Genomic sequence analysis of granulovirus isolated from the tobacco cutworm, Spodoptera litura

Background

Spodoptera litura is a noctuid moth that is considered an agricultural pest. The larvae feed on a wide range of plants and have been recorded on plants from 40 plant families (mostly dicotyledons). It is a major pest of many crops. To better understand Spodoptera litura granulovirus (SpliGV), the nucleotide sequence of the SpliGV DNA genome was determined and analyzed.

Methodology/Principal Findings

The genome of the SpliGV was completely sequenced. The nucleotide sequence of the SpliGV genome was 124,121 bp long with 61.2% A+T content and contained 133 putative open reading frames (ORFs) of 150 or more nucleotides. The 133 putative ORFs covered 86.3% of the genome. Among these, 31 ORFs were conserved in most completely sequenced baculovirus genomes, 38 were granulovirus (GV)-specific, and 64 were present in some nucleopolyhedroviruses (NPVs) and/or GVs. We proved that 9 of the ORFs were SpliGV specific.

Conclusions/Significance

The genome of SpliGV is 124,121 bp in size. One hundred thirty-three ORFs that putatively encode proteins of 50 or more amino acid residues with minimal overlap were determined. No chitinase or cathepsin genes, which are involved in the liquefaction of the infected host, were found in the SpliGV genome, explaining why SpliGV-infected insects do not degrade in a typical manner. The DNA photolyase gene was first found in the genus Granulovirus. When phylogenic relationships were analyzed, the SpliGV was most closely related to Trichoplusia ni granulovirus (TnGV) and Xestia c-nigrum granulovirus (XecnGV), which belong to the Type I-granuloviruses (Type I-GV).     

The storage of infective spores of Vairimorpha necatrix (Protozoa: Microsporida) in antibiotic solution at 4 degrees C.

The interactions between the Diacrisia virginica granulosis virus (DGV) and the Hyphantria cunea baculovirus isolates were determined, utilizing defined differences between the time-mortality responses of these viruses fed to H. cunea larvae. The DGV, having a prolonged incubation period, when given an advantage in time or in the number of capsules, was able to prevent the expression of the more lethal H. cunea granulosis virus (HcGV) isolate. The time-mortality response of test larvae simultaneously fed equivalent dosages of HcGV and DGV was intermediate to that achieved with HcGV alone or DGV alone. Larvae infected with the DGV isolate were still susceptible to double infection by the nucleopolyhedrosis virus. The time-mortality response demonstrated that the development of nucleopolyhedrosis was only partly inhibited by preinfecting the test larvae with the DGV isolate.     

Genome of Cnaphalocrocis medinalis Granulovirus,the First Crambidae-Infecting Betabaculovirus Isolated from Rice Leaffolder to Sequenced

Cnaphalocrocis medinalis is a major pest of rice in South and South-East Asia. Insecticides are the major means farmers use for management. A naturally occurring baculovirus, C. medinalis granulovirus (CnmeGV), has been isolated from the larvae and this has the potential for use as microbial agent. Here, we described the complete genome sequence of CnmeGV and compared it to other baculovirus genomes. The genome of CnmeGV is 112,060 base pairs in length, has a G+C content of 35.2%. It contains 133 putative open reading frames (ORFs) of at least 150 nucleotides. A hundred and one (101) of these ORFs are homologous to other baculovirus genes including 37 baculovirus core genes. Thirty-two (32) ORFs are unique to CnmeGV with no homologues detected in the GeneBank and 53 tandem repeats (TRs) with sequence length from 25 to 551 nt intersperse throughout the genome of CnmeGV. Six (6) homologous regions (hrs) were identified interspersed throughout the genome. Hr2 contains 11 imperfect palindromes and a high content of AT sequence (about 73%). The unique ORF28 contains a coiled-coil region and a zinc finger-like domain of 4–50 residues specialized by two C2C2 zinc finger motifs that putatively bound two atoms of zinc. ORF21 encoding a chit-1 protein suggesting a horizontal gene transfer from alphabaculovirus. The putative protein presents two carbohydrate-binding module family 14 (CBM_14) domains rather than other homologues detected from betabaculovirus that only contains one chit-binding region. Gene synteny maps showed the colinearity of sequenced betabaculovirus. Phylogenetic analysis indicated that CnmeGV grouped in the betabaculovirus, with a close relation to AdorGV. The cladogram obtained in this work grouped the 17 complete GV genomes in one monophyletic clade. CnmeGV represents a new crambidae host-isolated virus species from the genus Betabaculovirus and is most closely relative of AdorGV. The analyses and information derived from this study will provide a better understanding of the pathological symptoms caused by this virus and its potential use as a microbial pesticide.     

