Indian Phytopath. 50 (1) : 89-96 (1997)

Differentiation of potato virus Y strains by N-terminalserology and HPLC peptide profiling

A. ISHWARA BHAT, ANUPAM VARMA, R.K. JAIN and S.M.P. KHURANA*

Divisionof Plant Pathology, Indian Agricultural Research Institute, New Delhi 110 012"CemralPotato Research Institute, Shimla 171 001

ABSTRACT: An isolate of potato virus Y from Delhi (PVY-D) has been purified and antiserum produced. Inserologicaltests, unfractionated antiserum reacted strongly with different strains of PVY, eggplant mottle virus andpigweedmosaic virus indicating close relationship of these viruses with PVY. Henbane mosaic and cowpea aphidbornemosaic viruses were found to be distantly related. The fractionated antibodies specific to N-terminal regionofthe coat protein reacted only with strains of PVY and eggplant mottle virus but not with pigweed mosaic, henbanemosaicand cowpea aphid borne mosaic viruses showing differences in the N-terminal regions of the coat protein oftheseviruses. Reaction with fractionated antibodies specific to N- terminal region clearly showed that eggplant mottleisveryclosely related to PVY. The serological tests, however, failed to differentiate strains ofPVY. Peptide profilingof tryptic digests of coat proteins of the strains of PVY by HPLC, however, could clearly differentiate the strainsof PVY tested. PVY-D was found to be distinct strain of PVY.

Keywords : Potato virus Y strains, differentiation, N- terminal serology, peptide profiling

Potato virus Y (PVY), type member of thefamilyPotyviridae and the genus Potyvirus (Shuklaet al., 1994) is an important virus commonly foundinfecting potato wherever it is grown. Besides po-tato, PVY infects many cultivated and wild spe-cies (Nagaich et al., 1974; Solangi et aI., 1983),and causes major diseases in chilli, eggplant andtomato (Varma, 1988).

Three distinct strains of PVY (yo, yc and Y'')have been identified based on symptomatology andhost range (Debokx and Huttinga, 1981; Khuranaet aI., 1990). These strains, however, cannot bedistinguished by serology using un fractionatedpolyclonal antiserum. Recent studies have shownthat virus specific antibodies directed towards the

Received for publication August 5, 1996.Publication No. 2223, PP, IAR!, New Delhi 110 012

N- termini of coat proteins or cDNA probes di-rected against 3' non-coding regions of the viralgenome and high performance liquid chromato-graphic (HPLC) peptide profiling of coat proteinscan be used for distinguishing strains of potyviruses(Shukla et al., 1994). In the present investiga-tions, attempts have been made to distinguishstrains/isolates of PVY by N- terminal serologyand elution profiles of tryptic digests of their coatproteins by HPLC.

MATERIALS AND METHODS

Virus isolates

An isolate of PVY from potato was obtainedfrom ACPV, IARI, New Delhi and is referred toas PVY -D. The PVY strains (yo, yc and yn)from Shimla were obtained from CPR!, Shimla.These are referred as Pv'Y", PVyc and pvyn in

90 Indian Phytopathology

this paper. All these strains/isolates were main-tained and propagated on Nicotiana tabacum cv.White Burley through successive sap inoculationsand maintained in an insect-proof glasshouse inDelhi. Cultures of cowpea aphid borne mosaic(CABMV), eggplant mottle (EMo V), henbanemosaic (HMV), papaya ringspot (PRSV), peanutstripe (PStV), pigweed mosaic (PWMV), sugar-cane mosaic (SCMV) and sweat potato featherymottle (SPFMV) potyviruses were obtained fromACPV, IARI, New Delhi.

Virus purification

All the strains/isolates were purified usingthe protocol described by Moghal and Francki(1976) except that the extraction buffer contained0.2M urea and the first pelletizing run was car-ried out using 5 ml of 30% sucrose pad (w/v) percentrifuge tube. Virus yields were calculated onthe basis of E1cm 0.1% 260 nm=2.5.

Antiserum production

Antiserum was prepared against PVY-D iso-late in rabbits by eight intramuscular injectionsadministered at 10 days intervals.' On eachoecassion, 0.5 mg of purified virus emulsified withequal volume of Freund's complete adjuvant wasinjected. The animal was bled 15 days after lastinjection and antiserum collected.

