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Virus Research 183 (2014) 85–88

Contents lists available at ScienceDirect

Virus Research

j ourna l h o mepa ge: www.elsev ier .com/ locate /v i rusres

hort communication

he isolation and genetic characterisation of a South African strainf Phthorimaea operculella granulovirus, PhopGV-SA

ichael D. Jukesa, Caroline M. Knoxa,∗, Martin P. Hillb, Sean D. Moorec

Department of Biochemistry, Microbiology and Biotechnology, Rhodes University, PO Box 94, Grahamstown 6140, South AfricaDepartment of Zoology and Entomology, Rhodes University, PO Box 94, Grahamstown 6140, South AfricaCitrus Research International, PO Box 20285, Humewood 6031, Port Elizabeth, South Africa

r t i c l e i n f o

rticle history:eceived 30 October 2013eceived in revised form 9 January 2014ccepted 14 January 2014vailable online 3 February 2014

eywords:NA viruseshthorimaea operculellaranulovirus

a b s t r a c t

The Phthorimaea operculella granulovirus (PhopGV) is considered a promising biopesticide that can beincorporated into integrated pest management programmes for sustainable control of the potato tubermoth, Phthorimaea operculella (Zeller) (Lepidoptera: Gelechiidae), a major pest of solanaceous crops insub-tropical and tropical regions worldwide. Several PhopGV isolates recovered from geographicallydifferent insect populations have been genetically characterised, and the full genome of the TunisianPhopGV-1346 isolate has been sequenced, providing a reference strain for comparison of novel isolates.Here we report the identification and genetic characterisation of a South African PhopGV isolate recov-ered from a P. operculella colony held under laboratory conditions. Transmission electron microscopyexamination of purified occlusion bodies together with analysis of granulin and late expression factor-

aculovirusgt gene

8 (lef-8) gene sequences confirmed the identity of the virus as PhopGV. The sequenced ecdysteroidUDP-glucosyltransferase (egt) gene was 1353 nt in length, placing PhopGV-SA in egt group II. Finally,a phylogenetic analysis using a range of egt sequences grouped PhopGV-SA together with the Kenyan,Ecuadorian, Indonesian and Colombian isolates. The results are discussed with reference to the possibleorigin of PhopGV-SA, and provide a platform for future studies involving virulence evaluation againstgeographically different P. operculella populations with a view to biopesticide development.

Phthorimaea operculella granulovirus (PhopGV) has been iso-ated and genetically characterised in various countries includingeru, Guatemala, Tunisia, Ecuador, Kenya, Egypt and IndonesiaCarpio et al., 2013; Espinel-Correal et al., 2010; Taha et al., 2000;eddam et al., 1999). This virus infects the potato tuber moth, P.perculella (Zeller) (Lepidoptera: Gelechiidae), which is a majorest causing extensive damage to potato crops by mining intoubers. Broodryk and Pretorius (1974) isolated a granulovirus (GV)rom the larvae of P. operculella in South Africa but the virus wasever genetically characterised or formulated into a biopesticide

or the control of P. operculella. Genetic characterisation of bac-loviruses is important in both identifying viruses to species level,nd enabling genotypic comparisons between isolates especially ifhe virus is intended to be used as a biopesticide. Genetic character-

sation involves either the determination of the complete genomeequence (Lange and Jehle, 2003; Luque et al., 2001; Taha et al.,000) or sequencing and analysis of specific viral genes. Genetic

∗ Corresponding author at: Department of Biochemistry, Microbiology andiotechnology, Faculty of Science, Rhodes University, Grahamstown, 6140, Southfrica. Tel.: +27 46 6038023; fax: +27 46 6037576.

E-mail address: caroline.knox@ru.ac.za (C.M. Knox).

