- Research article
- Open Access
Evidence of recent interspecies horizontal gene transfer regarding nucleopolyhedrovirus infection of Spodoptera frugiperda
- Gloria Patricia Barrera†1,
- Mariano Nicolás Belaich†2Email author,
- Manuel Alfonso Patarroyo3, 4,
- Laura Fernanda Villamizar1 and
- Pablo Daniel Ghiringhelli2
© Barrera et al. 2015
- Received: 19 August 2015
- Accepted: 16 November 2015
- Published: 25 November 2015
Baculoviruses are insect-associated viruses carrying large, circular double-stranded-DNA genomes with significant biotechnological applications such as biological pest control, recombinant protein production, gene delivery in mammals and as a model of DNA genome evolution. These pathogens infect insects from the orders Lepidoptera, Hymenoptera and Diptera, and have high species diversity which is expressed in their diverse biological properties including morphology, virulence or pathogenicity. Spodoptera frugiperda (Lepidoptera: Noctuidae), the fall armyworm, represents a significant pest for agriculture in America; it is a host for baculoviruses such as the Spodoptera frugiperda multiple nucleopolyhedrovirus (SfMNPV) (Colombia strain, genotype A) having been classified as a Group II alphabaculovirus making it a very attractive target for bioinsecticidal use.
Genome analysis by pyrosequencing revealed that SfMNPV ColA has 145 ORFs, 2 of which were not present in the other sequenced genotypes of the virus (SfMNPV-NicB, SfMNPV-NicG, SfMNPV-19 and SfMNPV-3AP2). An in-depth bioinformatics study showed that ORF023 and ORF024 were acquired by a recent homologous recombination process between Spodoptera frugiperda and Spodoptera litura (the Oriental leafworm moth) nucleopolyhedroviruses. Auxiliary genes are numerous in the affected locus which has a homologous region (hr3), a repetitive sequence associated with genome replication which became lost in SfColA along with 1 ORF. Besides, the mRNAs associated with two acquired genes appeared in the virus’ life-cycle during the larval stage. Predictive studies concerning the theoretical proteins identified that ORF023 protein would be a phosphatase involved in DNA repair and that the ORF024 protein would be a membrane polypeptide associated with cell transport.
The SfColA genome was thus revealed to be a natural recombinant virus showing evidence of recent horizontal gene transfer between different baculovirus species occurring in nature. This feature could be the cause of its high insecticidal power and therefore SfColA becomes a great candidate for bioinsecticide formulations.
- Spodoptera frugiperda
- Horizontal gene transfer
- Homologous recombination
Baculoviruses are double-stranded DNA viruses which infect insects from the orders Lepidoptera, Diptera and Hymenoptera. Significant baculovirus characteristics include the presence of two phenotypes during the cell cycle: budded viruses (BVs), and occlusion derived viruses (ODVs) which are embedded into protein crystals called occlusion bodies (OBs); they display very high diversity expressed in hundreds of species spread worldwide having different host ranges and/or virulence. Four genera have been recognized: Alphabaculovirus (lepidopteran-nucleopolyhedroviruses), Betabaculovirus (lepidopteran-granuloviruses), Gammabaculovirus (hymenopteran-nucleopolyhedroviruses), and Deltabaculovirus (dipteran-nucleopolyhedroviruses) [1–3]. The Spodoptera frugiperda multiple nucleopolyhedrovirus (SfMNPV) has been classified into the Baculoviridae family within the Alphabaculovirus [1, 4] and has been extensively studied for its potential regarding the biological control of fall armyworm, an important pest causing economic losses regarding several American crops, mainly corn fields . Since the first reports about SfMNPV [6, 7], work has been focused on studying its genomic constitution and the inter- and intra-population diversity by comparing different isolates. Restriction profiles initially revealed genetic heterogeneity in field isolates, in addition to providing information for determining the first physical maps of the genome [8–10]. Sequencing of single genes and genomic variable regions [11–15] and subsequent analysis showed that SfMNPV phylogenetically clustered with other members of the Group II Alphabaculovirus clade .
