Inter esting ly, th ese gen es are exp ec ted to h ave a similar evolution ary. 25. history b ecau se they ar e involved in aphid tran smission (Urcuqui- Inchima, Haenni, and. 26. Bern ardi, 2001). How ever, HC-Pro is a multifunctio nal pro tein involved not only on 13.
(14) 1. aphid propag ation but also in long distanc e and cell to cell movemen t, silenc ing. 2. suppr ession, viru s synergism in co- infec tion and d evelop men t of sympto ms (Ballut et. 3. al., 2005; Urcuqu i-Inch ima, Haenni, and Bern ardi, 2001). To study th e coevolution. 4. betw een these two g en es, the conserved p arts involv ed in aphid tran smission should. 5. be used. In th is c ase th e reg io n s around th e DAG motif in CP (c lo se to th e N-. 6. ter minus), which are implicated in aphid tr ansmission (Shukla, W ard, and Brunt, 1994),. 7. have shown consider able co nservation (Bateson et al., 1994; Bateson et al., 2002). For. 8. HC-Pro th e regions around the KITC (N- ter minus) and PTK (C-ter min us) seemed to be. 9. involved in aphid tr ansmission (Rev er s et al., 1999) and so they also should be u sed. 10. for the study of coevolu tion between th ese two g enes. As th e HC-Pro g ene g ave a. 11. more r eso lv ed p hylogeny, this gen e could b e u sed to reso lve the relation ships among. 12. PRSV iso lates.. 13. Finally, th e PRSV evolutionary h istory is very complex, inc luding several changes in. 14. biotyp e, which in clud e th e change of ho st r ang e. Molecu lar studies should b e done to. 15. under stand why PRS V-P app ear s to have lo st its n atural ability to in fect cucurb its. 16. despite all th is ho st c han ges. For this r eason, this viru s could be used as a b iolo gic al. 17. model to study this process. Mor eover it could be the mod el to study differ ent selec tiv e. 18. pressures on vir us, including how a viru s popu lation is influenced by the presenc e o f. 19. resistant cultivar s.. 20 21. ACKNOWLEDGEMENT S. 22. To Adrian a Castañed a and her ICA par tner s and to Aníbal Tap iero and h is CORPOICA. 23. partners, for acco mp anying me and giving me the nec essary resourc es for th e pap aya. 24. collec tion in Valle del Cauc a and Casanare, respectively. To my uncle Jaime Olar te for. 25. helping me in th e adventurous collection in Santander.. 26 14.
(15) 1. Figure 1 . Collec tion places of papaya samples used in this stu dy. A. Colomb ian map. 2. show ing the d ep artmen ts wher e th e isolates w ere collec ted. Green: Casanar e, r ed:. 3. Cundinamarc a, p ink: Santander, violet: Caldas, yellow: Risar alda and or ang e: Valle del. 4. Cauca. The number insid e or near each depar tmen t indicates th e nu mb er o f co llec ted. 5. samp les. B. Close up of the map showing in gr een th e sp ecific co llec tion sites at each. 6. department.. 7. Figure 2. Annealin g sites o f primer s u sed in this study, over th e g eno me sequ enc e of. 8. th e P RSV isolate from Hawaii (PRS VP- HA). The primer sequences and melting. 9. temp erature is also shown. Primer s th at amplify the HC-Pro gen e ar e shown in gr een.. 10. Primers that amp li fy the CP gene are shown in p urple (I mage mod ified fro m Trip ath i,. 11. et al., 2008).. 12. Figure 3. Symp toms ob served in pap aya fie ld s in Colombia. A. P apay a tree show ing. 13. foliar ar ea r educ tion . B. Fruit ring spo t. C . Leaf Mosaic. D. Distortion of young leaves.. 14. E. Chloro sis.. 15. Figure 4 . Phylogen etic tree o f the CP gen e u sing an Uncorrelated LogNor mal r elaxed. 16. molecular clock mod el. Black do ts r epr esent P RSV-W isolates, gray dots r epr esent. 17. unknown isolates su spec ted to b elo ng to th e P RS V-W group. Black arrow indicate th e. 18. Mexic an isolate that gave origin to the Au stralian clad e. In p ink, Colo mbian isolates. In. 19. orang e, th e isolates fro m Venezuela. Blue horizon tal bar s represent the 95% HP D. 20. interval for th e d ivergenc e time estimates. Br anch es colored in r ed are not well. 21. supported (po sterio r probabilities lower th an 0,65).. 22. Figure 5 . A. ML tree under th e G TR+I +G for th e CP gene. B. ML tree und er th e. 23. TIM+I +G for the HC-Pro gene. Above the branch es, the boo tstr ap v alues for a 1000. 24. replic ates is sho wn. External gro up: Po tato Virus Y (P VY).. 25 26 15.