168-1702/$ – see front matter © 2014 Elsevier B.V. All rights reserved.ttp://dx.doi.org/10.1016/j.virusres.2014.01.013

© 2014 Elsevier B.V. All rights reserved.

analysis of various baculovirus isolates has shown several genes tobe prime targets for virus characterisation. A study by Jehle et al.(2006) described the identification of baculovirus species usingpairwise nucleotide distances of late expression factor-8 (lef-8),late expression factor-9 (lef-9) and polyhedrin (polh)/granulin genesequences producing similar results to an analysis based on fullgenome sequences. While these genes and others have been widelyused in genetic identification of baculovirus species (Herniou et al.,2004; Jehle et al., 2006), few lef-8, lef-9 and granulin gene sequencesare available for PhopGV.

Another gene of interest is the ecdysteroid UDP-glucosyltransferase (egt) gene present in many lepidopteranbaculoviruses (Clarke et al., 1996; Herniou et al., 2001; Tumilasciet al., 2003) which plays a unique role during virus infectionby interfering with host development as well as altering hostbehaviour (Cory et al., 2004; Hoover et al., 2011; O’Reilly andMiller, 1991). The gene has been sequenced in various baculovirusspecies providing a database for comparison and analysis. ForPhopGV alone, the egt gene has been sequenced and deposited

into the National Center for Biotechnology Information’s (NCBI)GenBank database 24 times. Furthermore, a study by Carpio et al.(2013) in which 20 egt gene sequences were analysed, identified3 common gene structures due to major deletions (or insertions)

8 Resea

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c(ctCitOimtbtspsMat(du

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6 M.D. Jukes et al. / Virus

n the 3′ moiety of egt, with these groups being named egtI, egtIInd egtIII. The availability of sequence data for this gene makest a prime target for sequencing and for use in phylogeneticnalysis.

A colony of P. operculella was acquired from field collectedarvae from a commercial potato plantation in Patensie in the East-rn Cape, South Africa (33◦45′20′′ S; 24◦49′6′′ E). The colony wasaintained on whole baby potatoes in plastic containers at room

emperature as described by King (2011). The colony was observedaily for dead larvae and all cadavers were collected and stored at20 ◦C.

Baculovirus occlusion bodies (OBs) were recovered from larvaladavers according to methods adapted by Opoku-Debrah et al.2013) with minor modifications. Approximately 0.35 g of larvaladavers was used. Centrifugation with JA-20 (Beckman) centrifugeubes was carried out at 7840 × g for 30 min at 4 ◦C in a Beckmanoulter Avanti® J-E centrifuge. Glycerol gradients were prepared

n two SW 41 Ti Beckman® Ultra-clear centrifuge tubes and cen-rifuged at 27,783 × g for 15 min at 4 ◦C in a Beckman CoulterptimaTM L-90 K ultracentrifuge. Final OB extracts were suspended

n 750 �l ddH2O and stored at −20 ◦C. OBs were visualised by trans-ission electron microscopy (TEM) on a Libra 120 (Zeiss) with

he MegaviewG2 (Olympus soft imaging solutions) camera. Car-on grids were prepared by applying 5 �l of purified OBs ontohe grid for 30 s, followed by drying with filter paper. Grids weretained for 30 s with 5 �l uranyl acetate (1%) and dried with filteraper. Images were captured using the iTEM (Olympus soft imagingolutions) software and processed with Microsoft® Office Pictureanager. GV mean size was determined by measuring the length

nd width of 50 OBs. Genomic DNA was extracted from OBs usinghe CTAB DNA extraction protocol described by Opoku-Debrah et al.2013). Centrifugation was carried out at 12,100 × g in an Eppen-orf MiniSpin® desktop centrifuge and DNA stored at −20 ◦C beforese.

PCR amplification of the granulin, egt and lef-8 genes wasarried out using specific oligonucleotide primer pairs based onhe PhopGV-1346 nucleotide sequence (GenBank: NC004062.1)amely, grnF (5′-ATG GGA TAC AAC AAA ACT CTG AG-3′) and grnR5′-TTA ATA AGC GGG TCC GGT GAA C-3′), egtF (5′-GAG TCG AGCAA TTT TGT TTG CG-3′) and egtR (5′-GCA ACG ATG ATC TCAAT ATG AGC-3′) and lef8F (5′-ATT GGA ATG AGA TCA AGC GCATG-3′) and lef8R (5′-CGT GCG TTT TAC AAC TAA TCG AAG-3′)