Four complete SfMNPV genomes have been reported recently, one from a field isolate (SfMNPV-19)  and another three from genotypic variants recovered using in vitro plaque assay techniques in insects cells (SfMNPV-3AP2, SfMNPV-NicB, SfMNPV-NicG) [17–19]. The SfMNPV-NicB isolate was the predominant genotype having the largest genome and both SfMNPV-3AP2 and SfMNPV-NicG had deletions regarding the former. Phylogenetic analysis revealed that they were very closely related and also closely related to Spodoptera exigua MNPV, Agrotis spp. NPVs and Mamestra spp. NPVs.
SfMNPV inter-population diversity evaluated in Colombia by analyzing 38 isolates from three different geographical regions revealed that one isolate (SfMNPV-Col or SfCol) had the highest prevalence (92 %). SfCol had minimal genetic differences compared to the SfMNPV isolate from Nicaragua (SfMNPV-NicB or SfNicB) based on restriction profiles; however, it had large differences regarding virulence against S. frugiperda larvae from Colombia, SfCol being more potent than SfNicB for the local insect population . Subsequent intra-population diversity studies have revealed 10 different genotypic variants within SfCol (SfColA to SfColJ), SfColA being the most prevalent (72 %) and having the largest genome, while the other variants had different sized deletions. SfColA was 4.4 times more potent than and as virulent as SfCol for local insect pests . Such biological differences should correlate with genome organization; structural mutations (replacements, inversions, insertions or deletions) would presumably be how baculoviruses evolve in nature and improve their fitness, not forgetting the importance of single nucleotide mutations. According to previous genome evidence, natural recombination events are probably one of the most important processes involved in baculovirus genome plasticity . DNA crossover may occur between two loci from one genome, between genotype variants of the same species, or between genomes from different virus species co-infecting the same host . In any case, the resulting recombinant genomes may be affected by their prior gene content. Baculovirus diversity has been associated with the ubiquitous presence of transposons, which may collaborate in horizontal gene transfer and insertion/deletion (indel) mutations. Different kinds of transposable elements have been detected in baculovirus genomes from many species, sometimes affecting gene functions [24, 25]. Baculoviruses should thus be efficient vectors between animals and such ability would have an important impact on gene content and genome organization because they can provide the sequence homology required for crossover events .
Baculovirus genome variability has an undeniable effect on the virus’ life-cycle in the host and affects different parameters such as pathogenicity, virulence and OB’s production (yield) [27, 28]. Some genome regions are more prone to sequence variation than others; these would include loci containing homologous regions (hr) and Bro genes, both being the kind of sequences recognized as target sites for intragenomic recombination because they are usually found in more than one copy . Moreover, most variability is concentrated in regions having auxiliary genes (encoding non-essential proteins) as they are more tolerant to mutations because sequence changes do not affect the production of essential factors needed to complete the viral cycle.
The SfColA isolate was molecularly characterized in the present work to provide extra evidence to explain biological activities and to further understand how baculoviruses evolve in nature, losing ancient sequences or gaining new regions and thereby altering virus fitness.
The virus used here [SfMNPV ColA (SfColA)] had been previously isolated by plaque purification in the Sf9 cell line exposed to a natural SfMNPV isolated in Colombia (SfMNPV-Col or SFCol) . SfColA was propagated in S. frugiperda fourth instar larvae reared in laboratory conditions (25 ± 1 °C, 75 ± 5 % relative humidity, 16 h light: 8 h dark photoperiod and a wheat germ-based semisynthetic diet) and maintained as OB suspension in sterile distilled water.
