ALMEIDA, G. F.

CASTRO, M. E. B. de

INGLIS, P. W.

PAULA, D. P.

RIBEIRO, G. M.

RIBEIRO, Z. M. de A.

SOUZA, M. L. de

English

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Identification and sequence analysis of the Condylorrhizavestigialis MNPV p74 geneMaria Elita B. Castro • Débora P. Paula •Geraldo F. Almeida • Zilda Maria A. Ribeiro •Marlinda L. Souza • Peter W. Inglis • Bergmann M. RibeiroReceived: 10 February 2011 / Accepted: 20 June 2011 / Published online: 1 July 2011 Springer Science+Business Media, LLC 2011Abstract The baculovirus Condylorrhiza vestigialis mul-tiple nucleopolyhedrovirus (CoveMNPV), isolated fromC. vestigialis infected larvae in Paraná (Brazil), was identi-fied in our laboratory. A full-length clone was obtained fromthe CoveMNPV genome, of the gene that encodes thehomolog to baculoviral p74, essential for oral infectivitywhich was then sequenced and characterized. The Cove-MNPV p74 gene (GenBank accession number EU919397)contains an ORF of 1935 bp that encodes a deduced proteinof 73.61 kDa. The phylogenetic affiliations of the Cove-MNPV gene were determined by a heuristic search of 40aligned baculovirus p74 nucleotide sequences using maxi-mum parsimony (PAUP 4.0b4a). The phylogenetic analysisplaced CoveMNPV within lepidopteran nucleopolyhedro-virus (NPV) Group I, Clade A, as being the closest toChoristoneura fumiferana defective NPV.Keywords Alphabaculovirus  Condylorrhiza vestigialisMNPV  p74 gene  Phylogenetic analysisBaculoviruses are widely used as bioinsecticides for thecontrol of agricultural and forest insect pests. In mostcases, these naturally occurring viruses are obtained fromfield-collected larvae that have been isolated and selectedbased on their infectivity and virulence. They comprise alarge family of insect-specific DNA viruses, divided intofour genera: Alphabaculovirus (subdivided into Group Iand Group II nucleopolyhedroviruses (NPVs)), Betabacu-lovirus, Gammabaculovirus, and Deltabaculovirus [1–4],(www.ictvonline.org).The baculovirus Condylorrhiza vestigialis multiplenucleopolyhedrovirus (CoveMNPV), recently identified byCastro et al. [5], was isolated from the defoliating cater-pillar C. vestigialis (Guenée) (Lepidoptera: Crambidae), animportant pest of Poplar (Populus spp. Salicaceae). Initialfield and laboratory tests to evaluate the infectivity ofCoveMNPV to C. vestigialis have revealed a considerablepotential for its use in the control of this important insectpest [6].The genome of CoveMNPV has not yet been sequenced,and very little is known about the host infection processand proteins related to its infectivity. Identification ofstructural proteins associated with occlusion-derived virus(ODV), the viral form responsible for horizontal trans-mission between insect hosts, has facilitated the study ofthe functional role of these proteins in viral virulence andhost specificity. In addition, studies based on sequences ofindividual genes have provided additional information ontheir molecular characteristics and phylogenetic relation-ships [7].The p74 gene encodes a protein that is conserved amongall the sequenced baculoviruses and is essential for per osinfection [8–10]. P74 was the first of the per os infectivityfactor (PIF) family to be identified among the baculovi-ruses. To date, this family is represented by six genes (thepifs): pif1, pif2, pif3, pif4, pif5, and p74 [11–14].In order to investigate the taxonomic and phylogeneticstatuses of the CoveMNPV, the viral p74 gene was cloned,sequenced, and characterized. Occlusion bodies (OB) wereM. E. B. Castro (&)  D. P. Paula  G. F. Almeida Z. M. A. Ribeiro  M. L. Souza  P. W. InglisEmbrapa Recursos Genéticos e Biotecnologia, Parque EstaçãoBiológica W5 Norte Final, Brası́lia, DF 70770-917, Brazile-mail: elita@cenargen.embrapa.brG. F. Almeida  B. M. RibeiroDepartamento de Biologia Celular, Universidade