espectively. Oligonucleotide pairs for egt and lef-8 flanked thearget gene producing complete coding sequences while oligonu-leotide pairs for granulin were internal and produced a partialoding sequence. Reactions comprised 12.5 �l Taq ReadyMix PCRit (KAPA Biosystems) for the granulin gene or 12.5 �l MaximaTM

ot Start Green PCR Master Mix (Thermo Scientific) for the egtnd lef-8 genes, 2 �l forward and reverse primers (10 �M), 8 ng ofemplate DNA for the granulin and egt, and 5 ng template DNA foref-8. Reactions were made up to 25 �l using ddH2O and the con-ents briefly centrifuged. The PCR protocol was: 95 ◦C for 5 min,ollowed by 25 cycles of 95 ◦C for 30 s, 55 ◦C for 30 s, 72 ◦C for5 s for the granulin gene or 72 ◦C for 1 min 30 s for the egt genend a final extension at 72 ◦C for 5 min. The PCR protocol usedor the lef-8 gene differed only slightly having a total of 45 cyclesnd an extension step at 72 ◦C for 2 min 45 s. Amplified prod-cts were visualised by 1% agarose gel electrophoresis at 90 V for5 min in 1× TAE buffer (40 mM Tris–acetate, 20 mM acetic acid,

mM EDTA) stained with ethidium bromide. Gel images were cap-ured with the UVIpro chemi (UVItec) UV trans-illuminator andoftware. Amplified genes were sequenced by Inqaba Biotechni-

al Industries (Pty) Ltd (South Africa) with sequence data beinganually checked and edited using Chromas Lite (Technelysium)

efore analysis with Basic Local Alignment Search Tool (BLAST)NCBI).

rch 183 (2014) 85–88

The egt gene sequence of PhopGV-SA together with those avail-able on GenBank (Table 1) were translated, aligned and analysed inMolecular Evolutionary Genetics Analysis 5.2 (MEGA 5.2) (Tamuraet al., 2011). The egt sequence of one isolate from groups A, B, C andD was selected from each to represent the group in the analysisalong with the remaining isolate sequences including three iso-late sequences as out-groups (CrleGV, PlxyGV and AdorGV). Thesewere used in the construction of a maximum likelihood tree. Ananalysis of the alignment was used to determine the best pro-tein model, all sites were used and a consensus bootstrap treewas produced from 100 replicates. Alignments of the egt geneswere used to detect SNPs present in PhopGV-SA. Alignments werefurther analysed in ClustalW2 to determine whether amino acidchanges were conserved between strongly similar properties orbetween weakly similar properties (Goujon et al., 2010; Larkin et al.,2007).

Transmission electron microscopy of purified occlusion bodies(OBs) showed the presence of rod shaped particles with a meanlength of 355.9 nm (±28.1 nm SD) and a mean width of 188.6 nm(±21.9 nm SD). To further identify the virus, lef-8 (GenBank:KF724712) and granulin (GenBank: KF724710) gene sequenceswere submitted to BLAST, and aligned most closely with the com-plete genome of the reference strain, PhopGV-1346 (GenBank:NC004062.1) with identities of 99% and 100% respectively.

The third PhopGV-SA gene analysed was the egt (GenBank:KF724711) gene which has been shown to have high variability inthe 3′ region resulting in the classification of sequences into threegroups egt I, egt II and egt III with characteristic lengths of 1305,1353 and 1086 nt respectively (Carpio et al., 2013). A nucleotidealignment of the egt gene sequence acquired in this study alongwith additional egt gene sequences from 24 PhopGV isolates onGenBank (Table 1) showed the presence of five different egt genestructures (Fig. 1). Groups I, II and III were identified along withtwo additional egt gene structures denoted as egt IV and egt V, withthese having lengths of 1092 and 861 nt respectively. The PhopGV-SA egt gene was shown to align with members of group II havingthe characteristic length of 1353 nt.