Sequencing, assembly and ORFeome determination
Baculoviruses used in bioinformatics studies including SfMNPV ColA
Antheraea pernyi MNPV Isolate L2
Antheraea pernyi NPV Isolate Z
Anticarsia gemmatalis MNPV
Autographa californica MNPV Clone C6
Bombyx mandarina NPV S1
Bombyx mandarina NPV S2
Bombyx mori NPV Isolate T3
Choristoneura fumiferana MNPV
Choristoneura fumiferana Defective MNPV
Choristoneura murinana NPV Strain Darmstadt
Choristoneura occidentalis NPV Isolate BC1
Choristoneura rosaceana NPV Isolate NB1
Epiphyas postvittana NPV
Hyphantria cunea NPV
Maruca vitrata NPV
Orgyia pseudotsugata MNPV
Philosamia cynthia ricini NPV
Plutella xylostella MNPV Isolate CL3
Rachiplusia ou MNPV
Thysanoplusia orichalcea NPV P2
Adoxophyes honmai NPV
Adoxophyes orana NPV
Agrotis ipsilon MNPV
Agrotis segetum NPV
Apocheima cinerarium NPV
Buzura suppressaria NPV Isolate Hubei
Chrysodeixis chalcites NPV
Clanis bilineata NPV Isolate DZ1
Ectropis obliqua NPV Strain A1
Euproctis pseudoconspersa NPV
Helicoverpa armigera MNPV
Helicoverpa armigera NPV Isolate Australia
Helicoverpa armigera NPV Strain C1
Helicoverpa armigera NPV Strain G4
Helicoverpa armigera SNPV Strain NNg1
Helicoverpa zea NPV
Hemileuca sp. NPV
Leucania separata NPV Strain AH1
Lymantria dispar MNPV
Lymantria xylina MNPV
Mamestra brassicae MNPV Isolate Chb1
Mamestra brassicae MNPV Isolate K1
Mamestra configurata NPV Strain 90-2
MacoNPV 90 2
Mamestra configurata NPV Strain A90-4
MacoNPV A90 4
Mamestra configurata NPV Strain B
Orgyia leucostigma NPV IsolateCFS77
Spodoptera exigua MNPV
Spodoptera frugiperda MNPV Isolate 3AP2
Spodoptera frugiperda MNPV Isolate Nicaraguan B
Spodoptera frugiperda MNPV Isolate Nicaraguan DefG
Spodoptera frugiperda MNPV Strain 19
Spodoptera frugiperda MNPV ColA
Spodoptera littoralis NPV Isolate AN1956
Spodoptera litura II MNPV
Spodoptera litura MNPV Strain G2
Trichoplusia ni SNPV
Adoxophyes orana GV
Agrotis segetum GV
Choristoneura occidentalis GV
Clostera anastomosis GV Strain Henan
Cryptophlebia leucotreta GV
Cydia pomonella GV
Epinotia aporema GV
Helicoverpa armigera GV
Phthorimaea operculella GV
Pseudaletia unipuncta GV Strain Hawaiin
Pieris rapae GV
Plutella xylostella GV
Spodoptera frugiperda GV
Spodoptera litura GV Strain K1
Xestia c-nigrum GV
Neodiprion abietis NPV
Neodiprion lecontei NPV
Neodiprion sertifer NPV
Culex nigripalpus NPV
For detecting homologous regions (hrs) in the SfColA genome, the SfMNPV NicB hr-1 sequence was used as computational probe. All individual palindromes (44 residue lengths) were then recovered from SfColA, SfMNPV Nic, SfMNPV 3AP2 and SfMNPV 19 genomes and multiple alignments were performed using the ClustalX algorithm with default parameters. Sequence logos were constructed using the WebLogo server (http://weblogo.berkeley.edu/) . The secondary DNA structure prediction was obtained using the Mfold server of Michael Zuker website  and using RNADraw program . A/T-content was profiled using a sliding windows strategy (window = 500 nucleotides, displacement = 50 nucleotides) . Relationships between each point and the A/T-content average were obtained and peaks of 1.12 or above were considered as A/T-rich regions.