de Brası́lia,Brası́lia, DF, Brazil123Virus Genes (2011) 43:471–475DOI 10.1007/s11262-011-0640-6isolated and purified from CoveMNPV-infected larvae. AllDNA manipulations described above, including restrictionenzyme digestion, agarose gel electrophoresis, Escherichiacoli transformation, and plasmid purification, were per-formed essentially as described by Sambrook et al. [15].Primers for PCR-based probe construction and p74 ORFsequencing were designed based on the conserved regions of ap74 sequence alignment of representatives from each of thebaculovirus groups obtained from GenBank/EMBL andaligned by CLUSTALX 1.81 [16]. The following primerswere used: p74/Cove-GI-Forward: 50-gtgtacagcgagctgctggc-30 (?348 to ?367 nucleotides relative to the start codon), p74/Cove-GI-Reverse: 50-tacaccttgcgtcccgcgtc-30 (?1905 to?1924), p74/Cove-GII-Forward: 50-agattgcgtttcatacccaaat-30(?185 to ?207), p74/Cove-GII-Reverse: 50-agatgagtgtacagagcgctgg-30 (?1835 to ?1857), p74-Forward: 50-aataccctgggcaaaccaat-30 (?1951 to ?1931), and p74-Reverse: 50-aattttgtcgtttagtta-30 (?1380 to ?1362).The polymerase chain reaction (PCR) was performedusing 10–20 gg of CoveMNPV DNA, 10 lM of forwardand reverse primers, and one unit of Taq DNA polymerase(Invitrogen) in 19 PCR buffer supplemented with200 lM of each dNTP and 2.0 mM MgCl2 was performedusing a MJ Research PTC-100 thermal cycler according tothe following steps: an initial denaturation of 94C for5 min, 30 cycles of 94C, 1 min; 64C (56C), 1 min; and72C, 1 min, followed by a final extension step of 72C for10 min.After obtaining the p74 [a-32P] dCTP labeled probe,Southern blots of restriction enzyme-digested CoveMNPVDNA were hybridized overnight at 68C, and the mem-branes were washed three times in 29 SSC (standard salinecitrate), 0.1% SDS (sodium dodecyl sulfate) for 30 mineach before the exposure of Kodak T-MATTMG/RA filmfor 48 h at -80C.Restriction fragments corresponding to the hybridizedbands were cloned in pBlueScript II KS? (Stratagene)and the PCR amplicons cloned in pGEM-T Easy (Pro-mega), and then employed to transform E. coli XL-1-Blue. Clones were sequenced using the BigDye 3.1 kit andthe ABI 3130 XL automatic DNA sequencer (AppliedBiosystems, Foster City, CA, USA).The detection of the p74 locus in the CoveMNPV gen-ome consisted of the hybridization of a p74 consensusbaculovirus probe (1,600 bp), generated by PCR amplifi-cation, and with the CoveMNPV DNA digested by therestriction enzymes HindIII, PstI, and EcoRI (Fig. 1a). Asingle, strongly hybridized band was detected in the threedigests respectively: a 1-kb fragment from HindIII diges-tion, a 4.2–4.3-kb fragment from PstI digestion, and a3.1-kb fragment from EcoRI digestion (Fig. 1b). To con-firm the identity of the gene, the restriction fragments wereeluted from the 0.8% low-melting agarose gel, and clonedinto pBlueScript II KS? for DNA sequencing. We foundthat the 1.0-kb HindIII fragment (pBS-CoveHindIII) hadpart of the p74 gene and was present on the EcoRI 3.1 kbfragment, which contained 75% of the gene (about1,500 bp). Since 25% of the p74 gene sequence was stillmissing, we performed a PCR amplification of this regionfrom the CoveMNPV genome using the specific primerspreviously cited. The amplified fragment was cloned intopGEM-T Easy for DNA sequencing. After the combina-tion of both strategies, the full length of the CoveMNPVp74 gene was successfully obtained.Fig. 1 a Agarose gel (0.8%)electrophoretic profile of theCoveMNPV genome digestedwith HindIII, PstI and EcoRI,respectively (lanes 2–4). Themolecular mass marker DNAk/PstI is contained in lane 1.b Autoradiogram of theSouthern blot assay to locate thep74 locus in the CoveMNPVgenome digested as mentionedin a, using a radioactive p74probe (32P). The white arrowsindicate the correspondingintensely hybridizing regions472 Virus Genes (2011) 43:471–475123The