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(20) Figure 1 .. 20.
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(22) Figure 3.. 22.
(23) Figure 4. South Asia. Americas and Australia. Asia. 23.
(24) Figure 5 .. 24.
(25) SUPPLEMENTA RY MATERIAL Table1 . Description and characteristics of collec tion s plac es. Location. Casanare Between Barranca de Upia (Meta) and Villa Nueva, (Casanare) Norte de Santa nder Risaralda. Caldas. Cundin amarca. Santan der. Georeference Finca 1 Finca El Estepo. 4°28’29’’N, 7 2°52’3 5’’W, 262 MASL. Finca 2 Finca Villa Arizona. 4°28’09’’N, 7 2°52’2 7’’W, 262 MASL. Finca 3 Finca 6 Finca La Carmelita. After Santa Rosa de Cabal. WP00 4 Over the road between Honda-La Dorada. WP00 6 Outside Honda , goin g to Mariquita, near the Gu alí river.. 05º12’0 9.0’’ N 74º 45’21.3’’ W, 270 MASL. 17 Near the Tolemaida military base. -------------. 13 Finca Girón On the road between Lebrija an d Girón. 07º05’1 5.15’’ N73º 11’13 .62’’ W, 940 MASL. 14 Outside Girón. 07º04’1 9.75’’ N 73º 10’38 .53’’ W, 751 MASL. 15 Finca de Cecilia Outside Floridablanca. 07º03’2 4.14’’ N 73º 05’22 .45’’ W, 751 MASL. 16Entering Giró n. 07º04’0 6.13’’ N 73º 10’21 .05’’ W, 897 MASL. Finca 5 21 Before Roldanillo 22 Before Roldanillo 23 After Roldanillo Valle del Cauca (Municipio de Roldanillo, Corregimiento de Santa Rita). 24 After Roldanillo 25 After Roldanillo 27 After Roldanillo, arriving to La Unión 28 After Roldanillo 26 After La Unión 29 Between Roldanillo – Buga o n the road to Buenavent ura Muestra de Adriana Bernal ICV Cachipay distributor. 25. April 2008. 05º 17’33.5’’ N74º44’46 .4’’ W, 227 MASL 05º 21’15.6’’N 74º 43’4 6.3’’ W, 214 MASL. Finca 4 Finca Santa Rosa. February 2008. ------------4°51’31’’N, 75°39’0 6’’W, 1466 MASL. WP00 5. 18 On the road to Bucaramanga’s Airport (Palonegro ). Collect ion date. 07º06’2 2.85’’ N 73º 11’03 .92’’ W, 1105 MASL 4°46’42’’N, 7 5°58’4 5’’W, 938 MASL 4°,45’4 4’’N, 75°5 8’3’’W, 939 MASL. July 2008. March 2009. April 2009. April 2008. 4°,08’5 1’’N, 76°1 6’47’’W, 935 MASL ------------4°,27’4 0’’N, 76°7’3 4’’W, 914 MASL ------------4°,30’1 4’’N, 76°5’2 9’’W, 925 MASL 4°,29’0 8’’N, 76°6’1 9’’W, 909 MASL 4°,28’2 6’’N, 76°6’4 8’’W, 912 MASL 4°,31’56’’N, 76°4’3’’W, 907 MASL 3°56’17’’N, 76°20’2 9’’W, 960 MASL -------------------------------------. April 2009. ------------February 2009.