Purification of antibodies specific to N-terminal regions

N- terminal specific antibodies from polyclonalantiserum were separated by affinity chromatog-raphy as per Shukla et al. (1989). Surface ex-posed N- terminal peptide region from 3 mg ofpurified virions ofPVY-D was removed with a 30min incubation with lysyl endoproteinase(Boehringer, FRG). Enzyme resistant particles (de-void of N- terminus) were later concentrated bycentrifugation and coat protein was dissociatedusing formic acid and coupled to 0.75 g of CNBr-activated sepharose 4B (Pharmacia, Sweden) asper manufacturers directions. PVY-D antiserum(200 Ill) was washed through the column and

[Vol. 50(1) 1997]

antibodies not bound to column (directed towardsN- terminus) was collected.

Serological tests

Electro-blot immunoassay (EBIA)

SDS-PAGE was carried out as described ear-lier (Laemrnli, 1970) using LKB 2001 apparatus.The stacking and separating gels contained 5 and12% acrylamide respectively. Samples were mixedwith an equal volume of the Laemrnli sample bufferheated for 3 min in boiling water was used forloading on the gel.

EBIA was performed after SDS-PAGE asdescribed by O'Donell et al. (1982). After electro-blotting at 20V for 2-4 h, the nitrocellulose mem-branes were put in blocking solution (5% spraydried milk in TBS) for lh. The membranes werethen transfered to petri dishes containing virus-specific antisera diluted 1:100 in antibody buffer,incubated on a shaker for 1 h and washed threetimes in TBS containing 0.05% Tween and there-after transfered to solution containing antirabbit-alkaline phosphatase conjugate (Sigma ChemicalCo.) diluted at 1:1000 in conjugate buffer. After1 h of incubation on a shaker, the membraneswere washed as described before and incubatedfor 15 to 20 min in a preparation of the substrate,nitroblue tetrazolium and bromochloroindolylphosphate at 0.33 and 0.175 mg/ml of substratebuffer. The membranes were then transferred tofixing solution before being dried under folds offilter paper and preserved. The Bio-Rad pre stainedmarker proteins used were phosphorylase b(130,000), bovine serum albumin (75,000), ovalbu-min (50,000), carbonic anhydrase (39,000), soy-bean trypsin inhibitor (27,000) and lysozyme(17,000).

Enzyme linked immunosorbent assay(ELISA)

Direct antigen coated ELISA (DAC-ELISA)was done on polystyrene plates (Coming, NewYork, USA) using the protocol described by Clarkand Bar-Joseph (1984). Sap preparation includedgrinding leaf tissue in 10 volumes of coating buff-

[Vol.50(1) 1997]

er containing 2% polyvinyl pyrrolidone (PVP),filteredthrough cheese cloth and stored at 4°Covernightor 1 h at room temperature followed bycentrifugationat 5000 rpm for 5 min. Supernatantobtainedwas used to load ELISA plates. Antigen,antibodyand antirabbit immunoglobulin-alkalinephosphatasewere used at 1:10, 1:1000 and 1:2000dilutionsrespectively.The reactions of ELISA wereread at 405nm l h after adding substrate (p-nitrophenyl phosphate) by using a DynetechELISAreader.

Peptideprofiling

This was carried out as described by Shuklaet al. (1988). Briefly, freeze-dried viral prepara-tionswere suspended in 0.05M NH HCO and• 4 3

digestedat 37°C for 16-18 h with trypsin (SigmaChemicalCo.) at an enzyme : protein ratio of1:50.Solutions were dried, vortexed in 0.1%trifluroaceticacid and centrifuged at 10,000g for2min. Peptides thus obtained were seperated onaCII reverse phase column (218TP lOum, 4.6mmx 30cm,Bondapack, Waters Associates) connectedtoa Maxima 820 chromatography system (WatersAssociates).Components were seperated in 0.1%TFAat a flow rate of 1 ml per min using a lineargradient of 0-35% acetonitrile over 70 min atambienttemperature. Chromatography was moni-toredat 214 nm.

RESULTS

Virus purification

The purification procedure resulted in a cleanvirus preparation as judged by electron micros-copy, spectrophotometry and SDS-PAGE analy-sis. The particle length distributions in purifiedviruspreparations were not different from thosein crude leaf extracts, indicating that purificationprocedures caused neither significant particlebreakagenor end to end aggregation. The A 260/280 ratio varied from 1.22 to 1.30 and yield ofvirus obtained per 100 g of tissue varied from0.20 to 3.45 mg depending on the harvest timeafter inoculation and season.

