Sequence and Organization of the Neodiprion lecontei

JOURNAL OF VIROLOGY, July 2004, p. 7023–7035 Vol. 78, No. 130022-538X/04/$08.00�0 DOI: 10.1128/JVI.78.13.7023–7035.2004Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Sequence and Organization of the Neodiprion leconteiNucleopolyhedrovirus Genome

Hilary A. M. Lauzon,1 Christopher J. Lucarotti,2 Peter J. Krell,3Qili Feng,1 Arthur Retnakaran,1 and Basil M. Arif1*

Canadian Forest Service, Great Lakes Forestry Centre, Sault Ste. Marie, Ontario, Canada P6A 2E51;Canadian Forest Service, Atlantic Forestry Centre, Fredericton, New Brunswick,

Canada E3B 5P72; and Department of Microbiology, University of Guelph,Guelph, Ontario, Canada N1G 2W13

Received 15 October 2003 /Accepted 3 February 2004

All fully sequenced baculovirus genomes, with the exception of the dipteran Culex nigripalpus nucleopoly-hedrovirus (CuniNPV), have previously been from Lepidoptera. This study reports the sequencing and char-acterization of a hymenopteran baculovirus, Neodiprion lecontei nucleopolyhedrovirus (NeleNPV), from the red-headed pine sawfly. NeleNPV has the smallest genome so far published (81,755 bp) and has a GC content ofonly 33.3%. It contains 89 potential open reading frames, 43 with baculovirus homologues, 6 identified byconserved domains, and 1 with homology to a densovirus structural protein. Average amino acid identity ofhomologues ranged from 19.7% with CuniNPV to 24.9% with Spodoptera exigua nucleopolyhedrovirus. Theconserved set of baculovirus genes has dropped to 29, since NeleNPV lacks an F protein homologue (ac23/ld130). NeleNPV contains 12 conserved lepidopteran baculovirus genes, including that for DNA bindingprotein, late expression factor 11 (lef-11), polyhedrin, occlusion derived virus envelope protein-18 (odv-e18), p40,and p45, but lacks 21 others, including lef-3, me53, immediate early gene-1, lef-6, pp31, odv-e66, few polyhedra 25k,odv-e25, protein kinase-1, fibroblast growth factor, and ubiquitin. The lack of identified baculovirus homologuesmay be due to difficulties in identification, differences in host-virus interactions, or other genes performingsimilar functions. Gene parity plots showed limited colinearity of NeleNPV with other baculoviruses, andphylogenetic analysis indicates that NeleNPV may have existed before the lepidopteran nucleopolyhedrovirusand granulovirus divergence. The creation of two new Baculoviridae genera to fit hymenopteran and dipteranbaculoviruses may be necessary.

Several lepidopteran baculovirus genomes and one dipteranbaculovirus genome have been fully sequenced, but none so farhave been reported from Hymenoptera. Hymenopterans areancient insects that have existed since the early to mid-Meso-zoic era (41, 54). Sawflies (Symphyta) are primitive hymeno-pterans (22) that have existed since the Triassic period (206 to248) � 106 years ago (54). Baculoviruses infecting the Hyme-noptera likely represent more ancient viruses than those in-fecting Lepidoptera, which first appeared in the Cretaceousperiod (65 to 144) � 106 years ago during the late Mesozoic toCenozoic eras (41, 54). Analysis of the polyhedrin gene fromvarious baculoviruses, including that from Neodiprion sertifer(NeseNPV), suggested that hymenopteran nucleopolyhedro-viruses (NPVs) may have diverged from the lepidopteran bacu-loviruses before the separation of the lepidopteran NPVs andthe granuloviruses (GVs) (55, 71). With the radiation of bothLepidoptera and Hymenoptera, their respective baculovirusesmay have undergone host-dependent evolution with their hosts(55, 71).

This study was undertaken to sequence and characterize thegenome of the hymenopteran nucleopolyhedrovirus NeleNPV,from the redheaded pine sawfly, Neodiprion lecontei. This in-sect is a pest of young, natural pine stands, plantations, andgreenhouse cultures and may cause complete defoliation and

death of small trees, reduced growth, branch mortality and treedeformity (18). NeleNPV was first identified from the red-headed pine sawfly in Ontario in 1950 and proved to be highlyinfectious (10). Sawfly NPVs replicate in the nuclei of midgutepithelium cells, causing infectious diarrhea and sloughing offof infected cells during the advanced stage of the disease. Thegregarious nature of sawflies leads to the rapid spread of thevirus through a population, with insects dying 4 to 7 days afterinfection (22). NeleNPV is available as a registered productcalled Lecontvirus and is an effective control agent for N.lecontei infestations (19). NeleNPV is now one of the first twofully sequenced hymenopteran baculoviruses, the second beingNeseNPV (24).

Baculoviruses are divided into NPVs or GVs based on theirocclusion body (OB) formation. NPVs are found mainly inlepidopterans but have been identified in other insect orders,including Hymenoptera, Diptera, Coleoptera, Thysanura,and Trichoptera, and contain multiple virions with eithersingle or multiple nucleocapsids (22, 66). All hymenopteranNPVs contain single nucleocapsids (22). GVs are occludedwithin granulin, each OB contains a single virion, and GVshave been found only in lepidopterans (22).

Baculoviruses contain a set of conserved genes that are in-volved in essential functions, such as viral replication, transac-tivation, production of structural proteins, assembly, and re-lease of progeny viruses. The evolution of large genomes,particularly in lepidopteran NPVs, has reduced viral depen-dency on the host cell machinery and led to an increased

* Corresponding author. Mailing address: Canadian Forest Service,Great Lakes Forestry Centre, 1219 Queen St. E., Sault Ste. Marie,Ontario, Canada P6A 2E5. E-mail: [email protected].

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number of auxiliary genes that are not essential but provide aselective advantage (49). To date, the complete genomes of 23baculoviruses are available in GenBank. As the number of se-quenced genomes has increased, the number of conservedgenes has decreased. Previous reports list 62 to 67 conservedbaculovirus genes (13, 31, 32, 36). With the publication of theCulex nigripalpus nucleopolyhedrovirus (CuniNPV) genome,that number decreased to 30 genes conserved in 13 baculovi-ruses (2, 33). The number of conserved baculovirus genesremained at 30 with the sequencing of Mamestra configurataNPV isolate 90/2 (MacoNPV A) (44), M. configurata NPV-96B(MacoNPV B) (43), Phthorimaea operculella GV (PhopGV)(GenBank accession number AF499596), Rachiplusia oumultiple NPV (RoMNPV) (27), Adoxophyes honmai NPV(AdhoNPV) (GenBank accession number AP006270), Ad-oxophyes orana granulovirus (AdorGV) (69), Choristoneurafumiferana MNPV (GenBank accession number NC_004778),Cryptophlebia leucotreta GV (CrleGV) (42), Helicoverpa ar-migera NPV (GenBank accession number NC_003094), and C.fumiferana defective NPV (GenBank accession numberAY327402).

Our early work on NeleNPV estimated its size to be in theorder of 82,000 bp based on restriction enzyme digestion, mak-ing it the smallest known baculovirus genome. We hypothe-sized that its small genome would consist mainly of essentialbaculovirus genes, that it would contain a smaller core ofconserved genes, and that its sequence would provide usefulinformation on the evolution of baculoviruses. Here we reporton the complete sequence and gene organization of NeleNPVand compare it with other baculovirus genomes.

MATERIALS AND METHODS

Virus production and DNA preparation. N. lecontei larvae infected withNeleNPV were collected between 1975 and 1980 from areas throughout Ontarioor from Christian Island in southern Georgian Bay in 1995. Insects were freeze-dried, ground to a fine powder, lyophilized, and stored at 4°C as previouslydescribed (18). The lyophilized powder was diluted in 0.5% sodium dodecylsulfate (final concentration), stirred overnight, filtered, and centrifuged (2,500 �g, 30 min). The OB-containing pellets were washed with double-distilled H2O,centrifuged three times (2,500 � g, 30 min), and then passed through two 60, 50,10% discontinuous sucrose gradients (40,000 � g, 90 min, 20°C). The OBsbetween the 50 and 60% sucrose cushions were removed, passed through 10 to45% continuous sucrose gradients (40,000 � g, 90 min, 4°C), diluted with Tris-EDTA (TE), and centrifuged (87,000 � g, 90 min). The pellets were resuspendedin 0.5 ml of TE, and DNA was extracted from the purified OBs using the Qiagengenomic tip 20/G DNA extraction kit using 1 ml of general lysis buffer (G2)supplemented with 100 �l of proteinase K (20 mg/ml) with overnight incubation(50°C). The standard Qiagen protocol was then followed. Yields of virus werelow, since NeleNPV infection is restricted to the midgut, nucleocapsids containonly a single virion, and the freeze-dried samples were heavily contaminated,with insect debris trapping much of the virus. Purified DNA was checked byrestriction analysis on agarose gels for purity and concentration.