Colinearity genome studies and phylogenetic analysis
Nucleotide synteny blocks were searched using BlastN routine with the following parameters: expected value = 0.1 (−e 0.1), penalty for a nucleotide mismatch = −2 (−q −2), reward for a nucleotide match = 1 (−r 1) and filter query sequence = false (−F F). Output files for each genome comparison were drawn using the GenomeComp v1.2 software . A color code was used for showing different ranges of nucleotide identity. Baculoviridae phylogeny was inferred using the 37 core genes in silico translated from 75 baculovirus genomes (Table 1) which were independently aligned using ClustalX program with the following parameters: Pairwise alignment (Gap Open Penalty = 10, Gap Extension Penalty = 0.1, protein weight matrix: Blosum 30); Multiple alignment (Gap Open Penalty = 10, Gap Extension Penalty = 0.05, protein weight matrix: Blosum series). A concatemer was then generated by adding complete individual alignments and phylogeny was inferred using MEGA 5 software  with the following parameters: UPGMA; Bootstrap with 1000 replicates; Gap/Missing data = pairwise deletion; Model = Amino (Dayhoff Matrix); patterns among sites = Same (Homogeneous); rates among sites = Different (Gamma Distributed); gamma parameter = 0.9839. Besides, a phylogeny inference was similarly performed but using only SfMNPV genomes and the most related baculoviruses (SeMNPV, SpltNPV-II, SpltMNPV-G2, SpliNPV AN1956). The concatemer of individual alignments derived from 100 genes translated in silico which were shared among baculoviruses considered for the study (indicated in Additional file 1: Table S1).
Interspecies horizontal gene transfer studies
The partial SfColA genome sequence, from chitinase ATG to the gp37 stop codon genes, was compared to corresponding SfMNPV-B, SfMNPV 3AP2, SfMNPV 19, SeMNPV and SpltNPV-II regions to detect potential recombination events by running alternative methods. In the first one , ClustalX (default parameters) was used for aligning sequence pairs, always involving the putative recombinant candidate (SfColA) and one of the other sequences. Relative similarities were calculated using the ClustalX consensus symbol (* and blank space) as the input sequence, in an overlapping windows-based strategy. Arbitrary values of +1 for identical (*) and −1 for non-identical residues (blank spaces) were set for obtaining similarity profiles. The sum of assigned values for each residue in each window (35 nucleotides) was divided by the window width and allotted to the central position to generate the plots. Profiles were drawn and analyzed for detecting crossover points. Different window lengths were scanned to find good relationships between graph complexity and crosspoint detection sensitivity. Bootscan analysis (Simplot program, version 3.5.1) [40, 41] was performed using the following parameters: Window = 500 residues; Step = 50 residues; Gaps strip = on; Replicates = 100; Model = Kimura 2-parameters; Transition and transversion ratio = 2.0; Phylogenetic method = Neighbor Joining). A G/C-content study was made for the same SfColA, SfNicB and SpltNPV-II genome region. G/C-contents were studied using a sliding window strategy (window = 65 nucleotides, displacement = 1 nucleotide). The profiles were adjusted using in-house software. For this a multiple alignment with the three nucleotide sequences was used as template and the adjustment of numerical profiles consisted in the introduction of blank positions interrupting the curve where there were indel events. SigmaPlot v9 was used for all studies where final numerical profiles were represented and the putative crossover breakpoints were estimated. Multiple alignment of homologous proteins was done to estimate Kimura 2-parameter distances  using MEGA 5 software with the following parameters: Scope = Pairs of taxa; Estimate Variance, Variance Estimation Method = Bootstrap method; N° of Bootstrap Replications = 1000; Substitution model, Substitution type = Nucleotide, Model/Method = Kimura 2-parameter model, Substitutions to include = d: Transitions + Transversions; Rates and Patterns, Rates among Sites = Gamma parameter, Gamma Parameter = 0.9839, Pattern among lineages = Same (Homogeneous); Data Subset to Use, Gaps/Missing Data Treatment = Pairwise deletion, Select Codon Positions = All + Noncoding Sites.