The primers used to amplify egt in PhopGV-SA were designedto flank the gene providing a short stretch of sequence data forthe region beyond the stop codon. Nucleotide alignment of the25 egt gene sequences revealed internal deletions of various sizespresent in many of the sequences. Short stretches in the 3′ region ofgroups I, III and IV were found to align with the region downstreamof the stop codon in the PhopGV-SA sequence (Fig. 1). The differ-ent gene structures of the various egt groups are a result of theseinternal deletions, with groups I, III, IV and V each having a uniqueinternal deletion compared to the egt gene sequence acquired forPhopGV-SA. In the case of the groups I, IV and V, this deletion resultsin frame shifts in the coding sequence, changing the amino acidsequence in the C terminal region of the protein. The additionalsequence downstream of the stop codon acquired for the SA isolaterevealed a possible explanation for the existence of the various egtgene structures. Groups I, III and IV appear to have originated fromthe group II egt gene structure with each group being a result of aunique internal deletion in the 3′ region of the egt gene. The regionaffected by these internal deletions is only present in the group II egtgene sequences indicating that three separate divergences from thegroup II isolates may have occurred. Group V, on the other hand,has more complex origins as the internal deletion observed cov-ers a region present in all the remaining groups, and consequentlymay have diverged from any isolate present in groups’ I–IV. Theegt group II gene is similar in size to the egt gene present in the

closely related AdorGV and CrleGV, and may represent an ancestralform of the gene. These results suggest that the internal deletionsobserved in groups I, III, IV and V may be a result of more recentgenetic changes in the evolution of PhopGV.

M.D. Jukes et al. / Virus Research 183 (2014) 85–88 87

Table 1List of egt gene sequences for Phthorimaea operculella granulovirus isolates available on GenBank and this study. CIP: International Potato Center, Lima, Peru. INIA: InstitutoNacional de Investigaciones Agrícolas, Venezuela.

Isolate Location Original host Size (nt) GenBank no. Source Groupa

SA South Africa Phthorimaea operculella 1353 KF724711 This studyVG003 Colombia Tecia solanivora 1353 HQ166268.1 Espinel-Correal et al. (2010)1346 Tunisia Phthorimaea operculella 1305 NC004062.1 CIP AVG004 Colombia Tecia solanivora 861 HQ166269.1 Espinel-Correal et al. (2010) B1390-2 Peru Phthorimaea operculella 1305 HQ317419.1 Vickers et al. (1991) ATUTA Peru Tuta absoluta 1305 HQ317412.1 Carpio et al. (2013) AJLZ9f Ecuador Tecia solanivora 1353 HQ317403.1 Carpio et al. (2013) CVZ Venezuela Tecia solanivora 1353 HQ317415.1 INIA CINDO Indonesia Phthorimaea operculella 1353 HQ317413.1 Zeddam et al. (1999)KEN Kenya Phthorimaea operculella 1353 HQ317414.1 CIPCHI Chile Phthorimaea operculella 1353 HQ317406.1 Carpio et al. (2013) CVG002 Colombia Tecia solanivora 1092 HQ166267.1 Espinel-Correal et al. (2010)TANG Ecuador Symmetrischema tangolias 1086 JX082398.1 Carpio et al. (2013) DGV5 Ecuador Tecia solanivora 1086 HQ317417.1 Carpio et al. (2013) DTESO Ecuador Tecia solanivora 1086 HQ317408.1 Carpio et al. (2013) DEUQU Peru Eurysacca quinoae 1086 HQ317409.1 Carpio et al. (2013) DHUAN Peru Phthorimaea operculella 1086 HQ317405.1 Carpio et al. (2013) DAUS Australia Phthorimaea operculella 1086 HQ317407.1 Briese (1981) DEGY Egypt Phthorimaea operculella 1086 HQ317420.1 Carpio et al. (2013) DPADE Bolivia Paraschema detectendum 1086 HQ317416.1 Carpio et al. (2013) DGUA Guatemala Tecia solanivora 1086 HQ317410.1 CIP DTUR Turkey Phthorimaea operculella 1086 HQ317404.1 Carpio et al. (2013) DYEM Yemen Phthorimaea operculella 1086 HQ317411.1 Kroschel et al. (1996)GV6 Ecuador Tecia solanivora 1086 HQ317418.1 Carpio et al. (2013) D

61

.