(26) Table 2 . P RS V sequences used in th is study. W hen known, th e year of collection is shown in parenthesis under each acronym or geographic or igin.. A cronym. A ccesion number. Reference. X67673. Wang et al, (1994). AF196839. Davis, M. J. & Ying, Z. (1 999). WUS_FL**. D00 594. Quemada et al . (1990). Aust1 Aust2 Aust3 WAust_NT** WAust_DB1** WAust_GAT** Thail1 Thail2 Thail3 Thail4 Thail5 Thail6 Thail7 WThail3** WThail4** WTahil6** WTahil5** WTahil7** WTahil8** UTahil10* UTahil9* Taiwan1 Taiwan2 WTaiwCI** Jap1 China Cuba Bangla WSKorea Sri1 Vietnam1 Vietnam2 Vietnam3 UVietnam50* UVietnam48* UVietnam46* UVietnam45* UVietnam43* UVietnam41* UVietnam40* UVietnam39* UVietnam3* UVietnam37* UVietnam36* UVietnam35* UVietnam32* UVietnam31* UVietnam30* UVietnam42* UVietnam47* UVietnam44* UVietnam33*. U147 36 U147 37 U147 38 U147 44 S89 893 U147 39 AB04434 0 U147 43 AF506898 AF506899 AF506900 AF506901 AF506902 AF506895 AF506894 AF506892 AF506893 AF506891 AF506890 AF506896 AF506897 AB04434 1 X78557 AY02781 0 AB04433 9 AF243496 S46 722 AY423557 AB36927 7 U147 41 U147 42 AF506862 AF506889 AF506841 AF506843 AF506903 AF506846 AF506848 AF506850 AF506851 AF506852 AF506853 AF506854 AF506855 AF506856 AF506859 AF506860 AF506861 AF506849 AF506844 AF506847 AF506858. USA_Haw USA_Florid a (199 7). Geographic origin USA. Australia. Thailand (199 2). Taiwan Japa n China Cuba Bangladesh South Korea Sri Lanka Vietnam. Vietnam (199 8). Vietnam (199 8). 26. Bateson et al. (1994). Unpublished Bateson et al. (1994). Bateson et al. (2002). Unpublished Wang et al, (1994) Unpublished. Jain et al. (2004) Bateson et al. (1994) Bateson et al. (1994). Bateson et al. (2002). Bateson et al. (2002).