analysis of the nucleotide sequence of the Cove-MNPV p74 gene revealed the presence of an ORF of1,935 bp that was deposited in GenBank under the acces-sion number EU919397. The CoveMNPV p74 ORFpotentially encodes a protein of 644 amino acids ofmolecular mass 73613.3 Da (pI 5.12) containing P74domains related to oral infectivity, and transmembranedomains (C-terminal region) potentially related toanchoring inside of ODV envelope (data not shown). Thededuced protein alignment revealed a high similarity toTable 1 p74 baculovirus sequences used for phylogenetic analysesGenBank accession number Baculovirus Genome position ORF Size (bp)NP_818674.1 Adoxophyes honmai NPV 23,773–25,788 27 2,016NP_872507.1 Adoxophyes orana GV 37,367–39,295 54 1,929YP_006288.1 Agrotis segetum GV 57,392–59,419 56 2,028YP_529814.1 Agrotis segetum NPV 135,968–137,902 144 1,935YP_610989.1 Antheraea pernyi NPV 107,641–109,563 129 1,923AAY19516.1 Anticarsia gemmatalis MNPV–2D 112,268–114,202 134 1,935AAY19519.1 Anticarsia gemmatalis NPV-SF 419–2,353 – 1,935AAA46729.1 Autographa californica MNPV 65–2,002 138 1,938NP_047536.1 Bombyx mori NPV 108,796–110,733 138 1,938NP_932741.1 Choristoneura fumiferana DEFMNPV 111,392–113,398 132 2,007AAL13071.2 Choristoneura fumiferana GV 337–2,331 – 1,995AF512031 Choristoneura fumiferana MNPV 110,746–112,683 130 1,938YP_249621.1 Chrysodeixis chalcites NPV 18,970–20,946 17 1,977YP_717552.1 Clanis bilineata NPV 18,543–20,522 14 1,980NP_891905.1 Cryptophlebia leucotreta GV 47,165–49,177 58 2,013NP_203378.1 Culex nigripalpus NPV 64,492–66,537 73 2,046NP_148844.1 Cydia pomonella GV 48,578–50,644 60 2,067YP_874207.1 Ecotropis obliqua NPV 16,243–18,201 14 1,959NP_203290.1 Epiphyas postvittana NPV 101,271–103,205 121 1,935NP_203576.1 Helicoverpa armigera NPV 16,224–18,290 20 2,067AF334030_89 Helicoverpa zea SNPV 16,195–18,261 19 2,067YP_473207.1 Hyphantria cunea NPV 19,839–21,773 19 1,935YP_758321.1 Leucania separata NPV 24,273–26,249 – 1,977NP_047663.1 Lymantria dispar MNPV 26,645–28,663 27 2,019NP_613243.1 Mamestra configurata NPV-A 144,165–146,138 160 1,974NP_689333.1 Mamestra configurata NPV-B 147,569–149,542 159 1,974ABM05422.1 Maruca vitrata MNPV 94,724–96,661 106 1,938YP_025247.1 Neodiprion lecontei NPV 41,772–43,673 47 1,902YP_025157.1 Neodiprion sertifer NPV 49,560–51,464 50 1,905YP_001650925.1 Orgyia leucostigma NPV 16,694–18,652 15 1,959O10365.1 Orgyia pseudotsugata MNPV 112,559–114,493 134 1,935NP_663220.1 Phthorimaea operculella GV 46,284–48,260 55 1,977NP_068268.1 Plutella xylostella GV 37,776–39,512 48 1,737YP_758602.1 Plutella xylostella MNPV (CL3) 119,644–121,581 134 1,938NP_703125.1 Rachiplusia MNPV 116,861–118,798 138 1,938NP_037891.1 Spodoptera exigua MNPV 124,099–126,060 131 1,962AAO45529 Spodoptera frugiperda MNPV 166–2,106 134 1,941CAA67755.1 Spodoptera littoralis NPV 148–2,121 – 1,974NP_258289.1 Spodoptera litura NPV 19,706–21,679 21 1,974NP_059225.1 Xestia c-nigrum GV 71,928–74,060 77 2,133Virus Genes (2011) 43:471–475 473123several other baculoviruses. The highest similarity valueswere observed among the P74 of CfDEFNPV (94% iden-tity) AgMNPV (90%), AnpeNPV (81%), OpMNPV (79%),and CfMNPV (79%).The CoveMNPV p74 sequence along with 39 otherbaculovirus p74 DNA sequences (Table 1) was alignedusing MUSCLE [17]. Phylogenetic analysis was per-formed under the maximum parsimony criterion usingPAUP 4.0b4a [18]. Heuristic searches comprised 1,000cycles of random taxon addition, with TBR branchswapping, holding five trees per cycle. The three mostparsimonious trees were obtained, one of which isdepicted in Fig. 2. The phylogenetic tree confirmed theclassification of the Baculoviridae family into four mod-erately to well-supported clades representing genera:Alpha-(lepidopteran NPVs), Beta-(lepidopteran GVs),Gamma-(hymenopteran NPVs) and Deltabaculovirus(dipteran NPVs) [2]. Within the