SfColA ORF023 and ORF024 transcription studies
Reverse transcription-PCR assays were performed to determine SfColA ORF023 and ORF024 transcription activities. Forty five S. frugiperda second-instar larvae were kept fasting for 8 to 12 h at 26 °C and then allowed to drink from an aqueous suspension containing 10 % (wt/vol) sucrose, 0.001 % (wt/vol) Fluorella blue, and 1x107 OBs/mL. Larvae that ingested the suspension within 10 min were then transferred to individual plastic cups with semisynthetic diet. Total RNA was extracted from two infected larvae at 0, 2, 4, 6, 12, 24, 48, 72, 96, 120 and 144 hpi using TRIzol reagent (Invitrogen), as described in the manufacturers’ protocol. RNA samples were then treated with RNase free DNase (Promega) prior to ensuring the absence of contaminant DNA. First strand cDNA synthesis was done using reverse transcriptase (Promega) and oligo-dT primer. The resulting cDNAs were amplified by PCR with specific primers: Sf23.1 (5′ GCTTGTGCGTTGTCGTTGAT 3′) and Sf23.2 (5′ TTGTAGTCGACTCGGTCCCA 3′) for ORF023; Sf24.1 (5′ TCGTCGGCATCATACTGCTC 3′) and Sf24.2 (5′ CACGTTCGCATGGTTTTCGT 3′) for ORF024; Sfpolh.1 (5′ TTGCGACCCTGACTACGTTC 3′) and Sfpolh.2 (5′ ACGAGCGACAGTTCAAGGAG 3′) for the very late transcribed polyhedrin gene (polh); Sfie-0.1 (5′ CATTTGCCAAGAGAGCAGCG 3′) and Sfie-0.2 (5′ TTTAGCGGCAGTGGGAGTTT 3′) for the early transcribed ie-o gene. The amplification products were resolved in 1 % agarose gels and visualized with ethidium bromide and UV exposure.
Characterization of SfColA ORF023 and ORF024 proteins
Different bioinformatics tools were used for determining the nature of SfColA ORF023 and ORF024 proteins. Hydrophobicity profiles were constructed using a sliding windows strategy (window = 21 amino acids; sliding 1 residue each time). Several hydrophobicity scales were assayed [43–47]. The presence of signal peptides was assessed by using SignalP (http://www.cbs.dtu.dk/services/SignalP/; ). Putative functions were predicted using the HHpred server (http://toolkit.lmb.uni-muenchen.de/hhpred; ). Secondary and tertiary structures were predicted using the LOcal MEta-Threading-Server (LOMETS; http://zhanglab.ccmb.med.umich.edu/LOMETS; ) and the I-TASSER server (http://zhanglab.ccmb.med.umich.edu/I-TASSER; ). SfColA ORF023 secondary and tertiary structures were also predicted using the QUARK (http://zhanglab.ccmb.med.umich.edu/QUARK; ) ab initio prediction server. Post-translational modifications were predicted by the INTERPROSCAN tool (http://www.ebi.ac.uk/interpro/; ).
The SfColA genome and gene content
Five genomes have been sequenced to date from baculoviruses isolated from Spodoptera frugiperda: 4 alphabaculoviruses (SfMNPV 3AP2, SfMNPV NicB, SfMNPV NicG, SfMNPV 19) and 1 betabaculovirus (SpfrGV) (Table 1). The aforementioned polyhedroviruses were sequenced using a molecular cloning strategy followed by an automated Sanger’s method; only the granulovirus involved using next generation-sequencing (NGS) . The Colombian isolate’s (SfCol) SfColA genotypic variant was molecularly characterized in view of its interesting biological properties to complete the study of the baculovirus group naturally infecting the armyworm and to provide extra information about baculovirus evolution. This genome (GenBank: KF891883) consisted of 134,239 bp, and was covered 64 times by 454 sequencing, this being the first Spodoptera frugiperda alphabaculovirus studied by the NGS approach. Its size was revealed to be the largest regarding previously reported genomes where SfMNPV-NicB (132,954 bp) was the previous head of the ranking. Group II alphabaculoviruses have a broad range of A + T content [42.53 % in Lymantria dispar MNPV  to 66.64 % in Apocheima cinerarium NPV (unpublished data)]. SfColA had 59.66 %, thereby agreeing with data published for the other Spodoptera frugiperda nucleopolyhedrovirus [SfNicB 59.72 %, SfMNPV-19 (Sf19) 59.74 %, and SfMNPV-3AP2 (Sf3AP2) 59.75 %].