CpssdtpTbw

FKfC3c

VG005 Colombia Tecia solanivora 8

a Groups with identical amino acid sequences are denoted as groups A, B, C and D

PhopGV-1346 was identified as a member of the egt I group byarpio et al. (2013) resulting in a highly variable 3′ end when com-ared to PhopGV-SA. For this reason, a comparison of the egt geneequences between PhopGV-1346 and PhopGV-SA was only pos-ible up to nucleotide 1262 (Fig. 1). Only one SNP, 1059A→G, wasetected in this region with this substitution being silent. SNPs forhe complete 1353 nt gene of PhopGV-SA were determined by com-

aring the aligned egt sequence against other group II members.his comparison showed the presence of several additional SNPsetween PhopGV-SA and individual group II members. Four SNPsere observed in an alignment with PhopGV-KEN at nucleotide

1 50 800Phop GV-SA

I

II

III

IV

V

1

840 1015

1

5’ AT G---AATGTG---TT TCCAC AGC GCGAA TTGTT AAAT---GGT GTT- M N V F P Q R E L L N G V

5’ AT G---AATGTG---TT TCCAC AGC GCGAA TTGTT AAAT---GGT GTT- M N V F P Q R E L L N G V

5’ AT G---AATGTG---TT TCCAC AGC GCGAA TTGTT AAAT---GGT GTT- M N V F P Q R E L L N G V

5’ AT G---AATGTG---TT TCCAC AGC GCGAA TTGTT AAAT---GGT M N V F P Q R E L L N G

5’ AT G---AATGTG---TT TCCAC AGC GCGAA TTGTT AAAT---GGT M N V F P Q R E L L N G

5’ AT G---AAT T TCCAC AGC GCGAA TTGTT AA M N F H S A N C *

PhopGV-SA

I

II

III

IV

V

ig. 1. Diagrammatic representation of a multiple nucleotide alignment of the five egt genEN, PhopGV-GUA, PhopGV-VG002 and PhopGV-VG005 respectively compared to the egt

or each gene sequence with stop codon positions for groups I–V given relative to PhopGarpio et al., 2013) are represented by a “V” shape. Alignment of the 3′ regions for group′ end of the deletion. The translated amino acid sequence is shown below the nucleotideonducted in MEGA 5.2 (Tamura et al., 2011). (For interpretation of the references to colo

HQ166270.1 Espinel-Correal et al. (2010) B

positions: 327G→A, 647C→T, 736G→A and 909C→T, the first threeresulting in amino acid changes at positions: 109M→I, 216A→Vand 246D→N. Alignment of the amino acid sequence in ClustalW2showed two of these changes were conserved between amino acidgroups of strongly similar properties while the amino acid changeat position 216 was conserved between groups of weakly similarproperties. When aligned with PhopGV-INDO, there were two SNPs

at positions: 615G→C and 646G→A, both resulting in amino acidchanges of strongly similar properties at positions: 205W→C and216A→T. In the case of PhopGV-CHI, there were two SNPs at posi-tions 969C→A and 1053T→C but these were silent mutations. Two

1457

05 3

05 3

135 3

139 1

138 6

139 1

1262

--TTGA AA---ACACT GTAAA TAAAC TTTTT GAC AAAAT TTATT GTTTT ATAAA ATGA 3’ L K T L *

--TTG AAT GTAAA TAAAC TTTTT GAC AAAAT TTATT GTTTT ATAAA ATGA 3’

L N V N K L F D K I Y C F I K *--TTGA AA---ACACT GTAA 3’

L K T L * A TAAAC TTTTT GAC AAAAT TTATT GTTTT ATAA 3’

I N F L T K F I V L * AA TAAAC TTTTT GAC AAAAT TTATT GTTTT ATAAA ATGA 3’

N K L F D K I Y C F I K *

3’

e sequences of PhopGV groups I–V represented by isolates PhopGV-1346, PhopGV-gene sequence of PhopGV-SA. Start codons (green) and stop codons (red) are shownV-SA (red text). Internal deletions present in groups I, III, IV and V (modified froms I–V against PhopGV-SA shows a frame shift occurring in groups I, IV and V at the

sequence for each codon. Nucleotide alignments and amino acid translations werer in this figure legend, the reader is referred to the web version of the article.)