DNA cloning and sequencing. Viral DNA was sheared into small fragments bynebulization, cloned, and sequenced by Qiagen Sequencing Services using acombination of shotgun cloning, primer walking, and PCR to generate gap-spanning fragments, for an average 12� coverage. An ABI PRISM 377XL orABI PRISM 3700 sequencer was used with Qiagen modified ABI sequencingchemistries and Qiagen purified DNA. The sequence data were manually editedand automatically assembled by using LASERGENE’s DNAStar Seqman pro-gram, version 4.06, for sequence assembly and contig management. Some poly-morphisms were found and were attributed to the virus being from field isolates.These ambiguities have been left and given standard codes as determined byprogram analysis (M � A,C; R � A,G; W � A,T; S � C,G; Y � C,T; K � G,T;V � A,C,G; H � A,C,T; D � A,G,T; B � C,G,T; N � A,C,G,T).

Sequence analysis. DNA sequence data were analyzed using DNAStarLASERGENE programs, version 4.06 or 5.05, and MacVector sequence analysissoftware, version 4.1.4. Open reading frames (ORFs) encoding more than 50amino acids (aa) with minimal overlap were accepted as putative genes; other-wise, the largest ORF was selected. Sequence data were submitted to GenBank,and database searches were performed using the National Center for Biotech-nology Information (NCBI) ORF Finder (http://www.ncbi.nlm.nih.gov/gorf/gorf.html) and BLAST (4). If difficulties were encountered identifying homologueswith standard protein-protein BLAST (blastp) searches, further analysis wasdone using blastp for short, almost exact matches, PSI BLAST (5) using E valuesof 10 and 0.01 for iterations, analysis using the Simple Modular ArchitectureResearch Tool (SMART), version 3.4 (58, 59), and Smith Waterman similaritysearches using DeCypher with default settings (coe02.ucalgary.ca). An ORF wasconsidered clearly identified if any of the following criteria were met. (i) A blastpsearch showed a baculovirus match with an E value of 0.1 or less. (ii) Amino acididentity to a baculovirus homologue was 20% or greater based on DNAStarMegalign ClustalW analysis of complete ORFs. (iii) A conserved domain wasfound. (iv) Matches were close but did not meet the first three criteria, but ORFsshowed a significant match to clearly identified ORFs in NeseNPV as determinedby A Garcia-Maruniak, et al. (24). The sequence was analyzed for repeats usingMacVector’s Pustell DNA matrix, Emboss Palindrome (23), and TandemRepeats Finder (9). Multiple alignments were performed using DNAStar’sMegalign ClustalW alignment, and percent amino acid identity indicates thepercentage of identical residues between complete ORFs. Phylogenetic treeswere constructed using ClustalW protein alignments and PAUP 4.0b10 (62),using maximum parsimony analysis with heuristic search and stepwise additionoptions, and were confirmed using bootstrap analysis with heuristic search and1,000 replicates. Gene parity plots were performed on the NeleNPV genomeversus the genomes of Autographa californica MNPV (AcMNPV) (7), Helico-verpa armigera single-nucleocapsid NPV (SNPV) (HaSNPV) (13), CuniNPV (2),and Plutella xylostella GV (PxGV) (28), using established methods (13, 34, 37).

Nucleotide sequence accession number. The NeleNPV genome sequence hasbeen deposited in GenBank under accession number AY349019.

RESULTS AND DISCUSSION

Nucleotide sequence analysis. The NeleNPV genome was81,755 bp in size, making it the smallest baculovirus genome sofar known, with others ranging from 99,657 bp for AdorGV(69) to 178,733 bp for Xestia c-nigrum GV (XcGV) (30). TheG�C content was 33.3%, with the lowest so far publishedbeing that of CrleGV at 32.4% (42) and the highest being57.5% for Lymantria dispar MNPV (LdMNPV) (40) (Table 1).

TABLE 1. Comparison of baculovirus genomes

Size(bp)

%G�C

No. ofORFs

No. ofhr’sa

No. ofbrob

genes

No. ofhomo-

logues inNeleNPVc

% Overallaa id withNeleNPVd

NeleNPV 81,755 33.3 89 0 0AcMNPV 133,894 40.7 154 8 (9) 1 41 23.2OpMNPV 131,993 55.1 152 5 3 42 23.0BmNPV 128,413 40.4 136 7 5 42 23.4LdMNPV 161,046 57.5 163 13 16 42 23.7SeMNPV 135,611 44.0 139 6 0 (1) 42 24.9EppoMNPV 118,584 41.0 135 5 1 42 24.0HaSNPV 131,403 39.1 135 5 3 42 24.7SpltMNPV 139,342 42.7 141 17 2 42 23.7CuniNPV 108,252 50.9 109 4 6 29 19.7XcGV 178,733 41.0 181 9 7 42 22.2PxGV 100,999 40.7 120 4 0 42 23.6CpGV 123,500 45.2 143 0 1 42 23.1AdorGV 99,657 34.5 119 0 0 42 23.1CrleGV 110,907 32.4 129 3 0 42 23.2

a Eight hr’s reported for AcMNPV (7), nine hr’s reported (53).b No bro genes reported in SeMNPV (37), one bro gene reported (13).c Only ORFs meeting criteria for clear identification included, ac27 iap not

included.d Calculated using ClustalW results for complete ORFs; average based on

shared ORFs. aa id, amino acid identity.

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The genome contained 89 potential ORFs, accounting for88.8% of the total sequence. Identifying homologues was dif-ficult due to the low similarity of the NeleNPV genome toother baculovirus genomes. Only 50 ORFs met our criteria forclear identification with 43 identified as baculovirus homo-logues, 6 recognized by the presence of conserved domains,and 1 as a potential match with a structural protein fromdensoviruses (Fig. 1; Table 2).

Repeat regions. Nine direct repeat regions were found inNeleNPV, but they showed little similarity to typical NPVhomologous regions (hr’s). NPV hr’s are characterized by thepresence of multiple tandemly repeated perfect or imperfectpalindrome sequences within a direct repeat and have beenimplicated as origins of DNA replication (39) and as en-hancers of transcription (26). Although NPV hr’s typicallyshow similarity to each other, differences have also beennoted in CuniNPV (2), HaSNPV (13), and Spodoptera lituraMNPV (SpltMNPV) (50).

NeleNPV direct repeat regions were up to 77% AT rich andcontained two or three copies of direct repeat sequences, rang-ing in size from 29 to 160 bp, located either in tandem orseparated by 30 to 439 bp (Fig. 2). Direct repeats within each

region were not the same as those in other regions, with theclosest match being 55.2% nucleotide identity between those inregions 3 and 5. Part of direct repeat 4 (31 bp), however, wasrepeated four times in nl2 and three times in nl35. Repeat 1,located between ORFs 7 and 8, contained two 43-bp directrepeats (97.7% nucleotide identity) separated by 28 bp. Repeat2, between ORFs 25 and 26, contained two 53-bp direct tan-dem repeats (100% nucleotide identity). Repeat 3, betweenORFs 38 and 39, had two 29-bp direct tandem repeats (93.1%nucleotide identity), and repeat 4, between ORFs 76 and 77,contained three 46-bp direct repeats separated by 45 bp (84.8to 80.0% nucleotide identity). Repeat 5 and 6 were both be-tween ORFs 78 and 79, with repeat 5 having three 67-bp directtandem repeats (88.1 to 90.8% nucleotide identity) and repeat6 having two 45-bp direct tandem repeats (93.3% nucleotideidentity) with one partially overlapping nl79. Repeat 7 had two30-bp direct repeats (80% nucleotide identity), and repeat 8had two 160-bp tandem direct repeats (95.6% nucleotide iden-tity), with the left direct repeat of region 7 overlapping nl85and separated from the right direct repeat by 439 bp. Bothtandem repeats in region 8 overlapped nl86. Repeat 9, between

FIG. 1. Linear map of the NeleNPV genome. Arrows indicate the position and direction of transcription for potential ORFs, with polyhedrin(ph) shown as ORF 1. NeleNPV ORF numbers and potential gene names are shown below the arrows, and AcMNPV ORF numbers (7) are shownabove the arrows.