Indian Phytopathology 91

Characterization of strains of PVY

Antiserum produced to purified PVY-D re-acted with homologus virus in tube precipitin testand had a titre of 1: 4096. In direct antigen coatedELISA (PAC-ELISA) and electro-blot immuno-assay (EBIA), PVY-D antiserum reacted stronglywith all the strains/isolates of PVY, and also withsome other potyviruses (Table 1). UnfractionatedPVY-D antiserum reacted strongly with otherpotyviruses like eggplant mottle (EMoV) and pig-weed mosaic (PWMV) and to a lesser extent withhenbane mosaic (HMV) and weakly with cowpeaaphid borne mosaic virus (CAMBV). In contrastto the reactions of unfractionated antiserum, af-fmity purified N- terminal specific antibodies ofPVY-D reacted only with strains of PVY andEMoV but not with other viruses used in thesetests (Table 1; Fig. 1). These results clearly indi-cate that serologically EMoV is closely related tothe various strains of PVY used in these experi-ments, although, EMoV differs from PVY as itcould not be transmitted by aphids in a non-persistant manner. The reaction with CABMV,HMV and PWMV is indicative of distant relation-ship as these viruses do not share N- terminalepitopes with PVY.

Differentiation of PVY strains by HPLCpeptide profiling

Elution profiles of tryptic digests of coat pro-teins ofPVYc, PVY-D and PVyo (Fig. 2) showedsubstantial similarity. Peaks (1, 2, 6, 8, 11, 13,15, 20, 23, 24, 25, 26, 28, 31 and 33) eluted atretention times of 14.50-14.75,16.00-16.25,19.50-19.75, 20.75-21.00, 23.50-23.75, 26.50"26.75,29.50-29.75, 36.50-36.75, 41.50-41.75, 44.25-44.50, 46.75-47.00, 48.25-48.50, 50.75-51.00,53.75-54.00 and 57.50-57.75 min were commonto all the three profiles (Fig. 2) indicating simi-larities between the three strains ofPVY. Pairwisecomparison of retention times of each peak clearlydemonstrate that per cent similarity between dif-ferent strains varied from 62-77% indicatingstrainal differences. PVyc and PVY-D showed

75

1 2 3 4130 50

75

SO

92 Indian Phytopathology

1 2 3 413075

50

39

27

17

3.27

17

A B

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2 3 4 5

130

39

27

...c

Fig. 1. Reactivities of PVY -D unfractionated (A) and fractionated antibodies specific to N- terminal region (B, C)in electro-blot immunoassay.Lane I shows bio-rad prestained protein standards and numbers on the left indicate MW of markerproteins. Lanes 2-4 in A and B show preparations ofPVY-D, HMV and EMoV while Lanes 2-5 in C showpreparations of PVY-D, Pv'Y", PVY" and PVyc. Membrane A is probed with unfractionated PVY-Dantiserum while B and C are probed with fractionated antibodies specific to N- terminal region.

77% similarity while, Pv'Y? and P'VY? 72% andPVY -D and PVYo 62% (Table 2).

Detailed comparison of the profiles of PVYstrains showed slight differences. Peak numbers 5and 21 eluted at retention times of 18.25-18.50and 37.50-37.75 min were unique to PVY? whilepeak numbers of 10, 14, 17,34 and 35 eluted atretention times of22.00-22.25, 28.50-28.75, 34.50-34.75, 59.75-60.00 and 61.50-61.75 min werefound to be unique to PVY -D (Fig. 2). Similarly,peak numbers 12, 18, 22 and 30 eluted at reten-tion times of 24.25-24.50, 34.50-34.75, 40.75-41.00 and 52.00-52.25 min were unique to Pv'Y?

profile. In addition, there were differences in theamounts of different peptides as judged by thesize of different peaks (Fig. 2).

Thus, peptide profiling study clearly demon-strated that PVyc, PVY-D and Pv'Y? are distinctstrains of PVY.

DISCUSSION

DAC-ELISA and EBIA tests withunfractionted PVY-D antiserum showed close re-lationships of PVY with EMo V and PWMV anddistant relationship with HMV and CABMV.Earlier fmdings have also suggested distant rela-

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tionships of PVY with HMV (Hollings and Brunt,1981). In contrast, fractionated antibodies specificto N-terminal region of the coat protein reactedonly with strains of PVY and EMo V but not withPWMV, HMV and CABMV indicating differencesin the N- terminal region of the coat proteins ofthese viruses. Recent studies on the structure andsequence of many potyviruses have revealed thatcore + C - terminal ends of coat protein ofpotyviruses carry group specific epitopes while N-terminal ends carry virus specific epitopes (Shuklaand Ward, 1989). Thus the high cross reactivityof PVY-D un fractionated antiserum observed inthe present investigations would be due to thepresence of more antibodies to the group specific