Genomic Sequencing and Analysis of Sucra jujuba Nucleopolyhedrovirus

The complete nucleotide sequence of Sucra jujuba nucleopolyhedrovirus (SujuNPV) was determined by 454 pyrosequencing. The SujuNPV genome was 135,952 bp in length with an A+T content of 61.34%. It contained 131 putative open reading frames (ORFs) covering 87.9% of the genome. Among these ORFs, 37 were conserved in all baculovirus genomes that have been completely sequenced, 24 were conserved in lepidopteran baculoviruses, 65 were found in other baculoviruses, and 5 were unique to the SujuNPV genome. Seven homologous regions (hrs) were identified in the SujuNPV genome. SujuNPV contained several genes that were duplicated or copied multiple times: two copies of helicase, DNA binding protein gene (dbp), p26 and cg30, three copies of the inhibitor of the apoptosis gene (iap), and four copies of the baculovirus repeated ORF (bro). Phylogenetic analysis suggested that SujuNPV belongs to a subclade of group II alphabaculovirus, which differs from other baculoviruses in that all nine members of this subclade contain a second copy of dbp.     

Sequence analysis of the genome of the Neodiprion sertifer nucleopolyhedrovirus

The genome of the Neodiprion sertifer nucleopolyhedrovirus (NeseNPV), which infects the European pine sawfly, N. sertifer (Hymenoptera: Diprionidae), was sequenced and analyzed. The genome was 86,462 bp in size. The C+G content of 34% was lower than that of the majority of baculoviruses. A total of 90 methionine-initiated open reading frames (ORFs) with more than 50 amino acids and minimal overlapping were found. From those, 43 ORFs were homologous to other baculovirus ORFs, and 29 of these were from the 30 conserved core genes among all baculoviruses. A NeseNPV homolog to the ld130 gene, which is present in all other baculovirus genomes sequenced to date, could not be identified. Six NeseNPV ORFs were similar to non-baculovirus-related genes, one of which was a trypsin-like gene. Only one iap gene, containing a single BIR motif and a RING finger, was found in NeseNPV. Two NeseNPV ORFs (nese18 and nese19) were duplicates transcribed in opposite orientations from each other. NeseNPV did not have an AcMNPV ORF 2 homolog characterized as the baculovirus repeat ORF (bro). Six homologous regions (hrs) were located within the NeseNPV genome, each containing small palindromes embedded within direct repeats. A phylogenetic analysis was done to root the tree based upon the sequences of DNA polymerase genes of NeseNPV, 23 other baculoviruses, and other phyla. Baculovirus phylogeny was then constructed with 29 conserved genes from 24 baculovirus genomes. Culex nigripalpus nucleopolyhedrovirus (CuniNPV) was the most distantly related baculovirus, branching to the hymenopteran NeseNPV and the lepidopteran nucleopolyhedroviruses and granuloviruses.     

Genome of a Bombyx mori Nucleopolyhedrovirus Strain Isolated from India

Bombyx mori nucleopolyhedrovirus (BmNPV), a member of the Baculoviridae, is a major pathogen of silkworm and has also been recently developed as an expression vector for heterologous gene expression in the silkworm larvae and pupae. To better understand the diversity of this important baculovirus, we sequenced the complete genome of the BmNPV strain isolated from India, where its host is available throughout the year due to its tropical climate. The genome of the Indian strain consists of 127,879 nucleotides, with a G+C content of 40.36%. There are 138 open reading frames (ORFs) encoding the predicted proteins of more than 50 amino acids. Genomic comparison of the Indian strain with 3 other reported BmNPV strains showed that the baculovirus repeat ORFs (bro) and homologous repeat regions (hr''s) are highly variable. These results suggest that the BmNPV strain heterogeneity is mainly caused by single-nucleotide polymorphisms (SNPs) and changes in the hr''s and bro genes.     

Interinstar susceptibility of the fall webworm,Hyphantria cunea,to its nucleopolyhedrosis and granulosis viruses

Comparative interinstar susceptibility of the fall webworm, Hyphantria cunea, to granulosis and nucleopolyhedrosis viruses was quantitated. As the larvae matured, susceptibility to either baculovirus preparation decreased. Mortality data converted to a dose per milligram body weight basis demonstrated that this decrease in susceptibility was only in part due to the difference in weight of the larvae tested. Lethal time data showed that incubation time for either baculovirus was dependent on the age of the larvae and the dose of virus assayed. A difference in interinstar susceptibility was observed between the granulosis virus and nucleopolyhedrosis virus.     