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TABLE 2. Characteristics of NeleNPV ORFse

ORFno. Name Position Size

(aa)

AcMNPVORF no.

(% aa id)a

HaSNPVORF no.(% aa id)

CuniNPVORF no.(% aa id)

PxGVORF no.(% aa id)

Comment(s)b

1 Polyhedrin 1>744 247 8 (46.7) 1 (45.9) X 4 (36.7) NeabNPV polyhedrin, e � e � 143, aa id 98%2 961�1287 108 Internal repeats3 1680�1922 804 1843�2841 3325 3049�3207 526 3671�4450 259 Trypsin-like serine protease, AAD21829 C. felis

CfSP-2, e � 8e � 46, aa id 38.7%7 4663�5058 131 Internal repeat

Direct repeat 1 5152–5194 2 43-bp direct repeats5223–5265

8 5279�6859 526 Internal repeat9 7567�9951 79410 10181�10345 5411 iap-3 10334<11116 260 X 103 (23.8) X 98 (17.6) Sf iap AF186378, e � 3e � 33, aa id 28.4%12 11194�11436 8013 11733�12476 247 Transmembrane domain, coiled coil14 dbp 12565<13275 236 25 (18.6) 25 (17.3) X 61 (14.3) HaSNPV ORF25 dbp, e � 0.94, aa id 17.3%c

15 lef-11 13256<13561 101 37 (16.7) 32 (23.5) X 46 (12.4) LsNPV lef 11, e � 0.014, aa id 23.5%16 13575<14345 256 92 (21.4) 80 (23.1) 14 (15.6) 76 (20.3) MacoNPV B ORF 92, e � 2e �22, aa id 22.5%17 14347>14859 170 93 (14.8) 81 (18.4) X 75 (15.9) LsNPV ORF93, e � 0.037, aa id 14.0%18 14868�15518 216 Signal peptide19 15584�15775 63 Transmembrane domain20 dna pol 15806<18577 923 65 (25.8) 67 (27.3) 91 (15) 93 (23.7) MacoNPV B ORF114 DNA polymerase, e � 2e � 61,

aa id 28.1%21 18576�20762 728 2 coiled coils22 20927�22069 38023 odv-e56 22321>23331 336 148 (32.6) 15 (38) 102 (20.8) 16 (37.7) PhopGV ORF 16 odv-e56, e � 1e � 54, aa id 37.7%24 23386�23964 19225 24120�24905 261

Direct repeat 2 24924–25029 2 53-bp direct tandem repeats26 25183�25863 22627 26082�26654 19028 p6.9 26772<27080 102 100 (30.4) 88 (23.3) 23 (34.8) 67 (40.4) AdorGV ORF 72 p6.9, aa id 64.3%d

29 p40 27110<28210 366 101 (9.4) 89 (10.4) X 66 (11.4) XcGV ORF 93 p40, aa id 13.9%30 28231�28581 11631 p48 28578<29750 390 103 (11.1) 91 (14.8) X 63 (12.7) MacoNPV A ORF 83 p48, e � 0.026, aa id 15.1%32 29769<30485 238 106/107 (8.1) 101 (18.4) X 40 (17.9) LdMNPV ORF140, e � 6e � 10, aa id 16.7%33 alk-exo 30588>31796 402 133 (24.3) 114 (19.6) 54 (16.8) 106 (23.0) PxGV ORF106 alk exo, e � 3e � 25, aa id 23.0%34 31793�32266 157 Transmembrane domain, coiled coil35 32364�32657 97 Internal repeats36 32787�33746 31937 lef-9 33771<35282 503 62 (31.5) 55 (33.5) 59 (17.1) 99 (32.3) PhopGV ORF 109 lef-9, e � 2e � 75, aa id 34.1%38 35308<35733 141 68 (15.5) 64 (21.6) 58 (18.1) 96 (17.8) SpltNPV ORF66, e � 2e � 04, aa id 21.8%, signal

peptide, transmembrane domainDirect repeat 3 35829–35886 2 29-bp direct tandem repeats

39 35984�37045 35340 37042�37419 12541 37416<37646 76 76 (18.2) 70 (19.5) X 91 (22.1) SeNPV ORF95, e � 1.4, aa id 24.7%d

42 vlf-1 37649<38713 354 77 (22.8) 71 (28.5) 18 (17.5) 89 (28.8) SpltNPV ORF74, vlf-1, e � 1e � 41, aa id 29.6%43 38682<38966 94 78 (14.7) 72 (20.0) X 88 (13.5) AdorGV ORF 90, aa id 20.9%d

44 gp41 38987<39799 270 80 (26.9) 73 (30.6) 33 (7.4) 87 (20.3) HaSNPV ORF 73 gp41, e � 2e � 1945 39821>40348 175 81 (34.1) 74 (35.2) 106 (17.0) 86 (34.7) LdMNPV ORF 89, e � 5e � 23, aa id 34.1%46 p47 40601<41770 389 40 (21.3) 35 (19.2) 73 (8.5) 51 (21.7) CpGV ORF68 p47, e � 9e � 25, aa id 22.8%47 p74 41772<43673 633 138 (38.3) 20 (36.4) 74 (34.4) 49 (32.8) RoMNPV ORF138 p74, e � e � 124, aa id 39.0%48 43651�44187 17849 44247>44903 218 Dm NP_609448, e � 1e � 13, aa id 17.4%, 4 C2H2

zinc fingers50 45012>46235 407 NeseNPV AF121349, e � 2e � 29, aa id 45.7%,

internal repeat51 46237�46815 19252 pif-2 46834>47988 384 22 (44.6) 132 (43.8) 38 (43.9) 37 (47.2) NeseNPV AF121349, e � e � 152, aa id 74.3%53 48058�48372 10454 lef-2 48374>48961 195 6 (13.8) 117 (16.3) 25 (14.8) 32 (11.2) NeseNPV AF121349 lef-2, e � 7e � 27, aa id 35.7%55 lef-5 48989<49696 235 99 (25.4) 87 (25.8) 88 (9.3) 69 (28.4) NeseNPV AF121349 lef-5, e � 2e � 66, aa id 58%56 38k 49687>50604 285 98 (30.1) 86 (27.3) 87 (25.5) 70 (30.4) OpMNPV ORF99 38k, e � 8e � 27, aa id 26.2%57 50590<51096 168 96 (25.4) 85 (27.2) 90 (26) 71 (23.5) CuniNPV ORF 90, e � 3e � 16, aa id 26.0%58 helicase 51083>54487 1134 95 (16.8) 84 (20.4) 89 (11.9) 72 (17.8) PxGV ORF72 helicase, e � 7e � 4559 lef-4 54484<55890 468 90 (20.9) 79 (22.1) 96 (16) 78 (25.9) PxGV ORF78 lef-4, e � 2e � 2860 p49 55904>57232 442 142 (20.5) 9 (22.1) 30 (12.4) 14 (16.9) HaSNPV ORF 9 p49, e � 1e � 2261 57244�57480 78 transmembrane domain62 odv-e18 57503>57760 85 143 (14.3) 10 (14.6) X 13 (24.4) XcGV ORF 12 odv-e18, aa id 27.4%d

63 odv-ec27 57775>58563 262 144 (21.3) 11 (18.3) 32 (9.1) 80 (16.0) BmNPV ORF120 odv-e27, e � 1e � 05, aa id 21.7%

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ORFs 87 and 88, had two 43-bp direct repeats (100% nucleo-tide identity) located 46 bp apart (Fig. 2).

Several ORFs (nl7, nl8, nl35, nl49, nl50, and nl87) containedinternal repeats. Interestingly, nl7 and nl8 bordered repeat 1,and nl87 was between repeats 8 and 9. None of the ORFs withinternal repeats showed homology to baculovirus repeat ORFs(bro’s); however, similarities may exist that were at too low alevel to identify. The ORFs containing internal repeats andfound near repeat regions may not be functional but may bepart of the repeat regions. Ambiguous bases did not appear tobe randomly distributed; instead, repeat regions contained ahigher number, with 12 of 162 or 7.4% of ambiguous basesbeing found in the direct repeats, while these regions ac-counted for only 1.4% of the genome.