(27) T able 2 . (Continued) A cronym. India WIndia* * India_AP India_CG India_DL India_HP India_JK India_KA1 India_KA3 India_ UPLK India_ UPV India_WB WIndia1** WIndia2** Mexico1 (1997) Mexico2 (1994) Mexico3 (1995) Mexico4 (1998) Mexico5 (2000) Mexico6 Mexico7 (2000) Jama1 (1990) Jama2 (1999) Jama3 (1999) Jama4 (1999) Jama5 (1999) Jama6 (1999) Jama7 (1999) JamWoodbur ne (1999) JamSpring_Fiel d (199 9) JamPondside (1999) JamPamphr et (1999) JamBelvedere (1999) Brasil1 Brasil2 Brasil_BP Brasil_PE Brasil_BA_CA Brasil_BA_IT1 Brasil_DF Brasil_SP Brasil_ES Brasil_PR WBras1** WBrasC** Ve_MerVigia (1993) Ve_MerLag unillas (1993) Ve_Lara (2004) Ve_Merida4 (20 04) Ve_Mer5 (200 4) Ve_Mer6 (200 4) Ve_Mer8 (200 4) Ve_Tachira (2004) Ve_Trujillo (2004) Ve_Tru2 (2004) Ve_Trujillo5 (2004) Ve_Zulia (200 4) Ve_Zul7 (20 04). Geographic origin. A ccesion number AF063220 AF063221 AY23888 0 AY49101 1 AY23888 3 AY45861 7 AY45861 9 AY23888 4 AY45861 8 AY45862 0 AY23888 2 AY23888 5 AF506845 EU47 5877 AJ012 649 AJ012 099 AJ012 650. India. Mexico. Reference Jain et al . (1998). Jain et al . (2004). Bateson et al. (2002) Silva-Rosales et al . (2000). AF309968 AF319493 AF319499 AF319502 DQ104823 DQ104822 DQ104821 DQ104820 DQ104819 DQ104818 DQ104817 DQ104816 DQ104815 DQ104814 DQ104813 DQ104812 AF344640 AF344647 AF344645 AF344646 AF344641 AF344639 AF344650 AF344642 AF344644 AF344643 DQ374153 DQ374152 DQ339576 DQ339577 DQ339580 EF18 973 0 EF18 973 1 EF18 973 6 EF18 973 3 DQ339579 EF18 973 4 DQ339578 EF18 973 5 DQ339581 EF18 973 2. Jamaica. Brasil. Brasil. Venezuela. *= Virus biotype no t con fir med , bu t PRSV-W is su spec ted. **= PRS V-W 27. Unpublished Tennant , (199 6). Chin et al. (2007). Lima et al. (2002). Lima et al. (2002). Chin et al. (2007).
(28) Table 3. Nu cleo tide diversity (π ), Segr egan t S ites (S), Watter son’s θ (θW) and Taj ima’s D (Taj ima, 1989). NC = No Calculated, due to the n eed o f a minimum of 4 sequ enc es to do that an aly sis. The nu mber of haplotypes is included in p ar enthesis.. Num ber of se que nce s*. S. π. θW. D. All P W. 13 (12) 11 (10) 2 (2). 101 92 46. 0,07977 0,07681 0,07199. 0,06823 0,06585 0,07199. -0,06992 -0,11355 NC. All P W. 12 (12) 10 (10) 2 (2) 13 (13). 76 68 16 107. 0,03052 0,03202 0,02492 0,04565. 0,03920 0,03744 0,02492 0,05371. -0,09384 -0,05404 NC -0,07820. 12 (5) 7 (7) 15 (15). 40 129 137. 0,01102 0,07177 0,05331. 0,02063 0,07587 0,06594. -0,10956 -0,12496 -0,06165. P W. 7 (7) 8 (8) 9 (9) 3 (3). 64 108 61 31. 0,03301 0,06455 0,03915 0,03219. 0,04088 0,06518 0,03507 0,03219. -0,05563 -0,05412 -0,09172 NC. All P W All. 6 (6) 3 (3) 3 (3) 25 (23). 29 12 17 138. 0,011508 0,01248 0,01765 0,03974. 0,01981 0,01248 0,01765 0,05693. -0,05823 NC NC -0,10078. P W. T aiwan. 3 (3) 22 (20) 3 (3). 27 132 21. 0,02804 0,03670 0,02191. 0,02804 0,05640 0,2191. NC -0,12481 NC. All P W Am ericas and A ustralia. 61 (57) 27 (26) 35 (33) 63 (56). 186 174 204 234. 0,06983 0,08401 0,05319 0,05322. 0,08332 0,07560 0,07752 0,07771. -0,14325 -0.02547 -0,10005 -0,09784. All sequences. 124 (112). 243. 0,07533. 0,09134. -0,11985. Popula tion. India Brazil V enezuela Jamaica Mexico. All T hailand C olom bia USA A ustralia. V ietnam. Asia. 28.
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