Research and application of Chouioia cunea Yang (Hymenoptera: Eulophidae) in China

Chouioia cunea Yang (Hymenoptera: Eulophidae) is an effective parasitoid of many lepidopteran pests in China. Specifically, C. cunea has successfully suppressed populations of the fall webworm, Hyphantria cunea (Drury) (Lepidoptera: Arctiidae), an invasive and quarantined pest in China. Fall webworm biological control programmes in China have been aided by the development of artificial rearing technology for C. cunea. While researchers have determined some aspects of this parasitoid’s biology, such as fecundity and ratio of female offspring, as well as rearing methods, there was less information on the behavioural and ecological mechanisms by which C. cunea regulates host populations. Here, we review the research and application of C. cunea since it was first discovered in China.     

The genome sequence of Pseudoplusia includens single nucleopolyhedrovirus and an analysis of p26 gene evolution in the baculoviruses

Background

Pseudoplusia includens single nucleopolyhedrovirus (PsinSNPV-IE) is a baculovirus recently identified in our laboratory, with high pathogenicity to the soybean looper, Chrysodeixis includens (Lepidoptera: Noctuidae) (Walker, 1858). In Brazil, the C. includens caterpillar is an emerging pest and has caused significant losses in soybean and cotton crops. The PsinSNPV genome was determined and the phylogeny of the p26 gene within the family Baculoviridae was investigated.

Results

The complete genome of PsinSNPV was sequenced (Roche 454 GS FLX – Titanium platform), annotated and compared with other Alphabaculoviruses, displaying a genome apparently different from other baculoviruses so far sequenced. The circular double-stranded DNA genome is 139,132 bp in length, with a GC content of 39.3 % and contains 141 open reading frames (ORFs). PsinSNPV possesses the 37 conserved baculovirus core genes, 102 genes found in other baculoviruses and 2 unique ORFs. Two baculovirus repeat ORFs (bro) homologs, bro-a (Psin33) and bro-b (Psin69), were identified and compared with Chrysodeixis chalcites nucleopolyhedrovirus (ChchNPV) and Trichoplusia ni single nucleopolyhedrovirus (TnSNPV) bro genes and showed high similarity, suggesting that these genes may be derived from an ancestor common to these viruses. The homologous repeats (hrs) are absent from the PsinSNPV genome, which is also the case in ChchNPV and TnSNPV. Two p26 gene homologs (p26a and p26b) were found in the PsinSNPV genome. P26 is thought to be required for optimal virion occlusion in the occlusion bodies (OBs), but its function is not well characterized. The P26 phylogenetic tree suggests that this gene was obtained from three independent acquisition events within the Baculoviridae family. The presence of a signal peptide only in the PsinSNPV p26a/ORF-20 homolog indicates distinct function between the two P26 proteins.

Conclusions

PsinSNPV has a genomic sequence apparently different from other baculoviruses sequenced so far. The complete genome sequence of PsinSNPV will provide a valuable resource, contributing to studies on its molecular biology and functional genomics, and will promote the development of this virus as an effective bioinsecticide.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1323-9) contains supplementary material, which is available to authorized users.     

Mass rearing and augmentative releases of the native parasitoid Chouioia cunea for biological control of the introduced fall webworm Hyphantria cunea in China

The fall webworm, Hyphantria cunea Drury, is an economically important introduced pest in China. A native pupal endoparasitoid, Chouioia cunea Yang (Hymenoptera: Eulophidae, Tetrastichinae) causes considerable mortality of H. cunea pupae in some areas of China. Alternative hosts of C. cunea were tested to find potential substitute hosts for mass rearing the parasitoid. Antheraea pernyi Guerin-Meneville (Lepidoptera: Saturniidae) was chosen as a surrogate host as a single pupa produced an average of 6552 wasps and a maximum of 11,256 per pupa. A mass-rearing technique and successful release program of this parasitoid is described. Parasitism by C. cunea in the areas where it was released averaged 67.74% and was usually over 80%. Other native parasitoids, such as Coccygomimus disparis (Viereck), C. parnasae (Viereck) (Hymenoptera: Ichneumonidae) and Exorita japonica Townes (Diptera: Tachnidae), caused the total average parasitism to exceed 90% on average, reaching a maximum of 96.28% in the release areas. These successful releases indicate that an introduced pest species can also be controlled by the mass-rearing and release of native parasitoids in the country of introduction.     