Perfect and imperfect palindromes ranging in size from 11 to40 bp were found in several of the repeat regions but were notnecessarily embedded within the direct repeats. Those foundwithin the direct repeats are shown in Fig. 2. The palindromes

showed limited similarity to each other and no similarity topalindromes in other baculovirus hr’s.

Repeat regions in GVs show more variability than those inNPVs, and most do not have a palindromic core (69). XcGVrepeat regions are AT rich and contain direct imperfect re-peats that are highly variable between each region and withineach region (30), similar to those in NeleNPV. Little sequencesimilarity, however, exists between the NeleNPV and XcGVrepeat regions. Typical NPV hr sequences are also not found inCydia pomonella GV (CpGV) (46); instead, one major repeatregion and 13 copies of a single repeated imperfect palindromeare found, with six of the repeats found within potential ORFs(46). Similarly, NeleNPV has a longer repeat region (repeat 8),and some of the NeleNPV direct repeats and palindromesoverlap or are within ORFs, but none exhibit sequence simi-larity to CpGV repeats.

It would appear that NeleNPV repeat regions show littlesequence similarity to baculovirus hr’s but that they share a

TABLE 2—Continued

ORFno. Name Position Size

(aa)

AcMNPVORF no.

(% aa id)a

HaSNPVORF no.(% aa id)

CuniNPVORF no.(% aa id)

PxGVORF no.(% aa id)

Comment(s)b

64 58590>58922 110 145 (24.4) 12 (23.7) X 12 (23.2) CpGV ORF9, e � 2e � 07, aa id 30.4%65 lef-1 58925<59560 211 14 (19.8) 124 (29.2) 45 (28.8) 55 (29.2) PhopGV ORF 66 lef-1, e � 9e � 16, aa id 30.7%66 59622>60203 193 115 (26.3) 98 (30.4) 46 (25.3) 29 (28.0) SpltNPV ORF107, e � 1e � 12, aa id 25.8%67 60357>61421 354 109 (20.0) 54 (18.0) 69 (16.1) 43 (17.7) MacoNPV A ORF 80, e � 1e � 13, aa id 22.5%68 61421�61630 69 Signal peptide69 61617>62027 136 RCC1/BLIP-II70 61993>62394 133 RCC1/BLIP-II71 62479>62778 99 RCC1/BLIP-II72 63034�63885 28373 63835�64128 97 Transmembrane domain74 64127�64924 26575 65298�65927 20976 pif 65909<67501 530 119 (26.9) 111 (28.5) 29 (28.2) 7 (28.1) AcMNPV ORF119 pif, e � 2e � 59

Direct repeat 4 67561–67606 3 46-bp direct repeats67652–6769767742–67787

77 67897<68346 149 53 (13.6) 43 (18.2) X 112 (11.6) CpGV ORF 134, e � 2.4 aa id 15.7%c

78 lef-8 68351>70882 843 50 (29.7) 38 (30.6) 26 (19.7) 109 (32.1) PhopGV ORF 121 lef-8, e � e � 113, aa id 33.9%Direct repeat 5 71364–71562 3 67-bp direct tandem repeatsDirect repeat 6 72017–72105 2 45-bp direct tandem repeats

79 72085�72279 6480 72547�72783 78 2 transmembrane domains81 72879>73325 148 Casphalia extranea densovirus NP_694840, e � 1e �

11, aa id 30.2%82 vp91 capsid 73334>75745 803 83 (22.8) 76 (22.9) 35 (21.6) 84 (23.4) HaSNPV ORF77 vp91 capsid, e � 3e � 5683 vp1054 75747<76688 313 54 (20.4) 47 (17.5) 8 (15.0) 115 (15.4) LdMNPV ORF57 vp1054, e � 2e � 09, aa id 19.1%84 76813�77022 6985 77030�77947 305

Direct repeat 7 77914–77943 2 30-bp repeats, one within nl8578383–78412

Direct repeat 8 78002–78161 2 160-bp direct tandem repeats overlapping nl8678162–78321

86 78101�78361 86 Signal peptide, two transmembrane domains87 78468�79898 476 Internal repeat

Direct repeat 9 79967–80009 2 43-bp direct repeats80056–80098

88 vp39 capsid 80133<81080 315 89 (18.7) 78 (17.7) 24 (11.0) 79 (14.2) SpltNPV ORF81 p39, e � 7e � 20, aa id 20.5%89 81089>81640 183 Phosphotransferase

Avg (23.2) Avg (24.7) Avg (19.7) Avg (23.6)

a % Amino acid identity (aa id) calculated for complete ORFs using ClustalW.b Top NCBI blastp 2.2.5 or SMART WU-BLAST 2.0 match with e-value and % aa id. If top % aa id is listed in columns 5 to 8, %, it is not repeated. In some instances

top blastp match and ORF with highest % aa id are not the same; if no baculovirus homologue was clearly identified, domains and features as identified with SMARTor NCBI blast conserved domain database are given.

c Accepted based on NeleNPV results.d Accepted based on aa id of �20%.e ORFS in bold are considered clearly identified using criteria given in Materials and Methods. � and � indicate direction of transcription. e values are given as

follows: e � 8e � 46, e � 8 � 10�46.


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closer similarity to the organization of repeat regions in GVsthan to those in NPVs. The lack of typical baculovirus hr’s inNeleNPV may reflect a different genome replication strategyfor the virus or its replication in a nonlepidopteran host.

Conserved ORFs. The number of conserved core baculovi-rus genes has decreased from 30 (2, 33) to 29, since an Fprotein homologue (ac23/ld130) was not found in NeleNPV.Of the 62 conserved lepidopteran baculovirus genes (32), only41 were clearly identified in NeleNPV. Three of these, nl14(ac25/dna binding protein [dbp]), nl29 (ac101/p40), and nl77(ac53) met our identification criteria using earlier blastp ver-sions but did not with blastp 2.2.5. They have been accepted,however, since they were strong homologues of ORFs foundindependently in NeseNPV that have been identified as bacu-lovirus homologues (24). NeleNPV contained 12 genes con-served in the lepidopteran baculoviruses but not found in thedipteran virus CuniNPV, including homologues to ac8 (poly-hedrin), ac25 (dbp), ac37 (lef-11), ac53, ac76, ac78, ac93, ac101(p40), ac103 (p45), ac106, ac143 (odv-e18), and ac145. Con-served lepidopteran baculovirus genes not clearly identified in

NeleNPV include homologues to ac10 (protein kinase 1 [pk1]),ac13, ac23, ac28 (lef-6), ac29, ac32 (fibroblast growth factor[fgf]), ac35 (ubiquitin), ac36 (pp31), ac38, ac46 (odv-e66), ac61(few polyhedrin 25K), ac66, ac67 (lef-3), ac75, ac82, ac94 (odv-e25), ac102, ac110, ac139 (me35), ac146, and ac147 (immediateearly gene-1 [ie-1]) (Table 3).

Replication genes. All the previously considered conservedbaculovirus replication genes, lef-2 (nl54/ac6), lef-1 (nl65/ac14),DNA-polymerase (nl20/ac65), and helicase (nl58/ac95), werefound in NeleNPV. The conserved lepidopteran baculovirusreplication gene dbp (nl14/ac25) was present, but lef-3 (ac67),me35 (ac139), and ie-1 (ac147) were absent. nl25 showed a verylow blastp match to lef-3 but was not close enough to meet ourcriteria. NeleNPV was also missing several variable DNA rep-lication genes or those involved in replication in some but notall baculoviruses, including proliferating cell nuclear antigen(ac49), lef-7 (ac125), p35 (ac135) (45), ie-2 (ac151), and pe38(ac153) (Table 4), as well as helicase-2, dna-ligase, RNase reduc-tase-1 (op32), RNase reductase-2 (op34), and dutpase (op31)(31).

FIG. 2. Alignment of NeleNPV direct repeat sequences. Direct repeat regions are numbered according to their order in the genome. Therepeat sequences are aligned to obtain maximum similarity, and their locations within the genome are indicated. Sequences within repeat 4 thatare found within nl2 and nl36 are bolded and underlined. Arrows indicate palindromes.


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Transcription-specific genes. All conserved transcription-specific genes, lef-5 (nl55/ac99), lef-4 (nl59/ac90), lef-8 (nl78/ac50), lef-9 (nl37/ac62), very late factor-1 (nl42/ac77), and p47(nl46/ac40) were found. Lef-11 (nl15/ac37) was present but isno longer considered a conserved baculovirus gene, since it isabsent from CuniNPV. Lef-6 (ac28) and pp31/39k (ac36) wereabsent, as in CuniNPV. Variable transcription-specific genes,including ac36, lef-12 (ac41), and lef-10 (ac53a), were notfound (Table 4).