Analysis of the SfColA genome led to detection of 145 putative open reading frames (ORFs) considering sequences encoding polypeptides having at least 50 amino acids, starting in an ATG codon and having minimal overlap with the closest ones. Thus the ORFeome would cover 92.5 % of the whole nucleotide sequence and each ORF was numbered, starting at the polyhedrin gene (ORF001) and continuing the numbering downstream polyhedrin stop codon. Promoter motifs were searched, an early CAKT initiator sequence (INR)  was found in 40 ORFs, irrespective of including the TATA-box, while 29 ORFs had a late INR motif  and another 59 had both early and late elements. As expected, the SfColA genome contained the 37 core genes present in all baculoviruses and these sequences were thus identified using current denominations. The other putative genes were mentioned using the most accepted names based on their Blast relationships regarding the annotated ORFs from other baculoviruses [3, 57, 58].
A very different situation occurred with SfColA ORFs 023 and 024 (Fig. 1b); both genes were not present in the other SfMNPV genomes, although these sequences have similarity with some Group II alphabaculovirus genes. By contrast, SfNicB, Sf19 and Sf3AP2 had one ORF in that locus (023, 022 and 023, respectively) that is not present in SfColA and had homology with other baculoviruses. This gene is present in the SpfrGV genome (ORF099) where the encoded polypeptide was hypothesized as being a soluble protein containing ring finger motifs . Such genome replacement (2 genes acquired compared to 1 lost) might thus be considered a recombination product as will be shown below.
Another genome location having differences was the region containing SfColA ORFs 027 and 028 because the theoretical polypeptides encoded by these genes had low identity and similarity values regarding the homologous Sf3AP2 ORFs 026 and 027 (Fig. 1c). This was due to deletion in the Sf3AP2 genome affecting the corresponding carboxy terminal of the ecdysteroid UDP–glucosyltransferase (egt) gene (Sf3AP2 ORF026) and the amino terminal of the other one (Sf3AP2 ORF027).
SfColA ORFs 033 and 124 also had lower similarity values than the expected ones when the in silico translated sequences were compared to the corresponding orthologs (Fig. 1d). Both putative genes encoded unknown proteins; the former only had differences with Sf19 because of a two single nucleotide deletion in this gene affected the reading frame annotated in the other SfMNPVs starting in a later ATG. SfColA ORF124 had differences with only SfNicB due to this sequence having a 5 bp microdeletion. The SfColA ORF131 did not present an annotated ortholog in SfNicB (Fig. 1e). Sequence analysis revealed 6 different nucleotides in the same stretch, including 1 nucleotide deletion affecting the reading frame, even though the region is present in SfNicB and other Group II alphabaculoviruses. In fact, in that location was annotated other ORF (SfNicB ORF130) with similarity with AcMNPV ORF29 and SeMNPV ORF128. It is important to note that homologs of SfNicB ORF130 are also present in the other genotypes of SfMNPVs, including SfColA, and were annotated as ORF130 in Sf3AP2 and ORF128 in Sf19. For these reasons, in the genome of SfColA both putative coding regions were included as ORF131 and ORF131a (Fig. 1e).