20 30

N

=

Indian Phytopathology 93

core epitopes in the antiserum which couldrecognise other distinct members of the potyvirusgroup. On the other hand, recognition of strains/isolates of PVY by fractionated N- terminal spe-cific antibodies of PVY-D in this investigationsclearly indicates that the isolates of PVY tested inthe present investigations are strains of PVY asall strains of an individual potyvirus are shown tohave similar N- terminus (Shukla and Ward, 1989).These findings confirm the earlier classification(Khurana and Singh, 1986) of the three isolatesfrom Shimla as strains (yo, yc and Y") based onthe biological reaction on diffemtial hosts. PVY-D is also a strain of PVY but whether it is differ-ent from the other three strains or not could not

... "N N

pVY-D

Ec<:t{\J

woZ<l PVyOCD Na::0(f)CD<l

10

RETENTION TIME (rnin )

40 50 60

Fig. 2. High performance liquid chromatography peptide profiles of tryptic digests of coat proteins of PVY strains.

PVyc PVY-D PVYO

94 Indian Phytopathology [Vol. 50(1) 1997]

Table 1. Reactivities" of polyclonal antiserum of PVY-D raised against intact and N- terminal specific region of coatprotein in DAC-ELISA

Detecting PVY-D antiserumAntigens

UnfractionatedFractionated antibodiesspecific to N- terminal

region

CABMV

EMoV

HMV

PRSV

PStV

PVY-D

PVyo

PVyc

+

+++ ++

++

+++ +++

+++ ++

++++ ++++

PVY· +++ ++

PWMV

SCMV

SPFMV

+++

"'Am Values ranked as >0.91(++++); 0.61-0.90 (+++); 0.31-0.60(++); 0.10-0.30(+); no reaction (-).

Table 2. Comparison of retention times of peaks from HPLC profiles of coat proteins from PVY strains

PVY strains Percentage of peaks with similar retention time"

PVyc

PVY-D

pvyo

100 77

100

72

62100

I Peaks with retention times 0.25 min of each other were considered to be similar.

b The first injection peak-and last column cleaning peaks, common to all profiles were omitted from comparisons.

be ascertained by these tests. The detection ofEMoV also by PVY-D N- terminal specific anti-bodies indicates that EMoV is a strain of PVY.But higher coat protein molecular weight of EMoVas compared to PVY and non-transmission ofEMoV by aphids (Aphis craccivora and Myzus

persicae) indicated that EMoV is a biologicallydistinct potyvirus (A. Varma, personal communi-cation). In addition, our studies through HPLCpeptide profiling also showed EMoV as a distinctpotyvirus. Similarities of N- terminal regions ofcoat protein of other biologically distinct

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potyviruses are also known (Shukla et al., 1989;Khan et aI., 1990; Fortass et aI., 1991). There-fore, differences/similarities in N-terminal regionsof coat proteins as determined by serology alonecannot be used as a criteria for the classificationof potyviruses and their strains.

Differentiation of PVY strains by HPLCpeptide profiling

Previous studies (Shukla et aI., 1988; Jain etal., 1992; McKern et aI., 1991; 1992; 1992a) haveshown that peptide profiles of coat proteins fromstrains of a single potyvirus were very similarwith major peaks having common retention timeswhile that from distinct potyviruses shared only afew of peaks with common retention times.Pairwise comparison of peptide profiles haveshown that between 57 and 100% of peaks withidentical retention times when strains of individualpotyvirus were examined in contrast to the 16 to42% peak similarity between distinct viruses(Shukla et aI., 1988). Comparison of peptide pro-files of PVY -D, PVyo and PVYc showed substan-tial similarity as many of the peaks were found tobe common to all the profiles (Fig. 2). High percent similarity of elution profiles (62-77%) ob-tained between strains/isolates of PVY clearlydemonstrate that they are strains of one virus(PVy), and that PVY-D is distinct from PVycand pvyo. In the absence of amino acid sequencedata peptide profiling provides a very useful toolfor differentiating strains of viruses.

ACKNOWLEDGEMENTS

The authors thank the Indian AgriculturalResearch Institute for providing Senior Research'Fellowship to A. Ishwara Bhat, and to the IndianCouncil of Agricultural Research for their supportto the Advanced Centre for Plant Virology.

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