Structural protein genes. Conserved baculovirus structuralgenes identified include odv-e56 (nl23/ac148), p6.9 (nl28/ac100),gp41 (nl44/ac80), p74 (nl47/ac138), odv-ec27 (nl63/ac144),vp91/p95 capsid (nl82/ac83), vp1054 capsid-associated protein(nl83/ac54), and vp39 capsid (nl88/ac89). Polyhedrin and odv-e18, present in all sequenced lepidopteran baculoviruses butnot in CuniNPV, were also found (Table 3).

Neither a GP64/67 homologue nor an F protein homologue(ac23/ld130) was found in NeleNPV. Membrane fusion pro-teins mediate the fusion of budded virus to cell membranesand the release of nucleocapsids (51). Previously, GP64/67homologues have been found in all group I NPVs, and Fprotein homologues (ac23/ld130) have been found in group IINPVs (56). Ld130 homologues are thought to be the primor-dial baculovirus envelope fusion protein (51). When blastpsearches failed to identify a GP64/67 or F protein (ac23/ld130)

homologue in NeleNPV, all unidentified ORFs were examinedmore thoroughly as listed in Materials and Methods.

F proteins are acid pH dependent and contain a predictedsignal peptide at the amino terminus, a transmembrane do-main near the carboxyl terminus, up to 11 conserved cysteines,and a furin cleavage site (40, 51, 56). nl38 (141 aa) and nl86 (86aa) contained signal peptides and transmembrane domains butwere much shorter than ld130 (676 aa) and lacked the con-served cysteines. Nl38 met our identification criteria as a ho-mologue to SpltMNPV ORF 66, further suggesting that it isnot an F protein homologue. nl18, nl62, and nl68 showed signalpeptides with possible overlapping transmembrane domains,but due to large size differences, the conserved cysteines weredifficult to assess. Nl87 contained five potential conserved cys-teines but did not have a signal peptide or transmembranedomain. Although ld130 homologues generally show low con-servation (56), the fact that we were unable to find matcheswith either BLAST searches, Smith Waterman searches, orsearches for conserved features suggests that NeleNPV may bethe first NPV found without an envelope fusion protein. Sim-ilar results have recently been found with another hymenopte-ran baculovirus, NeseNPV (24).

Analysis of AcMNPV exon0 (ac141) has shown that exon0 isexpressed as a late gene and all early transcripts from this generegion are spliced to form ie-0. exon0 knockouts produced with

TABLE 3. Conserved genes in baculovirus genomesd

Gene function Genes present in allbaculovirusesa

Genes present in alllepidopteran baculoviruses

and NeleNPV

Genes present in all lepidopteranbaculoviruses but not

in NeleNPV

Replication lef-2 (ac6), lef-1 (ac14), dnapol (ac65), heli-case (ac95)

dbp1 (ac25) lef-3 (ac67), me53 (ac139), ie-1 (ac147)

Transcription p47 (ac40), lef-8 (ac50), lef-9 (ac62), vlf-1(ac77), lef-4 (ac90), lef-5 (ac99)

lef-11 (ac37) lef-6 (ac28), pp31/39K (ac36)

Structural proteins vp1054 (ac54), gp41 (ac80), vp91/p95 (ac83),vp39 (ac89), p6.9 (ac100), p74 (ac138),odv-e27 (ac144), odv-e56 (ac148),

polh (ac8), odv-e18 (ac143) ac23/ld130b, odv-e66 (ac46), fp25K (ac61),odv-e25 (ac94), pk1 (ac10)

Per os infectivityfactorsc

pif (ac119)pif-2(ac22)

Auxiliary alk-exo (ac133) fgf (ac32), ubiquitin (ac35)Unknown ac68, ac81, ac92, ac96, 38K (ac98), ac109,

ac115, ac142ac53, ac76, ac78, ac93, p40 (ac101), ac106,

p45 (ac103), ac14538.7K (ac13), ac29, ac38, ac66, ac75,

ac82, p12 (ac102), ac110, ac146,

a Based on the genomes of 22 completely sequenced baculoviruses as listed in the text.b CuniNPV, a dipteran baculovirus, also has a potential ld130 homologue.c P74 is listed as a structural protein but is also necessary for per os infectivity.d Total number of genes present in all baculoviruses, 29; total number of genes present in all lepidopteran baculoviruses and NeleNPV, 12; total number of genes

present in all lepidopteran baculoviruses but not in NeleNPV, 21. Table 3 is modified with permission from the publisher of reference 33.

TABLE 4. Variable lepidopteran baculovirus genes found in AcMNPV but not in NeleNPV

Gene function Genes in AcMNPV but not NeleNPVa,b

Replication pcna (ac49), lef-7 (ac125), p35 (ac135), ie-2 (ac151), pe38 (ac153)Transcription 39k (ac36), lef-12 (ac41), lef-10 (ac53a)Structural ptp (ac1), orf1629 (ac9), p80/p87 capsid (ac104), gp67 (ac128), p24 (ac129), pp34-calyx (ac131), p10 (ac137)Auxiliary conotoxin (ac3), egt (ac15), iap-1 (ac27), sod (ac31), chitinase (ac126), vcath (ac127)Unknown ac4, ac5, ac7, ac11, ac12, ac16, ac17, ac18, ac19, ac26, ac30, ac33, ac34, ac39, ac43, ac44, ac45, ac51, ac52, ac55, ac56, ac57, ac58, ac59,

ac60, ac63, ac70, ac72, ac73, ac74, ac84, ac85, ac87, ac88, ac91, ac97, ac106, ac107, ac108, ac111, ac112, ac113, ac114, ac116, ac117,ac118, ac120, ac121, ac122, ac124, ac125, ac140, ac149, ac150, ac152, ac154

bro (ac2), arif-1 (ac20/21), pkip-1 (ac24), gta (ac42), ets (ac47), etm (ac48), gp37 (ac64), met (ac69), iap-2 (ac71), pnk/pnl (ac86), he65(ac105), pk2 (ac123), gp16 (ac130), p25 (ac132), p94 (ac134), p26 (ac136), exon0/ie0 (ac141)

a Variable genes are those found in some but not all baculovirus genomes.b Some missing genes may be present but due to low homology were not identified.


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AcMNPV BACmids have shown that exon0 is essential forbudded virus production (X. Dai and D. Theilmann, personalcommunication). The NeleNPV genome lacks exon0 and ie-0homologues. The absence of both ld130 and exon0 homologuesin NeleNPV suggests that the budded virus phenotype may notplay a role in the biology of hymenopteran NPVs or that otherunidentified proteins may be involved in budded virus produc-tion. It is also possible, however, that these homologues arepresent but their similarity was too low for clear identification.

Questions have been raised on how sawfly viruses might spreadfrom cell to cell, whether they would produce a budded virus orhave a GP64 homologue and, if not, what other mechanism theymight use for cell-to-cell transmission (22). The lack of an in-sect colony or a cell culture for NeleNPV has made the pres-ence or absence of budded virus in vivo difficult to determine.

Conserved lepidopteran structural genes pk-1 (ac10) (31, 33)and odv-e66 (ac46) were missing. Genes for two potential pro-teins, weakly matching protein kinases, were seen. Nl9 showeda low SMART match to a phosphotransferase with possible dualspecificity as a Ser/Thr/Tyr kinase (STYKc domain; SMARTaccession number SM0221). Smith Waterman searches withnl9 showed level 2 evidence for a cAMP- and cGMP-depen-dent protein kinase phosphorylation site (CAMP PHOSPHOsite) (PROSITE: PDOC00004) or a tyrosine kinase phosphor-ylation site (PROSITE: PDOC00007). By using a search forshort nearly exact matches, nl25 showed a low blastp match toa possible vaccinia virus protein kinase (AAA48288; e � 0.50),and Smith Waterman searches showed level 2 evidence for aCAMP PHOSPHO site. Compared to ac10 (pk1), however, nl9and nl25 showed very low amino acid identity and neither wasaccepted as a pk1 homologue. The presence of such a homo-logue, however, cannot be discounted at the present time.