Regarding non-encoding loci, baculoviruses homologous regions (hr) are sequence repeats which are dispersed throughout their genomes. All previously described SfMNPVs have 8 h interspersed in different locations; they are characterized by tandem repeats consisting of a 44 bp nucleotide stretch which include an imperfect 34 bp palindromic core. These sequences are variable; however, the local secondary structure motifs are very similar, constituting hairpin loops (see Additional file 2: Figure S1). The hr-1 has 7 repeats; hr-2, hr-3 and hr-6 have only 1 repeat; hr-4 and hr-7 have 6 repeats and hr-5 and hr-8 have 4 repeats. It should be noted that SfColA lacked hr-3 since this sequence was located in the region where gene replacement occurred (Fig. 1f). Sf19 lacked hr-5c and hr-5d, and SfNicB lacked hr-8a and hr-8b. Two unique ORFs (039a and 110a) were annotated in SfNicB but the corresponding sequences were also present in Sf19, Sf3AP2 and SfColA showing few single nucleotide polymorphisms. The locus containing SfNicB ORF039a was located close to hr-4 while the SfNicB ORF110a was close to hr-7 and both postulated encoding sequences were probably not real genes. All repeats from SfMNPV hrs can be summarized in a consensus sequence using the IUPAC ambiguity code: 5′ YNAWSTTDRCTTTYVDYNAHRHDYBTBRNBDAAAKYMAASWTBR 3′. Conserved nucleotides (bold) would be A or T and probably essential for their role as replication origins and/or as transcription enhancers.
Interspecies horizontal gene transfer
All the aforementioned analysis suggested that recombination occurred between recent SfMNPV and SpltNPV-II ancestors, involving the end of the chitinase gene and the start of the gp37 gene, causing the replacement of ~1470 bp (including hr-3 and SfNicB/Sf3AP2 ORF023 or Sf19 ORF022) for ~2970 bp carrying 2 complete genes having great similarity to SpltNPV-II 020 and 021 ORFs and a truncated gene similar to SpltNPV-II ORF019. Breakpoints seemed to be inside the reading frames for the chitinase gene (around the position 21,471 in SfColA) and the gp37 gene (around the position 24,443 in SfColA) restoring the continuity of both frames. Regarding the SpltNPV-II ORF019 homolog in SfColA, a sequence analysis revealed 8 different deletions (11 bp, 240 bp, 11 bp, 3 bp, 1 bp, 3 bp, 1 bp and 11 bp, ordered from ATG to stop codon) thereby resulting in a frame shift.
SfColA ORF023 and ORF024
The SfColA ORF024 theoretical protein consisted of 462 residues, 33 being negatively charged (Asp + Glu) and 41 positively charged amino acids (Arg + Lys + His). Based on sequence, the molecular weight was 52,210.0 Da and the theoretical isoelectric point was 7.60. The hydrophobicity profile suggested that this polypeptide would be a membrane protein having +0.08 average hydrophobicity and having at least 6 transmembrane regions containing 12 α-helices and a signal peptide detected by SignalP (Fig. 8c). The secondary structure predicted by LOMETS and I-TASSER servers gave 85.8 % coincidence, revealing the presence of 19 α-helices (47.8 % of residues), 11 β-sheets (12.5 % of residues), and the remaining amino acids constituting loops or turns. Coincidentally, the LOMETS server predicted a tertiary structure and a secondary motif distribution consistent with a transmembrane motif (Fig. 8d). HHpred identified one region (from amino acid 51 to 451) as a member of the Major Facilitator Superfamily (MFS); these proteins are permeases which act as secondary carriers in cell transport . INTERPROSCAN found several putative post-translational modifications, including 2 N-glycosylation sites (from amino acid 36 to 39 and from amino acid 317 to 320), 1 cAMP- and cGMP-dependent protein kinase phosphorylation site (from amino acid 3 to 6), 2 Protein kinase C phosphorylation sites (from amino acid 452 to 454 and from amino acid 455 to 455) and 1 Casein kinase II phosphorylation site (from amino acid 50 to 53). These post-translational modifications could form part of protein function but experimental confirmation is required. It should be mentioned that the homologous protein encoded by the Helicoverpa armigera nucleopolyhedrovirus (G4 strain) was not detected as a structural protein, suggesting that its role occurs within the infected cells [61, 62].
It is importante to note that all the data sets supporting the results of this article are included within the article and its additional files.