Susceptibility of Hyphantria cunea to a granulosis virus isolated from Diacrisia virginica

The dosage-mortality response of Hyphantria cunea larvae to a granulosis virus isolated from Diacrisia virginica was studied. Serial decimal dilutions of the D. virginica granulosis virus were fed to early second-instar H. cunea larvae. the LD₅₀ for this virus (7.9 × 10⁵ capsules/larva) was significantly greater than the LD₅₀ calculated for the H. cunea granulosis virus (7.06 × 10⁴ capsules/larva) against the same instar of H. cunea. Time mortality studies demonstrated that the LT₅₀ values for the D. virginica granulosis were four to six times greater than for comparable dosages of H. cunea granulosis virus. Based on the mortality response of assayed H. cunea, it is possible to distinguish between the D. virginica granulosis virus and the H. cunea granulosis virus.     

Horizontal Escape of the Novel Tc1-Like Lepidopteran Transposon TCp3.2 into Cydia pomonella Granulovirus

We characterized an insertion mutant of the baculovirus Cydia pomonella granulovirus (CpGV), which contained a transposable element of 3.2 kb. This transposon, termed TCp3.2, has unusually long inverted terminal repeats (ITRs) of 756 bp and encodes a defective gene for a putative transposase. Amino acid sequence comparison of the defective transposase gene revealed a distant relationship to a putative transposon in Caenorhabditis elegans which also shares some similarity of the ITRs. Maximum parsimony analysis of the predicted amino acid sequences of Tc1- and mariner-like transposases available from the GenBank data base grouped TCp3.2 within the superfamily of Tc1-like transposons. DNA hybridization indicated that TCp3.2 originated from the genome of Cydia pomonella, which is the natural host of CpGV, and is present in less than 10 copies in the C. pomonella genome. The transposon TCp3.2 most likely was inserted into the viral genome during infection of host larvae. TCp3.2 and the recently characterized Tc1-like transposon TC14.7 (Jehle et al. 1995), which was also found in a CpGV mutant, represent a new family of transposons found in baculovirus genomes. The occasional horizontal escape of different types of host transposons into baculovirus genomes evokes the question about the possible role of baculoviruses as an interspecies vector in the horizontal transmission of insect transposons. Received: 27 February 1997 / Accepted: 16 May 1997     

Genome sequencing and analysis of a granulovirus isolated from the Asiatic rice leafroller,Cnaphalocrocis medinalis

The complete genome of Cnaphalocrocis medinalis granulovirus(CnmeGV) from a serious migratory rice pest, Cnaphalocrocis medinalis(Lepidoptera: Pyralidae), was sequenced using the Roche 454 Genome Sequencer FLX system(GS FLX) with shotgun strategy and assembled by Roche GS De Novo assembler software. Its circular double-stranded genome is 111,246 bp in size with a high A+T content of 64.8% and codes for 118 putative open reading frames(ORFs). It contains 37 conserved baculovirus core ORFs, 13 unique ORFs, 26 ORFs that were found in all Lepidoptera baculoviruses and 42 common ORFs. The analysis of nucleotide sequence repeats revealed that the CnmeGV genome differs from the rest of sequenced GVs by a 23 kb and a 17 kb gene block inversions, and does not contain any typical homologous region(hr) except for a region of non-hr-like sequence. Chitinase and cathepsin genes, which are reported to have major roles in the liquefaction of the hosts, were not found in the CnmeGV genome, which explains why CnmeGV infected insects do not show the phenotype of typical liquefaction. Phylogenetic analysis,based on the 37 core baculovirus genes, indicates that CnmeGV is closely related to Adoxophyes orana granulovirus. The genome analysis would contribute to the functional research of CnmeGV,and would benefit to the utilization of CnmeGV as pest control reagent for rice production.     

The complete mitochondrial genome of the fall webworm,Hyphantria cunea (Lepidoptera: Arctiidae)

The complete mitochondrial genome (mitogenome) of the fall webworm, Hyphantria cunea (Lepidoptera: Arctiidae) was determined. The genome is a circular molecule 15 481 bp long. It presents a typical gene organization and order for completely sequenced lepidopteran mitogenomes, but differs from the insect ancestral type for the placement of tRNA^Met. The nucleotide composition of the genome is also highly A + T biased, accounting for 80.38%, with a slightly positive AT skewness (0.010), indicating the occurrence of more As than Ts, as found in the Noctuoidea species. All protein-coding genes (PCGs) are initiated by ATN codons, except for COI, which is tentatively designated by the CGA codon as observed in other lepidopterans. Four of 13 PCGs harbor the incomplete termination codon, T or TA. All tRNAs have a typical clover-leaf structure of mitochondrial tRNAs, except for tRNA^Ser(AGN), the DHU arm of which could not form a stable stem-loop structure. The intergenic spacer sequence between tRNA^Ser(AGN) and ND1 also contains the ATACTAA motif, which is conserved across the Lepidoptera order. The H. cunea A+T-rich region of 357 bp is comprised of non-repetitive sequences, but harbors several features common to the Lepidoptera insects, including the motif ATAGA followed by an 18 bp poly-T stretch, a microsatellite-like (AT)₈ element preceded by the ATTTA motif, an 11 bp poly-A present immediately upstream tRNA^Met. The phylogenetic analyses support the view that the H. cunea is closerly related to the Lymantria dispar than Ochrogaster lunifer, and support the hypothesis that Noctuoidea (H. cunea, L. dispar, and O. lunifer) and Geometroidea (Phthonandria atrilineata) are monophyletic. However, in the phylogenetic trees based on mitogenome sequences among the lepidopteran superfamilies, Papillonoidea (Artogeia melete, Acraea issoria, and Coreana raphaelis) joined basally within the monophyly of Lepidoptera, which is different to the traditional classification.     