lepidopteran NPVs, thereis a division comprising two groups: Group I and GroupII were observed (Fig. 2).300 stepsNeseNPVLdMNPVHaNPVSfMNPVXcGVPlxyMNPVCfDEFNPVPhopGVOlNPVHcNPVSpliNPVCfGVCcNPVBmNPVMacoNPV AEpobNPVLsNPVRoMNPVPlxyGVOpMNPVMacoNPV BCbNPVApNPVAgMNPV 2DNeleNPVSeMNPVAcMNPVAgMNPV SFMaviNPVCfMNPVAsNPVAdorGVAdhoNPVCuniNPVCrleGVSpltNPVAsGVCpGVEppoNPVCoveMNPVHzSNPV996610081869510010079851001007492999770100659799100901001001008455Lepidoptera NPV Group II AlphabaculovirusLepidoptera NPV Group IAlphabaculovirusLepidoptera GVBetabaculovirusHymenoptera NPVGammabaculovirusDiptera NPV Deltabaculovirus (Outgroup) Clade AClade BFig. 2 One of three mostparsimonious trees resultingfrom a heuristic search of 40aligned baculovirus p74 DNAsequences using PAUP 4.0b4a[18]. The tree was rooted usingthe sequence from CuniNPV,and the branch support is givenas a percentage of 1,000bootstrap pseudoreplicates. Thecircle indicates the CoveMNPVbaculovirus474 Virus Genes (2011) 43:471–475123The Group I Alphabaculovirus presented a topology thatfavors its division into two subgroups: Clade A and CladeB, with the CoveMNPV baculovirus appearing in Clade A.The Group II Alphabaculovirus possessed a largely linearhierarchy in the tree, where many internal branches pos-sessed poor bootstrap support. Compared with Group I,Group II has a greater and more ancient diversificationamong the species from their common ancestor [19].Herniou et al. [20] found that the most topologicalvariations of the family Baculoviridae were within the GVand NPV Group II. These variations may be because somerepresentatives of these groups have very large genomes,creating an imbalance when phylogenetic analyses arebased on gene content and gene order. Another explana-tion, discussed in their study, suggests that some specieswere very similar and others, more divergent, thus com-plicating the choice of appropriate markers to establishtheir phylogenetic relationships.According to the topology shown for the cladesbelonging to Group I, Clade A, and Clade B, CoveMNPVis the most closely related to CfDEFNPV (Choristoneurafumiferana defective NPV), but is also closely related toAgMNPV (Anticarsia gemmatalis MNPV), both belongingto Clade A. These relationships were supported by highbootstrap values. The phylogenetic data reported in thisstudy indicate that the CoveMNPV baculovirus belongs toGroup I, which was previously classified only in terms ofgenus.The p74 sequence is the first gene of the baculovirusCoveMNPV to be described until now, and provides a firstapproach for CoveMNPV phylogeny. Our findings are inagreement with the current classification and phylogeneticrelationships within the family, Baculoviridae [2] andprovide new insights on CoveMNPV taxonomy reinforcingthe view that this virus is a distinct baculovirus species.Acknowledgments The authors thank Dr. Filipe R. da Silva(EMBRAPA/CENARGEN) for his comments and suggestions. To Dr.Nilton J. Sousa (Universidade Federal do Paraná, Curitiba-PR) andEdilene B. Machado (Swedish Match do Brasil S.A., Curitiba-PR) forproviding the CoveMNPV infected larvae. The authors also thank thePlataforma de Sequenciamento de DNA of Embrapa Recursos Gen-éticos e Biotecnologia (CENARGEN), especially to Luciana B.D.Labuto. This study was supported by EMBRAPA/CENARGEN, andGeraldo F. Almeida was supported by an MSc fellowship fromCAPES-Coordenação de Aperfeiçoamento de Pessoal de Nı́velSuperior.References1. D.A. Theilmann, G.W. Blissard, B. Bonning, J.A. Jehle, D.R.O’Reilly, G.F. Rohrmann, S. Thiem, J.M. 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Identification and sequence analysis of the Condylorrhiza vestigialis MNPV p74 gene.

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Identification and sequence analysis of the Condylorrhiza vestigialis MNPV p74 gene.

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