Baculoviruses and other viruses having large dsDNA genomes mainly evolve due to the accumulation of structural mutations (insertions, deletions, replacements, inversions, translocations) affecting gene content, where recombination or transposition appear to be the most relevant examples of mechanisms occurring in nature affecting DNA integrity. Analysis of complete baculovirus genomes has revealed a “core genome” represented by 37 genes encoding essential factors accumulating sequence variability since the last virus ancestor . Such pathogens carry sequences acquired from other entities defining a “plastic genome” which contains sequences included in all members of each genus and other regions present in only some species or variants. Core genes usually produce key factors needed to complete a virus cycle, by contrast many encoding sequences in the plastic genome produce auxiliary proteins collaborating in virus processes even though not being essential for producing infectious progeny increasing fitness for them to perpetuate in nature. New technologies available for acquiring whole genome information have facilitated associating phenotype characteristics with gene content. The SfMNPV ColA genome (from a particular Colombian isolate having better biological properties than others)  was thus sequenced having high coverage and compared to other genotypes isolated from other geographical regions.
The most relevant differences occurred in a locus where SfColA underwent recent sequence replacement, losing 1 gene and gaining 2 new encoding sequences. The genome location where recombination occurred has been described as hypervariable since SfMNPV variants have different deletions [17, 18, 21]. These regions include auxiliary genes such as ecdysteroid UDP–glucosyltransferase (egt), protein-tyrosine-phosphatase (ptp), chitinase and cathepsin whose products have activity affecting insect host physiology, development, behavior and integrity . Interestingly this location also contains hr-3, a kind of sequences recognized as being a recombination facilitators [27, 28]. By contrast, most of the other hrs were closer to core genes, thereby decreasing the fitness of natural recombinant viruses due to possible loss of essential functions. It is worth stressing that hr-1 was close to odv-56 (pif5) and f genes, hr-2 was near lef-1, hr-4 next to alk-exo, hr-6 was close to lef-9 and hr-7 was near lef-8 and u-box/ring. The locus containing hr-3 thus seemed to be a hot genome region prone to undergoing structural mutations.
Recombination is an important evolutionary mechanism which might be used as a viral strategy to gain advantage for maintaining adaptability to changing environments . Recombination could facilitate resistance to host range expansion [63–65]. These kinds of interactions between genomes occur if two types of DNA coexist in the same cell, have sequence similarities and they are replicating. Artificial coinfections with AcMNPV and BmNPV in larvae and in cell culture have shown that homologous recombination can occur between viruses belonging to two different species . It has been reported that some SpfrGV genes were acquired by horizontal gene transfer from other baculovirus species including SpltNPV-II , such genome having been identified as DNA donor for SfColA ORFs 023 and 024. SpfrGV contains an orthologous gene for SfNicB ORF023, Sf19 ORF022 and Sf3AP2 ORF023 (SpfrGV ORF099), the encoding sequence lost in SfColA. This would suggest that recombination occurred when these viruses co-infected Spodoptera frugiperda larvae.
Spodoptera litura and Spodoptera frugiperda are polyphagous insect pests living on crops such as rice, corn, cotton and tobacco; they have been reported in subtropical locations in both the Old and New world, although cross migration of both insects has been reported . S. litura have been recorded in 80 species of host plant  while S. frugiperda has been described in 186 such plants , many of them shared between both lepidoptera. Natural coinfection involving circulating variants of SpltNPV and SfMNPV could thus occur in the same host. Both species have similar sequences and genome organization, and it has been reported that SpltNPV can infect Spodoptera frugiperda-derived cells, such as Sf9 and Sf21 [65, 68]. The above and bioinformatics evidence provided here support the hypothesis that homologous recombination is used by baculoviruses in nature to acquire variability. The SfColA genome would thus seem to provide natural proof for affirming that horizontal gene transfer is exploited by organisms and viruses to increase their fitness and thus acquire a reproductive success ensuring their permanence in nature.
This work was supported by research funds provided by COLCIENCIAS (Colombia) and MinCyT (Ministry of Science and Technology; Argentina) Cooperation Program.
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