Variable structural genes few polyhedra 25K (ac61) andodv-e25 (ac94), as well as protein tyrosine phosphatase (ptp)(ac1), orf1629 (ac9), p80/p87 capsid (ac104), gp64/67 (ac128),p24 (ac129), pp34 calyx (ac131), p10 (ac137), and enhancin (31)were not seen (Table 4).

P10 (ac137), considered either a structural protein (31) oran auxiliary protein (33, 49), was not clearly identified inNeleNPV. P10 is a very late protein that is generally poorlyconserved at the amino acid level, but its size, hydrophilicitydistribution, and secondary structure are conserved (72). Fourstructural domains implicated in various functions are usuallyfound, including a coiled-coil domain at the amino-terminalend, followed by a proline-rich domain, a variable region notfound in all P10s, and a positively charged carboxy terminusoften containing serine or threonine residues (63, 64, 65, 68).Known P10 proteins range in size from 70 aa for Bombyx moriNPV (BmNPV) (25) to 105 aa for SpltMNPV (50) and areusually located between p26 and p74 (65). Nl53 showed aweak blastp match with P10 from Buzura suppressaria NPV(BusuNPV) (34), showing 47% identity over 26 of 54 aminoacids, but amino acid identity over the entire ORF was muchlower. Nl53 had two potential start sites, giving a size of either104 or 80 amino acids, both within the expected P10 size range.A putative late baculovirus transcriptional start site, TAAG(12), was found at position �85 to �82 with respect to thetranslational start codon of the smaller transcript and �10 to�7 of the putative larger transcript. The conserved A at �3(65) was seen only with the larger transcript. Nl53 was located

near the p74 homologue (nl47) and a weak match to a poten-tial RNA binding protein (nl48) that could be related to p26.SMART analysis did not indicate a coiled-coil domain, but byusing MacVector’s combined Chou-Fasman and Robson-Gar-nier method for determining secondary structure, nl53 wasdetermined to have two areas of helix-forming residues. Onlyone proline was found in the expected proline-rich domain.Hydrophilicity profiles (Kyte-Doolittle scale) showed that nl53had a positively charged carboxy terminus and had a serinein this area, as expected for P10 proteins. With ClustalW,nl53 showed the highest amino acid identity to the Epiphyaspostvittana MNPV (36) P10, but this was only 13.8%. Aminoacid identity, however, is usually low for P10 proteins, andAcMNPV P10, for example, shares only 20.1% amino acididentity with SpltMNPV P10 (65). We cannot, therefore, ruleout the possibility that nl53 may be a P10 homologue.

Per os infectivity factors. To date, the products of threegenes have been identified as essential for per os infectivity ofbaculoviruses. These include structural protein P74 (ac138) (20),PIF (ac119) (38), and PIF-2 (ac22) (52), all of which have beenidentified in NeleNPV, as nl47, nl76, and nl52, respectively (Ta-ble 3). The presence of genes involved in per os infectivity in anancient virus such as NeleNPV strengthens the idea of thesegenes being essential (20, 38, 52) and that per os infectivity isa conserved process in baculoviruses. While early baculovirusesmay not have needed budded viruses, they could not have sur-vived without an efficient means of insect-to-insect transmission.

Auxiliary genes. Analysis of the NeleNPV genome has sup-ported the hypothesis that small baculovirus genomes wouldcarry few auxiliary genes (49), since most baculovirus genes inthe auxiliary class (2, 22, 31, 33) were not found in NeleNPV(Table 4). Only alkaline exonuclease (alk-exo) remained as aconserved baculovirus auxiliary gene (nl33/ac133). Conservedlepidopteran auxiliary genes, fgf (ac32) and ubiquitin (ac35),were not found (Table 3).

Inhibitors of apoptosis proteins. NeleNPV contained oneiap-like gene (nl11) but its top eight blastp matches were toinsect iap genes, including those from Spodoptera frugiperda(GenBank accession number AAF35285) (e � 3 � 10�33),Trichoplusia ni (accession number AAF19819) (e � 4 �10�32), Anopheles gambiae (accession number EAA04007) (e� 5 � 10�32), and B. mori (accession number AAK5760) (e �10�31). Top blastp insect virus matches included BusuNPViap-1 (e � 10�28), Amsacta moorei entomopoxvirus (EPV) iap(AmEPV) (8) ORF 21 (e � 5 � 10�6), and MacoNPV A iap-3(e � 2 � 10�24). BusuNPV iap-1 is designated by its order inthe viral genome and not by homology and is actually aniap-3 (op35) homologue (34). ClustalW alignments withnl11 showed the highest amino acid identity to an iap fromA. gambiae (accession number EAA04007) (32.2%), withthe closest baculovirus match being CpGV ORF 17 (iap-3)and LdMNPV ORF 139 (iap-3) at 28.7% amino acid identity.

Nl11 contained two baculovirus inhibitor of apoptosis pro-tein repeats (BIRs), an internal repeat between the BIRs anda transmembrane domain at the C termini, but did not have aRING finger. It is not yet known if nl11 is a functional iap gene.While BIRs are critical for iap activity (11, 16, 17) and may besufficient for antiapoptotic activity in mammalian cells (57), theRING finger may also play an important role in inhibition ofapoptosis, especially in baculoviruses (47).


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It has been suggested that viral iap genes might have origi-nated from their hosts, since S. frugiperda iap genes share se-quence and functional similarity with their baculovirus coun-terparts (35). This hypothesis is supported by the fact that nl11shows a closer identity to insect iap genes than to baculovirusiap genes.

Conserved baculovirus ORFs of unknown function. Con-served baculovirus ORFs of unknown function found inNeleNPV included homologues to ac68, ac81, ac92, ac96, ac98(38K), ac109, ac115, and ac142. Homologues of conserved lep-idopteran baculovirus ORFs of unknown function, includingac53, ac76, ac78, ac93, ac101 (p40), ac103 (p45), ac106, andac145, were also found in NeleNPV, but homologues to ac13(38.7K), ac29, ac38, ac66, ac75, ac82, ac102 (p12), ac110, andac146 could not be identified by our criteria (Table 3).

Other clearly identified ORFs. Blastp searches showed thatnl50 was similar to a protein found in NeseNPV (accessionnumber AAF24987) (e � 3 �10�29; amino acid identity,45.7%). No baculovirus matches were found for these ORFs,suggesting that hymenopteran baculoviruses may containORFs unique to that group.

Six ORFs were accepted as clearly identified based on thepresence of conserved domains but did not show baculovirusmatches. Nl6 had a conserved trypsin-like serine protease do-main identified with the NCBI conserved domain search (e �2 � 10�61) and with SMART (e � 8.4 � 10�83). Trypsin-likeserine proteases are enzymes that exploit serine in their cata-lytic site, and they include a wide range of peptidase activities,such as exopeptidase, endopeptidase, oligopeptidase and ome-ga-peptidase activities (SMART accession number SM0020).The top blastp match for nl6 was a Ctenocephalides felis (catflea) trypsin-like serine protease (GenBank accession numberAAD21829) (e � 8 � 10�46), and their amino acid identity(38.7%) was much higher than the average amino acid identityof NeleNPV ORFs to NPV homologues. Other top matcheswere to trypsin-like serine proteases from B. mori (accessionnumber VDP_BOMMO) (e � 4 � 10�45) and Drosophila mela-nogaster (GenBank accession number NP_523518) (e � 10�43).

Nl49 contained four clearly identified C2H2 zinc finger do-mains. Zinc finger domains are nucleic acid binding proteinstructures composed of 25 to 30 amino acid residues in aC-X2-C-X12-H-X3-H-type motif in which zinc binds in a tetra-hedral array to yield a finger-like projection that interactswith nucleotides in the major groove of the nucleic acid(pfam00096). No insect virus matches were found for nl49;instead, the highest blastp match was to a zinc finger proteinfrom D. melanogaster (GenBank accession number NP_609448)(e � 10�13; amino acid identity, 17.4%).

BLAST analysis showed that nl69, nl70, and nl71 containeda conserved �-tubulin suppressor domain (ATSI) and relatedregulator of chromosome condensation factor (RCC1). SMARTanalysis also showed the presence of RCC1/-lactamase inhib-itor protein II domains. There were no significant blastpmatches to insect viruses, but all three ORFs showed signifi-cant matches to proteins in A. gambiae (nl69 [accession num-ber EAA06079; e � 10�20; amino acid identity, 36.5%], nl70[accession number EAA06079; e � 4 � 10�11; amino acididentity, 27.6%] and nl71 [accession number A04764; e � 2 �10�4; amino acid identity, 23.0%]). Nl70 also showed a matchto chromatin-binding protein BJ1 in D. melanogaster (acces-

sion number S15028; e � 10�10; amino acid identity, 23.9%), ahomologue of the vertebrate RCC1 gene.