88 M.D. Jukes et al. / Virus Resea

INDO

KEN

VG003

SA

Group C

egt II

Group D

YEMegt III

egt V Group B

egt IV VG002

egt I Group A

AdorGV

PlxyGV

CrleGV

67

11

32

10

100

100

41

85

100

90

0

Clade 1

Clade 2

Fig. 2. Phylogenetic reconstruction inferred by using maximum likelihood methodbased on the Whelan and Goldman model (Whelan and Goldman, 2001) forPhopGV egt amino acid sequences (see Table 1 for isolate data). Bootstrap con-sensus values (100 replicates) are shown next to branches with values less than50% being collapsed (Felsenstein, 1985), Thaumatotibia (Cryptophlebia) leucotreta(NE

iti4g

tgpiaiwi1gCblPoem

(baipaeSehilgmd

A

t

derived from multiple protein families using a maximum-likelihood approach.

CrleGV; GenBank: NC005068.1), Plutella xylostella granulovirus (PlxyGV; GenBank:C002593.1) and Adoxophyes orana granulovirus (AdorGV; GenBank: NC005038.1).volutionary analyses were conducted in MEGA 5.2 (Tamura et al., 2011).

solates PhopGV-VZ and PhopGV-VG003 had one SNP each at posi-ion 969C→A and 1282C→G respectively, with the latter resultingn an amino acid change of weakly similar properties at amino acid28P→A. A final SNP was observed at position 1059A→G in all egtroup II members which did not result in an amino acid change.

Multiple alignment of egt showed groups of isolates with iden-ical amino acid sequences (e.g. isolates TANG & AUS) which wererouped together and denoted as groups A, B, C and D (Table 1). Ahylogenetic analysis of PhopGV using the egt sequences is shown

n Fig. 2. Analysis of the dataset indicated the best protein models the WAG model with invariant sites selected. Isolates with sim-lar egt structures were found to be more closely grouped together,

ith egt groups I, III, IV and V forming clade 2 (C2) and egt IIsolates found in clade 1 (C1) supported by a bootstrap value of00%. Within clade 1, PhopGV-SA formed a branch with isolates inroup C, which includes PhopGV-JLZ9f, PhopGV-VZ and PhopGV-HI each of which have identical amino acid sequences, howeverootstrap support for this grouping was low (32%). Other iso-

ates also found in clade 1 include the remaining group II isolateshopGV-INDO, PhopGV-KEN and PhopGV-VG003. The separationf egt II into C1 and the other groups into C2 supports the hypoth-sis that groups I, III, IV and V may have diverged from group IIembers.P. operculella was first described in Western South America

WSA) alongside its solanaceous host, the potato, in 1873 (reviewedy Rondon, 2010). Since then the pest has spread across the worldnd is believed to have been accompanied by the granulovirus. Its possible that PhopGV was introduced into Africa on two inde-endent occasions in Tunisia (isolate 1346) and South Africa. Thisssumption is based primarily on each isolate belonging to differentgt groups, both of which frequently occur in WSA. Since PhopGV-A and PhopGV-KEN belong to egt group II, the Kenyan isolate (KEN)ither represents a third separate introduction into Africa, or mayave migrated north from its point of origin in Southern Africa. It is

nteresting to note that the amino acid sequence of PhopGV-SA isess similar to that of the Kenyan isolate than to those of the WSAroup II members. The reason for this observation is not clear butay relate to evolution of the different isolates in geographically

istinct insect populations in Kenya and South Africa.

cknowledgements

The financial support provided by River Bioscience (Pty) Ltd andhe Rhodes University Research Council is gratefully acknowledged.

rch 183 (2014) 85–88

The authors would also like to thank John Opoku-Debrah, CraigChambers, Fatima Abdulkadir and Tanya Fullard for their technicalassistance.

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