A Betabaculovirus-Encoded gp64 Homolog Codes for a Functional Envelope Fusion Protein

The GP64 envelope fusion protein is a hallmark of group I alphabaculoviruses. However, the Diatraea saccharalis granulovirus genome sequence revealed the first betabaculovirus species harboring a gp64 homolog (disa118). In this work, we have shown that this homolog encodes a functional envelope fusion protein and could enable the infection and fusogenic abilities of a gp64-null prototype baculovirus. Therefore, GP64 may complement or may be in the process of replacing F protein activity in this virus lineage.     

Partial sequence and characterization of nitric oxide synthase gene from Hyphantria cunea

Nitric oxide synthase (NOS) gene has been partially sequenced from Hyphantria cunea and compared with those already determined from insects. Hyphantria cunea NOS possesses putative recognition sites for co‐factors heme, BH₄, CaM, FMN, FAD, and NADPH common to NOS. The deduced amino acid sequence of H. cunea NOS cDNA showed 70.3% identity to Manduca sexta NOS and 57.6–69.5% identity to NOS sequences from other insects. Nitric oxide synthase is expressed in all tissues of H. cunea, except in hemocytes. The NOS expression in midgut, fat body, epidermis, and Malpighian tubule strongly increased against Gram‐positive and Gram‐negative bacterial infection. These results suggest that NOS may play an important role in insect defense system against bacterial infection.     

Characterization of a Granulovirus Isolated from Epinotia aporema Wals. (Lepidoptera: Tortricidae) Larvae

A granulovirus (GV) isolated from Epinotia aporema (Lepidoptera: Tortricidae)—a major soybean pest—was studied in terms of its main morphological, biochemical, and biological properties. The ovoidal occlusion bodies were 466 by 296 nm in size, and their most prominent protein had an apparent molecular mass of 29 kDa. Its amino-terminal sequence was remarkably homologous to that of the granulins of other GVs. The DNA genome size was estimated to be 120 kbp. The high specificity and pathogenicity of this newly described granulovirus (EpapGV) indicate that it is indeed a good candidate for the biological control of this pest.     

Baculovirus VP1054 Is an Acquired Cellular PURα, a Nucleic Acid-Binding Protein Specific for GGN Repeats

Baculovirus VP1054 protein is a structural component of both of the virion types budded virus (BV) and occlusion-derived virus (ODV), but its exact role in virion morphogenesis is poorly defined. In this paper, we reveal sequence and functional similarity between the baculovirus protein VP1054 and the cellular purine-rich element binding protein PUR-alpha (PURα). The data strongly suggest that gene transfer has occurred from a host to an ancestral baculovirus. Deletion of the Autographa californica multiple nucleopolyhedrovirus (AcMNPV) vp1054 gene completely prevented viral cell-to-cell spread. Electron microscopy data showed that assembly of progeny nucleocapsids is dramatically reduced in the absence of VP1054. More precisely, VP1054 is required for proper viral DNA encapsidation, as deduced from the formation of numerous electron-lucent capsid-like tubules. Complementary searching identified the presence of genetic elements composed of repeated GGN trinucleotide motifs in baculovirus genomes, the target sequence for PURα proteins. Interestingly, these GGN-rich sequences are disproportionally distributed in baculoviral genomes and mostly occurred in proximity to the gene for the major occlusion body protein polyhedrin. We further demonstrate that the VP1054 protein specifically recognizes these GGN-rich islands, which at the same time encode crucial proline-rich domains in p78/83, an essential gene adjacent to the polyhedrin gene in the AcMNPV genome. While some viruses, like human immunodeficiency virus type 1 (HIV-1) and human JC virus (JCV), utilize host PURα protein, baculoviruses encode the PURα-like protein VP1054, which is crucial for viral progeny production.