Nl89 appeared to be a phosphotransferase, since BLASTsearches showed two potential phosphotransferase domains,DUF60 (pfam01885) and KptA (COG1859), and SMART anal-ysis also showed a DUF60 conserved domain. Two members ofthe DUF60 family of proteins have been annotated as phos-photransferases, although this has not been supported experi-mentally, and KptA is a probable RNA 2-phosphotransferase.

Nl81 showed significant blastp matches to proteins fromdensoviruses, including viral protein 1-4 from Casphalia extra-nea densovirus (accession number NP_694840; e � 10�11;amino acid identity, 30.2%), a structural protein from Peripla-neta fuliginosa densovirus (accession number BAA82965; e �5 � 10�11; amino acid identity, 24.2%), and a capsid proteinfrom Bombyx mori densovirus (accession number NP_694837;e � 10�9; amino acid identity, 24.8%). Neither nl81 nor itspotential densovirus homologues showed blastp matches toany other baculoviruses. A potential densovirus homologuehas recently been reported for CrleGV (42), but Crle9 showedlittle similarity to nl81. Densoviruses replicate in the nu-cleus, and it is conceivable that a horizontal gene transfer toNeleNPV could have taken place via recombination or trans-position. It is not known if the potential densovirus homologueencodes a functional protein in NeleNPV.

Other possible homologues. Sequence analysis providedclues to other possible homologues or functions, but resultswere often inconclusive, and 39 ORFs remained unidentified(Fig. 1; Table 2). Potential features, such as transmembranedomains, signal peptides, coiled-coil domains, and internal re-peats, are listed in Table 2.

Several unattributed ORFs showed acceptable matches toORFs in EPVs by using earlier blastp analysis but did notwith blastp 2.2.5. Others had acceptable E values with blastp2.2.5 for short, nearly exact matches, had amino acid identitiesgreater than 20%, or had protein domains similar to those ofpotential homologues. Nl68, for example, had a blastp E valueof 0.008 with Melanoplus sanguinipes EPV ORF 186, a memberof the leucine-rich-repeat family (1), when blastp was used forshort, nearly exact matches; nl80 had an E value of 2.2 withAmEPV ORF 208, but their sizes were similar and both con-tained transmembrane domains; nl84 had an E value of 0.036with AmEPV ORF 051 with use of blastp for short, nearlyexact matches; nl86 had a blastp E value of 0.55 with AmEPVORF 196, and both contained transmembrane domains, weresimilar in size, and shared 23.0% amino acid identity. SmithWaterman searches showed that nl21 was a potential homo-logue of MSV156, with an E value of 0.000004.

NeleNPV DNA had a high A�T content (66.5%), and theA�T content of ORFs with potential EPV matches was evenhigher, ranging from 68.0 to 79.7%. The possible homology ofNeleNPV ORFs with those from EPVs may be due to randomsimilarities, since MsEPV and AmEPV also have a very highA�T content (1, 8). NeleNPV ORFs with low identity to EPVORFs, however, were clustered in two areas. NeleNPV is notthe only baculovirus that may contain ORFs similar to thosefound in EPVs. Spindlin or gp37 (ac64), a gene rich in A�Tresidues and found in many NPVs, is a homologue of fusolinfrom EPVs (6, 70).


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Gene content. It has been suggested from the analysis of upto 13 baculovirus genomes that 27 genes specific to GVs and 14genes specific to lepidopteran NPVs might distinguish thesetwo groups (32, 33). None of the unique NPV or GV genes wasclearly identified in NeleNPV, making it distinct from eithergroup. Low potential matches to the NPV-specific genes pkip(ac24) and p26 (ac136) were possible for NeleNPV, as was alow potential match with a metalloproteinase gene unique toGVs, but they were not considered clearly identified. An iapgene was found but it appeared closer to iap genes present ininsects and to iap-3 in baculoviruses than to iap-5, founduniquely in GVs and iap-2 in NPVs. A list of 20 genes distin-guishes group I from group II NPVs, 17 of which are found ingroup I (32, 33). NeleNPV did not appear to contain any of thegroup I- or II-specific NPV genes, which again suggests that itmay not be a typical NPV.

It is possible that NeleNPV may contain other baculovirusgenes not yet identified or that NeleNPV may not require themissing genes due to differences in host-virus interactions. Thelepidopteran conserved genes and some auxiliary genes mayhave been acquired when baculoviruses evolved to infect tis-sues beyond the midgut and may not be necessary in hymeno-pterans. Cathepsin and chitinase, for example, are needed tobreak down the host cuticle after death, liquefy the cadaver,and release occlusion bodies (29, 60). Neither gene was foundin NeleNPV, and they may not be necessary, since the virus isspread via sloughed-off intestinal cells, and infectious diarrheaand infected insects do not liquefy (22). Another gene missingin NeleNPV but conserved in all lepidopteran baculoviruses isfgf, a homologue of an insect fgf gene involved in tracheal

development (61). Such an FGF protein might be involved inspread of NPVs through the trachea but would not be neededby NeleNPV, whose replication is restricted to the midgut. It isalso possible that some unique hymenopteran baculovirusgenes are functionally equivalent but do not show close se-quence identity. No ie-1 homologue was found in NeleNPV,for example, but perhaps one or more other unidentified genesmay be involved in the transactivation of early and late genesas a substitute to ie-1, or NeleNPV may depend on host factorsfor early transcription, as suggested for CuniNPV (2).

Factors affecting genome size. NeleNPV is the smallest bac-ulovirus genome so far reported. Despite its small size, how-ever, there were still intergenic areas in the genome in whichORFs or repeat regions were not found. The lack of manybaculovirus ORFs is an important factor contributing to thesmall size of NeleNPV, but another is the lack of repeatedgenes that account for the large size of many baculovirus ge-nomes. The XcGV genome, for example, contains 30 repeatedgenes, accounting for 37.5 kb or 20% of the genome, andLdMNPV contains 32, accounting for 27.5 kb or 17% of thegenome (30, 40). The type of repeated gene varies but may in-clude bro genes, among others. NeleNPV had no clearly iden-tified bro genes and contained no repeated baculovirus ORFs.

Size differences can also be attributed to numerous inser-tions or deletions in hr’s. The Helicoverpa zea SNPV genome,for example, is larger than that of HaSNPV due mainly toinsertions in hr’s (14). SpltNPV contains the largest number ofhr’s with 17, each containing 2 to 29 palindromic repeats withan average length of 534 bp (50), greatly adding to its size.NeleNPV lacked typical NPV hr’s, and its repeat regions were

FIG. 3. Gene parity plots. Comparison of NeleNPV with AcMNPV (A), HaSNPV (B), CuniNPV (C), and PxGV (D). AcMNPV ORFs arerenumbered, with polyhedrin as ORF 1. The CuniNPV gene order is as listed in GenBank and not by ORF number for lef-5, 38k, ac96, and helicase.The graphic representations display the colinearity of gene arrangement between two genomes. ORFs unique to each virus are shown on the x axisand y axis, respectively. Most conserved clusters are circled: cluster 1, p6.9 (ac100), p40 (ac101), and p48 (ac103); cluster 2, ac76, vlf-1 (ac77), ac78,and gp41 (ac80); cluster 3, lef-5 (ac99), 38k (ac98), ac96, and helicase (ac95); cluster 4, p49 (ac142), odv-ec27 (ac143), odv-ec27 (ac144), and ac145.


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short, containing a maximum of three copies of direct repeatunits, also contributing to its small size.

Comparison of NeleNPV with other baculoviruses. Poly-hedrin is generally considered one of the most highly conservedof baculovirus proteins (55), but nl1 (polyhedrin) shared only 35.5to 49.2% amino acid identity with lepidopteran baculovirus poly-hedrins versus 98% with Neodiprion abietis NPV polyhedrin and82.1% with NeseNPV polyhedrin. Overall, polyhedrin was themost conserved baculovirus gene in NeleNPV, followed by pif-2(nl52/ac22), DNA polymerase (nl20/ac65), and p6.9 (nl28/ac100).

The average overall amino acid identity of clearly identifiedNeleNPV homologues with other baculoviruses ranged from19.7% with CuniNPV (29 common ORFs) to 24.9% with Spo-doptera exigua MNPV (SeMNPV) (37) (42 common ORFs).The closest GV was PxGV, with 42 common ORFs averaging23.6% amino acid identity, followed by CrleGV at 23.2% (Ta-bles 1 and 2).

Although the majority of ORFS were closer to those inNPVs, many, including nl23 (odv-e56), nl28 (p6.9), nl29 (p40),nl33 (alk-exo), nl37 (lef-9), nl46 (p47), nl58 (helicase), nl59(lef-4), nl62 (odv-e18), nl64, nl65 (lef-1), nl77, and nl78 (lef-8),showed a closer blastp match to GV homologues. The highestamino acid identity difference for a NeleNPV ORF comparedto an NPV and GV homologue was seen with p6.9, with nl28showing 30.4% amino acid identity with p6.9 in AcMNPV (ac100)and 64.3% amino acid identity with p6.9 in AdorGV (Ador72).

If NeleNPV ORFs were recently acquired from GVs, onewould expect them to be clustered in both genomes and theiramino acid identities to be close. It is unlikely that recentrecombination events have occurred between NeleNPV andGVs, since NeleNPV homologues with higher identity to GVORFs were spread throughout the genome, and in most casesthere was little difference in amino acid identities betweenNPV and GV homologues. This may mean that NeleNPV isindeed an older baculovirus that existed before the GV/NPVspilt and thus shows similarity to both types of virus.

Gene parity plots. Gene parity plots are used to display thegene order of genomes from any two viruses. Closely relatedbaculoviruses generally show colinear arrangements of genes,and colinearity decreases with increased divergence betweenbaculoviruses (34). Gene parity plots showed that gene orderwas not highly conserved between NeleNPV and a represen-tative type I NPV (AcMNPV), type II NPV (HaSNPV), a GV(PxGV), or CuniNPV, except for the central portion of thegenome containing lef-5 (ac99), 38k (ac98), ac96, and helicase(ac95), as has been reported with other baculoviruses (13) (Fig.3). This region may be highly conserved as a result of tran-scriptional or regulatory constraints and may have been main-tained over long evolutionary periods (13, 33). Three otherclusters in the central region appeared to be conserved be-tween NeleNPV and the lepidopteran NPVs, with some con-taining a single insertion or deletion, including p6.9 (ac100),p40 (ac101), and p48 (ac103) in one grouping, ac76, vlf (ac77),ac78, and gp41 (ac80) in a second, and p49 (ac142), odve-18(ac143), odv-ec27 (ac144), and ac145 in a third (Fig. 3).

Phylogeny of NeleNPV. Baculoviruses may have had a com-mon evolutionary origin in an ancestral virus that attacked themidgut or hepatopancreas of ancient arthropods and co-evolved with their hosts, eventually spreading to Lepidoptera(22). Midgut cell sloughing may have been an important host-

mediated selection pressure influencing NPV evolution, withvirion production in the midgut epithelium being selectedagainst due to its devastating effects on gregarious host popu-lations. The success of lepidopteran baculoviruses might bedue to their selection for ability to invade internal tissues sothat a larger inoculum of virus could be produced (21, 22, 67).

NeleNPV replicates only in the midgut and so may representan early stage in baculovirus evolution. A GV isolated from theWestern grapeleaf skeletonizer Harrisina brillians (HbGV), amember of a primitive lepidopteran family (Zygaedidae), is theonly GV restricted to midgut cells and may similarly representan early GV. H. brillians larvae also lead a gregarious lifestyle,feed in groups, and transmit virus via infectious diarrhea and

FIG. 4. Baculovirus phylogeny based on complete genome data.Most-parsimonious tree based on analysis of the combined sequencesof 29 conserved genes found in 24 fully sequenced baculovirus ge-nomes. Bootstrap values for 1,000 replicates are given. Sequences in-clude those for AcMNPV (7), Orgyia pseudotsugata MNPV (OpMNPV)(3), BmNPV (25), LdMNPV (40), SeMNPV (37), XcGV (30), PxGV(28), HaSNPV G4 (13), Helicoverpa armigera NPV (HaNPV) (Gen-Bank accession number NC_003094), SpltMNPV (50), CpGV (46),CuniNPV (2), E. postvittana MNPV (EppoMNPV) (36), H. zea SNPV(HzSNPV) (14), MacoNPV A (44), MacoNPV B (43), PhopGV (Gen-Bank accession number AF499596), RoMNPV (27), AdhoNPV (Gen-Bank accession number AP006270), C. fumiferana defective NPV (Cf-DEFNPV) (GenBank accession number AY327402), Choristoneurafumiferana MNPV (CfMNPV) (GenBank accession numberNC_004778), CrleGV (42), AdorGV (69), and NeleNPV (GenBankaccession number AY349019).


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sloughed-off midgut epithelium cells (21). It would be inter-esting to compare the gene content and phylogeny of HbGVwith NeleNPV, but only a few HbGV genes have been se-quenced.

Phylogenies based on the combined sequence of sharedgenes have been found to be more robust than those based onthe sequences of individual genes (32, 33). The most parsimo-nious tree produced by using the combined data set of 29conserved baculovirus proteins from 24 baculovirus genomesplaced NeleNPV and CuniNPV as separate branches that ex-isted before the split of the lepidopteran GVs and NPVs (Fig.4). This suggests that both NeleNPV and CuniNPV do not fitin the current NPV designation of group I or II NPVs. Theseparation of NeleNPV and CuniNPV into separate generawas corroborated with trees constructed by using individualgenes and with a combined concatemer of 10 genes conservedin all fully sequenced baculoviruses and the Hz-1 virus, whichinfects Helicoverpa zea (15), although the divisions withingroup II NPVs varied in some cases (data not shown). It hasalready been proposed that CuniNPV is a baculovirus withunusual characteristics that may represent a new genus withinthe family Baculoviridae (2, 33, 48). Our phylogenetic analysissuggests that NeleNPV may also belong to a genus separatefrom the dipteran and lepidopteran baculoviruses.

There are many factors supporting the idea that NeleNPVrepresents a new baculovirus genus. Members of the Hy-menoptera family are more ancient than Lepidoptera, andNeleNPV was isolated from a primitive hymenopteran. Thevirus showed low identity to all fully sequenced lepidopteranNPVs and GVs. Many genes conserved in lepidopteran bacu-loviruses, as well as genes identified as unique to lepidopteranNPVs and GVs, were not found in NeleNPV. Many ORFsshowed no baculovirus matches, and some had significantmatches to other insect viruses or to insect genes. With theexception of the lef-5-to-helicase region, gene order was nothighly conserved in NeleNPV relative to other baculoviruses,and repeat regions lacked sequence homology to hr’s found inNPVs or GVs. Phylogenetic trees suggest that NeleNPV, likeCuniNPV, does not fit into the present NPV designation butmay represent an early baculovirus that existed before thelepidopteran GVs and NPVs diverged from each other.

The present classification criteria encompassing only twogenera are clearly insufficient to accommodate the hymenopte-ran and dipteran baculoviruses. Moreover, it is conceivablethat baculoviruses from other insect orders will be discoveredthat also may not fit into the present genera. Other genera willhave to be created and must be flexible enough to accommo-date all baculoviruses, even ones yet to be discovered. We sug-gest adding two new genera to accommodate the hymenopte-ran and dipteran viruses and changing the names of theBaculoviridae genera to Alphabaculovirus, Betabaculovirus,Gammabaculovirus, and Deltabaculovirus. The proposed gen-era will accommodate not only the hymenopteran and dipteranbaculoviruses but also the present groups of NPVs and GVs.This system has the flexibility for adding more genera in thefuture and is consistent with the nomenclature of viruses inother families, including the Herpesviridae, Retroviridae, andEntomopoxvirinae.

ACKNOWLEDGMENTS

We thank David Theilmann for his help in the search for an ie-1gene and Theilmann and Xiaojiang Dai for personal communications;Gary Blissard for his help in the search for an F-protein homologue;Elizabeth Herniou for her advice on phylogenetic analysis; andAlejandra Garcia-Maruniak and James Maruniak for sharing theNeseNPV sequence prior to publication. We also thank Kees Van-Frankenhuyzen for the stock of NeleNPV and Lillian Pavlik for herexcellent work in extracting viral DNA.

The research was supported by grants from Genome Canada, theCanadian Biotechnology Strategy Fund, and the NSERC BiocontrolNetwork.

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